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Heterologous expression and purification of bovine rod photoreceptor glutamic acid rich protein Mah, Nancy Lynne 1999

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H E T E R O L O G O U S EXPRESSION AND PURIFICATION O F BOVINE R O D P H O T O R E C E P T O R G L U T A M I C ACID R I C H P R O T E I N by N A N C Y L Y N N E M A H B . S c , University of Alberta, 1993 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Biochemistry and Molecular Biology) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRIT ISH C O L U M B I A July 1999 © Nancy Lynne Mah, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of BlOoWidrj flivl M^e6M/(q\r ]^c?(oCj ^ The University of British Columbia Vancouver, Canada Date / W * f * ir. 171*1 DE-6 (2/88) A B S T R A C T Glutamic A c i d R ich Protein exists in three alternately spliced forms within the bovine rod photoreceptor: 1) as part of the unusual bipartite structure of the p subunit of the c G M P -gated cation channel; 2) as a 590 amino acid polypeptide ( f -GARP) ; and 3) as a 299 amino acid polypeptide ( t -GARP) . The function of G A R P in photoreceptors is unknown. Although it is not visible by Coomassie Blue staining, t - G A R P appears to be the most abundant form of G A R P as seen by western blotting. Such an abundant protein must have a role in the function or structure o f the photoreceptor. As a first step in examining the function o f G A R P , a method for the recombinant production and purification o f t - G A R P was developed in this study. Heterologous expression o f t - G A R P was attempted in three different systems— bacterial, yeast and mammalian systems. t - G A R P could not be expressed in E. coli, but it was expressed by mammalian COS cells and in the yeast Pichia pastoris. COS cells produced 23 R O S Units of t - G A R P (1 R O S Unit = amount of t - G A R P in 12.5 ug of ROS) per 10 cm plate. From Pichia, t - G A R P was both secreted and produced intracellularly at 280 and 700 R O S units per 500 mL of culture, respectively. Considering the cost, ease of production, quality and yield of recombinant t - G A R P , the intracellularly produced t - G A R P was selected as the system from which to purify recombinant t - G A R P . Using affinity chromatography, 9% of the t - G A R P produced intracellularly was isolated from French-pressed, detergent solubilized yeast lysates. i i T A B L E OF C O N T E N T S Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgements Dedication 1. I N T R O D U C T I O N 1.1 The Retina 1.2 The Photoreceptor 1.3 Visual Transduction and Photorecovery 1.4 Overview of Glutamic Ac i d R ich Proteins 1.5 Thesis Investigations 2. M A T E R I A L S A N D M E T H O D S 2.1 Materials, Growth Media and Solutions 2.2 General Molecular Biology Techniques 2.2.1 Isolation of D N A Fragments 2.2.2 Ligation and Transformation of Plasmid D N A 2.2.3 Screening of Plasmid Constructs 2.2.4 D N A Sequencing i i i 2.2.5 Electroporation of Pichia 21 2.3 General Protein and Immunological Methods 22 2.3.1 Protein Electrophoresis and Western Blotting 22 2.3.2 Isolation of Monoclonal Antibody 23 2.3.3 Preparation of G A R 4B1 Coupled Sepharose Beads 24 2.4 Expression of t - G A R P in COS Cells 24 2.4.1 Maintenance o f COS Cells 24 2.4.2 Transfection of COS Cells 25 2.4.3 Analysis of Total Cel l Lysates 25 2.4.4 Immunofluorescence and Immunoperoxidase Labeling 26 2.5 Expression of t - G A R P in E. coli 28 2.5.1 Cloning of Bacterial Expression Construct 28 2.5.2 Induction of t - G A R P in E. coli 29 2.5.3 Analysis of Expression 31 2.6 Intracellular Expression of t - G A R P in Pichia pastoris 32 2.6.1 Cloning of t - G A R P into Expression Vectors 32 2.6.2 Growth and Analysis of Transformants 33 2.7 Secreted Expression of t - G A R P in Pichia pastoris 33 2.7.1 Cloning of t - G A R P into Extracellular Yeast Expression Vector 33 2.7.2 Growth and Screening of Transformants 35 2.7.3 Enzymatic Analysis of Secreted t - G A R P 36 2.8 Characterization of G A R 4B1 Antibody Epitope 36 2.9 Synthesis of Peptide 38 iv 2.10 Isolation of Bovine Rod Outer Segments 38 2.11 Purification of G A R P from R O S 39 2.12 Purification o f t - G A R P from Pichia pastoris 40 2.12.1 Mechanical Disruption of Yeast Cells 40 2.12.2 Aff inity Purification of G A R P 41 2.12.3 Quantitative Analysis o f Recombinant t - G A R P 41 3. R E S U L T S 43 3.1 Expression of t - G A R P in E. coli 43 3.2 Expression o f t - G A R P in COS cells 46 3.3 Expression of t - G A R P in Pichia pastoris 54 3.3.1 Extracellular Expression of t - G A R P in Pichia 54 3.3.2 Intracellular Expression in Pichia 56 3.4 Development of a Purification Protocol 63 3.4.1 Preparation of an Aff inity Column 63 3.4.2 Characterization o f G A R 4 B 1 Epitope 63 3.5 Purification of t - G A R P from R O S 67 3.6 Purification of t - G A R P from Pichia 67 3.6.1 Disruption of the Yeast Ce l l 67 3.6.2 Aff inity Purification of t - G A R P from Pichia 72 4. D I S C U S S I O N 79 4.1 Purification of t - G A R P from R O S 79 4.2 Heterologous Expression of t - G A R P in E. coli and in COS cells 79 4.3 Heterologous Expression of t - G A R P in Pichia 82 v 4.3.1 Secreted Expression of t - G A R P 4.3.2 Intracellular Expression of t - G A R P 4.4 Purification of t - G A R P from Pichia 4.5 Summary and Future Directions R E F E R E N C E S A P P E N D I X 1 Nucleic A c i d and Protein Sequence of Bovine Rod Photoreceptor t - G A R P A P P E N D I X 2 Codon Usage in Bovine Rod Photoreceptor t - G A R P A P P E N D I X 3 Clustal Alignment of f - G A R P and t - G A R P A P P E N D I X 4 List of Suppliers LIST OF T A B L E S Table 1. Vectors and Ce l l Strains 17 Table 2. Primers Used for D N A Sequencing 20 Table 3. Primers Designed for P C R Amplif icat ion o f t - G A R P 29 Table 4. Results of Electroporation of Pichia with Intracellular Construct p G A P Z B 6 C l 62 Table 5. Comparison of the Amino Acids Responsible for Determining Protein Concentration in B S A and t - G A R P 87 v i i L IST OF F I G U R E S Figure 1. Cross-section of a vertebrate eyeball 2 Figure 2. The cell layers of the vertebrate retina 3 Figure 3. The rod and cone photoreceptors 5 Figure 4. Phototransduction in the rod outer segment 7 Figure 5. Protein sequence of bovine f - G A R P 10 Figure 6. Mode l of a subunit of the cyclic nucleotide-gated channel 12 Figure 7. Schematic of the a subunit of the cyclic nucleotide-gated channel and the three forms of bovine rod outer segment G A R P 13 Figure 8. Map of truncated G A R P in pET 30 Figure 9. Map of truncated G A R P in Pichia p G A P Z vectors 34 Figure 10. Overlapping series of peptides generated by SPOTs kit 37 Figure 11. Coomassie stained S D S - P A G E gel of selected clones containing bacterial expression vector pET246C 1 44 Figure 12. Western blot of total cell extracts of selected clones containing bacterial expression vector pET246C 1 45 Figure 13 Detergent solubilized COS cell extracts containing t - G A R P 47 Figure 14. Immunoperoxidase labeling of COS cells transfected with various expression constructs. 49 Figure 15. Immunofluorescence labeling of transfected COS cells 51 Figure 16. Screening of Pichia transformants for extracellular expression of t - G A R P 55 v i i i Figure 17. Ammonium sulfate fractionation of culture supernatants containing secreted t - G A R P 57 Figure 18. Enzymatic digestion of t - G A R P from Pichia culture supernatant #2 58 Figure 19. Intracellular expression of t - G A R P in Pichia using expression vector p G A P Z B 6 C l 61 Figure 20. Intracellular expression of t - G A R P in Pichia using p G A P Z B 6 C 1-10 61 Figure 21. Purification of G A R 4 B 1 mouse monoclonal antibody by Protein G Agarose column chromatography 64 Figure 22. Characterization of G A R 4 B 1 epitope by scanning peptide analysis 65 Figure 23. Competition of 4B1 Peptide for binding to G A R 4 B 1 antibody 66 Figure 24. Purification o f G A R P from R O S using G A R 4 B 1 affinity chromatography 68 Figure 25. Flowchart of steps involved in purification of t - G A R P from sonicated Pichia cells 70 Figure 26. Extraction of recombinant t - G A R P from sonicated Pichia extracts 71 Figure 27. Extraction of recombinant t - G A R P from Pichia cells by French press 73 Figure 28. Aff ini ty purification o f t - G A R P from Pichia extracts produced by sonication 74 Figure 29. Ammonium sulfate fractionation of French pressed Pichia lysates 76 Figure 30. Purification of recombinant t - G A R P from French pressed detergent solubilized Pichia lysates using G A R 4 B 1 affinity chromatography 77 ix LIST OF A B B R E V I A T I O N S A R R P autosomal recessive retinitis pigmentosa B C A bicinchoninic acid B E S N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid bp base pairs B S A bovine serum albumin c D N A D N A reverse-transcribed from an m R N A template (coding D N A ) c G M P guanosine 3,5'-cyclic monophosphate C H A P S 3-[(cholamidopropyl)-dimethylammonio]-l-propanesulfonate D A B 3,3' -diaminobenzidine tetrahydrochloride D M E M Dulbecco's modified Eagle medium D N A deoxyribonucleic acid dNTP deoxyribonucleoside tri-phosphate D T T dithiothrieitol E C L enhanced chemiluminescence E D T A ethylenediamine tetraacetate E G T A [ethylenebis(oxyethylenenitrilo)] tetraacetic acid g gravity G A R P glutamic acid rich protein G C A P guanylyl cyclase activating protein h hour I P T G isopropyl-beta-D-thiogalactopyranoside x kb kilobase kDa kilodalton L B Luria-Bertani broth L S L B low salt L B min minute nt nucleotide P A G E polyacrylamide gel electrophoresis P C R polymerase chain reaction P D E phosphodiesterase P B S phosphate buffered saline R N A ribonucleic acid rpm revolutions per minute R O S rod outer segment s second SDS sodium dodecyl sulfate Tris Tris [hydroxymethyl]aminomethane U unit Y P D yeast peptone dextrose x i A C K N O W L E D G E M E N T S I am very grateful to Dr. Robert Molday for giving me the opportunity to work in his lab and for his guidance throughout my project. I would also like to thank Dr. Ph i l Bragg and Dr. Dana Devine for taking the time to be members o f my committee. Special thanks to Laurie Molday for her help with the antibodies and to Dr. Mark Brown for helping me with the Pichia system. Finally, I would like to thank the members o f the Molday lab, past and present: Dr. Andy Goldberg, Dr. Orson Mori tz, Dr. Carol Colv i l le , Dr. Michel le Il l ing, Dr. Tom K i m , Chris Loewen, Rene Warren, Igor Nasonkin, Natalie Rundle, Dr. Jinhi Ahn , Az ien Safarpour, Dr. Jason Wong, Dr. L i Zhang, Dr. Heidi Stohr, and Dr. Arnold Rabin. x i i To: my family xiii INTRODUCTION 1.1 The Retina The retina is borne of the brain. In early development, the neural tube forms two optical cups whose inner walls develop into the multi-layered structure, the retina (Dowling, 1987). In the vertebrate eye, the retina is the thin, innermost layer of the eyeball wal l (Figure 1). It is protected by a tough outer layer, known as the sclera, and a middle layer, called the choroid, which supplies the eye with nutrients. Both layers form a light-tight sphere only allowing light to enter through the cornea and lens (Fatt et al, 1992). The retina is a multi-layered cellular network which converts light into an electrical signal which is transmitted to the brain via the optic nerve (Dowling, 1987 and Ko lb , 1994). The cell layers within the retina (Figure 2) are arranged in what seems to be a backward order. Light must pass through transparent ganglion and nuclear cell layers before being finally detected by the photoreceptors partly embedded in the outermost layer o f the retina, the pigment epithelium. Signal initiated by the photoreceptor is transferred to a complex network of bipolar, horizontal, and amacrine neurons in the inner nuclear layer. Then the signal is relayed to the ganglion cells whose axons line the vitreous humor. These axons form bundles which compose the optic nerve and channel the signal to the brain. 1.2 The Photoreceptor The photoreceptor is a highly specialized neuron where the vision cascade begins. 1 sclera I choroid Figure 1. Cross-section of a vertebrate eyeball. The eyeball is encased in an outer layer composed o f an opaque sclera and a transparent cornea, where light enters the eyeball. The conjunctiva forms a thin, transparent membrane over the cornea and also lines the eyelid. The choroid layer, made mostly of blood vessels and connective tissue, provides nutrients to the eye. Towards the front of the eye, the iris controls the amount of light entering the eye, much like a camera shutter. The lens serves to focus the image. The innermost layer o f the eyewall is the retina, which serves as the light sensitive surface, analogous to the f i lm of a camera. Figure modified from Dose, 1995. 2 Choroidal Border Figure 2. The cel l layers of the vertebrate retina. The retinal pigment epithelium (RPE) forms the outermost layer of the retina. The rods (R) and the cones (C) form the photoreceptor layer (Ph). The photoreceptor layer is subdivided into the outer segment (OS) and the outer nuclear layer (ONL) . The synaptic region between the photoreceptors and the inner nuclear layer (LNL) is defined as the outer plexiform layer (OPL) . The inner nuclear layer contains a variety of cells, including horizontal (H), bipolar (B), amacrine (A), and Mi i l ler (M) cells. The inner plexiform layer (IPL) forms the synaptic region between ganglion cells (G) and the axons of the cells in the inner nuclear layer. Axons from the ganglion cell layer (GCL) bundle together to form the optic nerve. Light must pass through many cell layers before reaching the photoreceptor. Figure modified from Farber and Adler , 1986. 3 There are two types of photoreceptors in vertebrates, descriptively known by their shape (Figure 3). Cone cells are responsible for vision in bright light and color vision, while rod cells are functional in dim light only. Both cell types have similar cellular organization, consisting of a synaptic terminal, nucleus, and regions known as the inner and outer segments (reviewed by Hargrave, 1986; Berman, 1991). The synaptic terminal releases the neurotransmitter glutamate, whose steady f low is reduced by the detection o f light. The inner segment, proximal to the nucleus, contains cellular organelles such as the endoplasmic reticulum, golgi apparatus and many mitochondria for the energy needs of the cel l . The outer segment is the heart of the phototransduction process. This segment is densely packed with thousands of flat membranous disks, much like a tall stack of pancakes. In the rod cel l , the disks are closed, but in the cone cel l , the disks are continuous with the plasma membrane. Within these disks lie the visual pigments that absorb light and initiate the phototransduction pathway. Rods have one visual pigment (rhodopsin), while cones have three pigments (blue, green and red opsin). A l l visual pigments consist of a seven-transmembrane protein, opsin, which is linked covalently to the chromophore, 11-as-retinal (Nathans, 1987). Amino acid sequence differences within opsin impart sensitivity to different wavelengths of light. There are many advantages to working with rod cells as opposed to cone cells. First, rod cells are more abundant and are larger than cone cells in most animals. Second, rod outer segments are relatively easy to isolate on sucrose gradients (Molday and Molday, 1993). Final ly, bovine eyes are readily available from a local slaughterhouse. For these reasons, most studies, including this one focus on the rod photoreceptor. 4 Disk Plasma Rod Outer Segment Cone Figure 3. The rod and cone photoreceptors. The photoreceptors share similar structure. The outer segment contains the phototransduction machinery. The inner segment contains vital components for cell metabolism. The signal generated in the photoreceptors is propagated to other neurons through the synaptic terminal. Figure modified from Mori tz , 1996. 5 1.3 Visual Transduction and Photorecovery The dissection of the visual system has served as an accessible model system in which to study neuronal processes. Through its utility as an ideal model, the process o f visual transduction has been well-investigated (reviewed by Yau , 1994; Jindrova, 1998). The process of phototransduction initiates in the disk of the rod outer segment (Figure 4). Light, having passed through the lens and penetrated many layers o f neuronal cells in the retina, is absorbed by one of the many thousands of molecules o f rhodopsin in the rod disks. Within the core of this seven-transmembrane protein lies the chromophore 11-cis-retinal. Upon absorbing a photon of light, the chromophore is isomerized to all-£raws-retinal, thus causing a conformational change which activates rhodopsin to its catalytic form metarhodopsin II (M*) . The cytoplasmic side of M * then interacts with the peripheral membrane protein transducin, a trimeric GTP-binding protein (T ap r). Upon activation by M * , transducin exchanges G D P for G T P , forming an activated T a p Y -GTP complex. The complex then dissociates into T a - G T P , Tpy and M * . The signal is amplified hundreds of times as M * continues to activate other G-proteins. Meanwhile, activated T a - G T P activates c G M P phosphodiesterase (PDE) by interacting with the inhibitory y subunits of P D E . P D E goes on to hydrolyse thousands of molecules o f c G M P to 5 ' G M P in the cytoplasm, thus amplifying the signal further. The decrease in intracellular [cGMP] causes the cGMP-gated channels in the plasma membrane to close, because there are fewer c G M P molecules available to hold the channels open. The inward current of N a + and C a 2 + through the channel is halted, and continuous extrusion of intracellular C a 2 + through the N a + / K + / C a 2 + exchanger further decreases the intracellular [Ca 2 + ] . As a consequence, the plasma membrane becomes 6 Photoreceptor Disk Plasma Membrane Figure 4. Phototransduction in the rod outer segment. Light isomerises 11-cw-retinal to l l - / ro«5 - ret inal within rhodopsin (R) to produce the activated form, metarhodopsin II (M* ) . M * activates transducin (T) by causing the exchange of G D P for GTP. The activated Tct-G T P interacts with the inhibitory y subunit o f c G M P phosphodiesterase (PDE) , al lowing P D E to hydrolyse c G M P to 5'GMP. The resulting decrease in [cGMP] causes the c G M P -gated channel to close, preventing Ca 2 +and N a + from entering the photoreceptor. Continuous extrusion o f C a 2 + by the N a + / K + / C a 2 + exchanger causes intracellular Ca 2 + levels to drop. 7 hyperpolarized. This electrical signal causes a reduction in the release of glutamate from the synaptic terminal and the signal is forwarded to the brain through bipolar, horizontal and ganglion cells. Whi le visual excitation is fairly well-characterized, photorecovery is less defined. Restoration to the dark state requires re-opening of the channel and the inactivation of rhodopsin. Both of these processes involve calcium, whose concentration is reduced below 100 n M by the photoresponse. To re-open the channel, P D E must be inactivated and retinal guanylate cyclase (RetGC) must be activated. T a - G T P deactivates itself through its own intrinsic GTPase activity, which is enhanced by P D E y subunit (Arshavsky and Bownds, 1992) and a GTPase activating protein R G S - 9 (Wei et al, 1998). Thus, T a - G T P is converted to T a - G D P , which can no longer activate P D E . Re tGC is activated through the action o f guanylate cyclase activating proteins (GCAP) . Act ive only at low [Ca 2 + ] , G C A P s stimulate retGC to synthesize c G M P , thus raising [cGMP] and re-opening the cGMP-gated channel (Gorczyca et al, 1994; Palczewski et al, 1994). Moreover, decreased C a 2 + levels cause the cGMP-gated channel to become more sensitive to c G M P . This is accomplished through the release o f a Ca 2 + /calmodul in complex which reduces the affinity o f the channel for c G M P (Hsu and Molday, 1993). The return of the photoreceptor to the dark state also requires inactivation of rhodopsin, since activated rhodopsin wi l l continue to set off the visual cascade. Rhodopsin is inactivated by phosphorylation by rhodopsin kinase and binding of arrestin (Wilden et al, 1986). This effect is thought to be mediated through C a 2 + levels, since S-modulin (also known as recoverin), inhibits phosphorylation of rhodopsin at high [Ca 2 + ] (Kawamura, 1993) . Once rhodopsin is inactivated, the photoreceptor is further quenched by the 8 regeneration of the chromophore (Palczewski et al, 1997). The membrane-associated enzyme, all-frans-retinal dehydrogenase, reduces all-?ra«s-retinal to all trans-VQtmo\, a form which cannot bind opsin. In a yet-to-be defined mechanism, the retinol makes its way to the R P E , where the retinol is converted into 1l-c/s-retinol. 11-c/s-retinol can then be stored or oxidized to 1 l-c/s-retinal by its dehydrogenase. B y an unknown mechanism, 11-c/s-retinal is transported from the R P E back to the disks of the photoreceptor, where it can re-bind opsin to form rhodopsin. 1.4 Overview of Glutamic A cid Rich Proteins Glutamic acid rich protein (GARP) exists in three alternatively spliced forms within the rod photoreceptor. The first discovered form is now known as full G A R P ( f -GARP) . It was first cloned by Sugiomoto et al. (1991) using a polyclonal antibody raised against a phosphodiesterase-enriched retinal extract from bovine rod outer segments (ROS). The protein was named G A R P due to its abnormally high content of glutamic acid residues. In the carboxy-terminal half of f - G A R P , there is a region of 109 amino acids where 68 are glutamic acid residues. Within this region, 28 of the glutamic acids are included in four repeats o f an 11-amino acid glutamic acid rich peptide. There are also two repeats of a 26-amino acid peptide in the carboxy-terminal half (Figure 5). The importance of these two peptide repeats is unknown. Overall, f - G A R P contains 24 % glutamic acid and 10% proline. G A R P was also identified as part of the cyclic nucleotide-gated channel. For some time, it was thought that the channel consisted of a homooligomer of the first discovered 9 MLGWVQRVLPQPPGTPQKTKQEEEGTEPEPELEPKPETAPEETELEEVSLPPEEPCVGKE 60 VAAVTLGPQGTQETALTPPTSLQAQVSVAPEAHSSPRGWVLTWLRKGVEKVVPQPAHSSR 120 PSQNIAAGLESPDQQAGAQILGQCGTGGSDEPSEPSRAEDPGPGPWLLRWFEQNLEKMLP 180 QPPKISEGWSDEPTDAALGPEPPGPALEIKPMLQAQESPSLPAPGPPEPEEEPIPEPQPT 24 0 IQASSLPPPQDSARLMAWILHRLEMALPQPVIRGKGGEQESDAPVTCDVQTISILPGEQE 300 ELHLILEEVDPHWEEDEHQEGSTSTSPRTSEAAPADEEKGKVVEQTPRELPRIQEEKEDE 360 EEEKEDGEEEEEEGREKEEEEGEEKEEEEGREKEEEEGEKKEEEGREKEEEEGGEKEDEE 4 2 0 GREKEEEEGRGKEEEEGGEKEEEEGRGKEEVEGREEEEDEEEEQDHSVLLDSYLVPQSEE 4 80 DQSEESETQDQSEVGGAQTQGEVGGAQALSEESETQDQSEVGGAQDQSEVGGAQAQGEVG 54 0 GAQEQDGVGGAQDQSTSHQELQEEALADSSGGSFQMSPFEALQECEALKR 590 Figure 5 . Prote in sequence of bovine f - G A R P . Double underlined sequence indicates the 11-amino acid repeat. Single underlined sequence indicates the 26-amino acid repeat. Arrowheads enclose a 109 amino acid region where 68/109 residues are glutamic acid. 10 subunit, now known as the a subunit. The a subunit from the cyclic nucleotide-gated channel was described first in bovine photoreceptors (Kaupp et al, 1989) and later in human photoreceptors (Dhallan et al, 1992). Based on homology to voltage-gated K + and C a 2 + channels and the c G M P binding domains of c G M P dependent protein kinases, the a subunit was modeled to contain six transmembrane domains, a voltage sensor-like motif, a pore region, and a c G M P binding domain (Figure 6). But then in 1993, Chen et al. reported the existence of a protein from human retina which had only 30% identity to the human a subunit. This protein, now called p', did not form functional channels when expressed alone in human embryonic kidney 293 cells. Upon co-transfection of both a and P' subunits, the channels displayed characteristics similar to native rod channels. These characteristics included activation by c G M P , rapid flickering in channel opening, sensitivity to the channel blocker L-c/s-dil it iazem, and modulation by Ca 2 + /calmodul in (Chen et al, 1993,1994; Hsu et al, 1993, 1994). However, the story was not finished. The P' subunit was discovered to be incomplete (Korschen et al, 1995; Colvi l le et al, 1996). It was shown in both bovine and human retina that the complete p subunit was a 240 kDa protein o f unusual bipartite structure. The C-terminal part of the p subunit was homologous to the a subunit and contained two calmodulin binding sites (Chen et al, 1994; Grunwald et al, 1998; Weitz et al, 1998). The N-terminal part o f the bovine p-subunit was virtually identical to bovine f-G A R P (Figure 7), while the N-terminal part of the human p-subunit was homologous to bovine f - G A R P . The third and final form of G A R P is a 299 amino acid polypeptide named truncated or t - G A R P . There are few published studies on rod photoreceptor t - G A R P . In one study, 11 COOH Figure 6. Model of a subunit of the cyclic nucleotide-gated channel. The channel contains six transmembrane domains (S1-S6) and a pore region, all of which are comparable to those found in the Shaker potassium channel. S4 contains a voltage sensor-like motif similar to that found in voltage-activated channels. A lso shown is the cycl ic nucleotide binding site (CNB) , homologous to cGMP-binding domains of cGMP-dependent protein kinase, (reviewed in Finn et al, 1996). 12 CNB 690 • c 92 N 1 2 B GARP Part © 0 © p' Part 572 N I 1 13 CNB I 1394 He GAR4B1 1 © © © 590 Nl I [C G A R 4 B 1 '—GSFQMSPFEALQECEALKR D 1 299 NI n c GAR4B1 I—RVVAAGSL Figure 7. Schematic of the a subunit of the cyclic nucleotide-gated channel and the three forms of bovine rod outer segment GARP. (A) The a subunit of the channel consists of six transmembrane regions (shaded boxes 1-6), a pore region (arrowhead) and the cycl ic nucleotide binding domain (CNB) . The first 92 amino acids are cleaved off in native a subunits. (B) The p subunit consists of a G A R P part and a P' part. The G A R P part contains repeats of a glutamic acid rich peptide. The P' part contains all the elements of the a subunit and additionally contains two calmodulin binding sites (hatched boxes). (C) Ful l G A R P is identical to the G A R P part of the P subunit of the channel, except for the last 19 amino acids, which are indicated as offshoots. (D) Truncated G A R P is about half the length o f f - G A R P and is completely identical to f - G A R P except for the last eight amino acids. The position of the epitope of the mouse monoclonal antibody G A R 4 B 1 is indicated for all three G A R P variants. 13 human t - G A R P was isolated from a retinal c D N A library using a D N A probe from the original bovine f - G A R P . Through further screening of a retinal genomic library, the genomic organization of the human t - G A R P was determined (Ardell et al, 1995; Ardel l et al, 1996). The gene mapped to 16ql3 and was originally suggested to be a candidate gene for Bardet-Biedl syndrome, a disorder which is associated with retinal degeneration and coincidentally maps within a broad region including 16ql3 to 16q22 (Kwitek-Black et al, 1993). However, Bardet-Biedl has since been linked to 16q21 (Bruford et al, 1997), so G A R P is no longer a candidate gene. There are mutations in the a subunit of the C N G channel (Dryja et al, 1995) which are linked to autosomal recessive retinitis pigmentosa ( A R R P ) , a set of genetically heterogeneous diseases in which rod and cone photoreceptors progressively degenerate and lead to blindness. Conversely, there are no known diseases linked to mutations in the p-subunit. Linkage between mutations in the G A R P portion o f the P subunit and A R R P is under investigation (Bharadwaj et al, 1999). A l l three forms of G A R P were shown to be present in human, rat and bovine rod outer segments (Figure 7) using an antibody made against the first 16 conserved amino acids of G A R P (Colvi l le and Molday, 1996). Bovine t - G A R P was subsequently isolated from a retinal c D N A library. It shares 60% identity with the human homologue and is 299 amino acids long. Unl ike f - G A R P , t - G A R P does not contain C-terminal repeat elements. Nevertheless, t - G A R P is rich in glutamic acid (12.7%) but more so in proline (17%). The predicted M W from the primary sequence is 32 kDa, yet it runs at about 60 kDa on an S D S -P A G E gel. The anomalous migration of t - G A R P on gels has also been observed for the p subunit and f - G A R P , and its migration has been attributed to the high content of glutamic acid and proline residues (Korschen et al, 1995). 14 Since the first a subunit of a cyclic nucleotide gated channel was first cloned by Kaupp et al. (1989), cyclic nucleotide channels have been found in many tissues, including skeletal muscle, colon, kidney, testis, and heart (reviewed in Kaupp, 1995; F inn et al., 1996). These channels have been found in sensory and central nervous tissue such as the cone photoreceptors, the olfactory epithelium, hippocampal and cerebellar neurons, and the pineal gland. The fact that many cyclic nucleotide-gated channels exist in other tissues lends to the possibility that G A R P may also be associated with other P subunits of the channel. To date, P t subunits have been detected in olfactory epithelium (Sautter et al, 1998), pineal gland (Sautter et al., 1997), testis, retina, kidney, heart and brain (Biel et al., 1996). However, G A R P has only been found as part of the P subunit in pineal gland and to a lesser extent, in testis. The only other tissue in which t - G A R P has been detected is the pineal gland. Since both the photoreceptor and the pineal gland respond to light stimuli, it is possible that G A R P has a particular function in these cells. Then again, G A R P may exist in other tissues and may yet be discovered. 1.5 Thesis Investigations Invisible by Coomassie staining, t -GARP appears to be present in abundant amounts in R O S as seen by western blotting (Colvi l le and Molday, 1996). Despite its apparent abundance, the function o f G A R P is unknown, whether it exists as part o f the p-subunit o f the channel or as a separate polypeptide. The presence or absence of the G A R P moiety on the P-subunit of the channel does not appear to affect channel properties (Korschen et al., 1995), so G A R P may have a structural function. The goal of this study was to develop a high 15 level cell expression system of t - G A R P , as a first step in studying its structure and function in photoreceptors. 16 M A T E R I A L S AND M E T H O D S 2.1 Materials, Growth Media and Solutions Unless otherwise noted, all lab chemicals were obtained from Fisher Scientific, Sigma, B D H Inc., Gibco B R L , Bio-Rad, or Di fco Laboratories. Vectors and strains used in this study are described in Table 1. Table 1. Vectors and Cel l Strains Vector E. coli Host and Genotype Media/Selection for Cloning Expression Host Media/Selection for Expression pBluescriptSK+ (Stratagene) X L 1-Blue: r ecA l endA l gyrA96 thi-1 hsdR17 supE44re lA l lac [FproAB lacPZ&M15 TnlO (TetR)] LB /Ampic i l l i n (50 ug/mL) E. coli LB /Amp ic i l l i n p c D N A l / A m p (Invitrogen) X L - 1 Blue L B / A m p COS cells D M E M / n o antibiotic selection pET24b (Novagen) TOPI OF: ¥-{lacr TnlO (TetR)} mcrA A(mrr-hsdRMS-mcrBC) (|)80/acZAM15 AlacX74 deoR rec A l araD139 A(ara-leu)1691 galU galK rpsL (StrR)endAl nupG) JM110: rp lL(Str R ) / /zr leu thi-1 lacYgalK galT ara tonA tsx dam dcm suppE44 A(lac-proAB) [F' traD36 proAB lacfZAM15] LB/Kanamycin (50 ug/mL) BL21(DE3) : F- ompT hsdSB(vB-m B -) gal dcm (DE3) LB/Kanamyc in p G A P Z a and p G A P Z B (Invitrogen) T O P I OF LSLB/Zeocin™ (25 ug/mL) X 3 3 : wi ld type SMD1168: pep4 his4 YPD/Zeocin™ (100-1000 ug/mL) 17 One litre recipes for frequently prepared growth media and solutions are as follows. A l l agar plates contained 15 g agar/L. L B : 10 g tryptone, 10 g N a C l and 5 g yeast extract dissolved in water, autoclaved to sterilize. L S L B : 10 g Tryptone, 5 g N a C l and 5 g yeast extract dissolved in water, autoclaved to sterilize. Y P D : 10 g yeast extract, 20 g peptone, and 20 g glucose, autoclaved to sterilize. Towbin: 3.03 g Tris base, 14.4 g glycine, 100 m L methanol in water. P B S : 80 g N a C l , 2 g KC1, 2 g K H 2 P 0 4 , 11.5 g N a 2 H P 0 4 , p H adjusted to 7.4 with HC1. 2.2 General Molecular Biology Techniques 2.2.1 Isolation of DNA fragments. Plasmid D N A was cut using restriction enzymes and 10X buffers from Pharmacia, New England Biolabs, or Gibco B R L . Typical ly, 1 - 5 ug of plasmid D N A , 1 U of restriction enzyme per pg D N A , and 10X restriction buffer was mixed in a total volume of 20 - 100 uL and incubated at 37°C for 1 - 2 h. If necessary, the D N A fragments were dephosphorylated by adding 0.5 U of calf intestinal phosphatase (Gibco B R L ) and further incubated at 37°C for 30 min. D N A fragments were separated by 1 - 2% agarose gel electrophoresis (Sambrook et al., 1989). D N A was stained by including 50 ug/mL ethidium bromide in the running buffer and gel. Gels were observed on a U V transilluminator at 254 nm and photographed by Gel Documentation System ( U V P Inc). Desired fragments were cut from the gel and purified using a GeneClean K i t (B io lO l ) . 18 2.2.2 Ligation and transformation of plasmid DNA. Vector and insert D N A were added in a molar ratio of 1 : 3 to a reaction mixture containing T4 D N A ligase (Pharmacia), 5 X ligation buffer (Gibco B R L ) , and sterile water to a final volume of 20 - 30 uL. The ligation mixture was incubated at 14°C overnight and then diluted five times for subsequent transformation into the appropiate E. coli strain. Competent E. coli cells were prepared according to the C a C l 2 method (Sambrook et ah, 1989). Aliquots of competent cells were stored in 0.1 M C a C l 2 , 15% glycerol and flash frozen in l iquid nitrogen for storage at -80°C. Plasmid D N A was transformed into competent cells by heat shock as follows. Usually, 100 -200 p L of ligated plasmid D N A was mixed with 200 uL of thawed competent cells on ice. Cells were heat shocked at 42°C for 2 min, then left on ice for 5 min. Appropiate sterile l iquid media (Table 1) was added to the cells and D N A , and the mixture was incubated at 37°C for 1 h to allow for expression of the antibiotic resistance. Cells were then plated on plates containing the appropiate antibiotic. Plates were incubated overnight at 37°C. 2.2.3 Screening of plasmid constructs. For a mini preparation of plasmid D N A , 2 m L of l iquid media containing the appropiate antibiotic was inoculated with a single colony and grown overnight at 37°C with shaking. For a maxi preparation, 100 - 500 m L of l iquid media was inoculated with 50 - 100 uL o f an overnight bacterial pre-culture. Plasmid D N A was isolated from these cultures using a mini- or maxi-prep kit (Qiagen) according to the kit instructions. The integrity of the plasmid constructs was initially checked by restriction digest as described in Section 2.2.1 and confirmed by D N A sequencing (Section 2.2.4) i f the fragment was a polymerase chain reaction (PCR) product. 19 2.2.4 DNA sequencing. D N A sequencing was done in two ways. For shorter lengths of D N A , sequencing was done manually by dideoxy chain termination method using the T7 Sequenase Quick Denature plasmid sequencing kit (Amersham) according to the manufacturer's instructions. Any new primers required for sequencing were synthesized by Nucleic A c i d and Protein Service (NAPS) at the University of Brit ish Columbia (Table 2). Selected constructs were sequenced on both strands to reduce the possibility of error. For longer lengths of D N A , sequencing was done by N A P S at the University of Brit ish Columbia. Table 2. Primers Used for D N A Sequencing Primer Name Direction Sequence Description T7~ Forward 5 ' -GTA A T A C G A C T C A C T A T A G G G C-3' sequence from T7 promoter, 5' to multicloning site C A C 3 " Forward 5 ' -CCC A C T A T T C A A G C C T C C - 3 ' sequence from t - G A R P c D N A at nt 765-783 T7tr Reverse 5 ' -GCT A G T T A T T G C T C A G C G G-3' sequence from T7 transcription terminator in pET24b, 3' to multicloning site 52150r Reverse 5 ' -CCT G G A G G G T T C A C T G G G TTC-3 ' sequence from t - G A R P c D N A at nt 521-501 p G A P F " Forward 5 ' -GTC C C T A T T T C A A T C A A T T G A A - 3 ' sequence from p G A P Z vectors, 5' to multicloning site 153176'" Forward 5'-GTT G G A G C C G A A G C C T G A A A C A G C T-3' sequence from t - G A R P c D N A atnt 153-176 328346 T Forward 5 ' - A C A G C A G T C C C A G A G G C T G-3' sequence from t - G A R P c D N A at nt 328-346 501523" Forward 5 ' -GGG C T C A G A T G A A C C C A G T G A A C - 3 ' sequence from t - G A R P c D N A atnt 501-523 myc Reverse 5 ' -ATG A T G G T C G A C G G C G C T A T T C-3' sequence from p G A P Z vectors, 3' to multicloning site 887867" Reverse 5 ' -CCC T T C C C A C G G A T C A C T GGC-3* sequence from t - G A R P c D N A at nt 867-887 C A C 2 " Reverse 5 ' -CCC A A G G C A G C A T C T G T A GG-3 ' sequence from t - G A R P c D N A at nt 646-627 20 282266" Reverse 5 ' - G A G T G A G A G C A G sequence from t - G A R P c D N A T C T C C - 3 ' at nt 282-266 *obtained from N A P S "previously made by Dr. Carol Colvi l le + newly synthesized for this study 2.2.5 Electroporation of Pichia. To prepare D N A for electroporation into Pichia, 10 pg of the construct was linearized with Bsp HI . The enzyme was heat-inactivated and then protein in the sample was removed by phenokchloroform extraction. Plasmid D N A was precipitated from the aqueous phase by adding 3 M sodium acetate to 0.3 M and 2.5 volumes of ethanol. D N A was pelleted by centrifuging 15 min at 14000 rpm. The pellet was'washed in 80% ethanol, air-dried, and resuspended in 10 uL of sterile water. Pichia strains X33 or SMD1168 were prepared for electroporation according to the Pichia expression vector manual (Invitrogen). Brief ly, 500 m L of Y P D in a 2 L flask was inoculated with 100 uL of an overnight pre-culture. The 500 m L culture was incubated at 30°C with shaking (250 rpm) until ODgQO =1 .3 -1 .5 , approximately 22 h. The cells were then pelleted by centrifugation and washed twice in sterile, ice-cold water and once in ice-cold 1 M sorbitol. Final ly, the cells were resuspended in 1 m L of cold 1 M sorbitol. To electroporate the yeast cells, 80 uL of the sorbitol-resuspended cells was mixed with 10 uL of linearized construct and transferred into an ice-cold 0.2 cm electroporation cuvette. The cells were pulsed by an Invitrogen Electroporator using a charging voltage of 1500 V , a capacitance of 50 uF, and a resistance of 200 Q. One m L of ice-cold sorbitol was quickly added to the cuvette and the cells were transferred to a sterile 15 m L polypropylene tube. Cells were allowed to recover at 30°C for 1 h prior to plating 50 - 200 m L on Y P D 21 plates containing 100 pg/mL or 1000 ug/mL Zeocin™. After incubating 3 - 7 days at 30°C, colonies were picked and patched to fresh YPD/Zeocin™ plates. 2.3 General Protein and Immunological Methods 2.3.1 Protein electrophoresis and western blotting. Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( S D S - P A G E ) was routinely carried out as described by Sambrook et al. (1989) using a gel apparatus from Hoefer or Bio-Rad. Samples were usually prepared by mixing with an equal amount of 2 X SDS sample buffer (5% SDS, 10 m M Tris HC1 p H 6.8, 40 % sucrose, 0.1 % bromophenol blue and 8% p-mercaptoethanol). Typical ly, 10-40 uL of sample was applied to each lane of a 10% polyacrylamide gel and electrophoresis was carried out using the Laemmli buffer system (1970). Gels were stained using two different methods. For general purposes, an S D S - P A G E gel was stained 30 min in Coomassie Blue stain (25 % isopropanol, 10 % acetic acid, 0.025 % Coomassie Bril l iant Blue R-250). Gels were destained overnight in 10 % acetic acid. For more sensitive detection of proteins, gels were silver stained as described by Wray et al. (1981). Gels were preserved by drying between two sheets o f cellophane (Bio-Rad). Western blotting and detection was typically performed as follows. Protein gels were transferred to Immobilon-P (Mill ipore) in Towbin buffer using a Bio-Rad semidry transfer cell for 30 min at 350 mA. The blots were blocked in a 1% milk solution in P B S . To detect G A R P , G A R 4 B 1 mouse monoclonal antibody was used. Supernatants from a hybridoma cell line producing the G A R 4 B 1 antibody were diluted 1:30 in 0.1% milk in P B S and incubated with the blots at room temperature for 1 h. The blot was washed three times for 10 min each 22 with P B S . The secondary antibody, a sheep antimouse Ig linked to horseradish peroxidase (Amersham), was diluted 1:5000 in 0.1% milk in P B S . After a 30 min incubation at room temperature in secondary, antibody, the blot was washed in P B S as before. Proteins on the blots were detected by the E C L Western blotting detection system (Amersham) according to the manufacturer's instructions. 2.3.2 Isolation of monoclonal antibody. The mouse monoclonal antibody (GAR4B1) had been previously generated by L. Molday against the first 16 amino acids of G A R P (Colvi l le and Molday, 1996). G A R 4 B 1 antibody was isolated from the ascites of Balb-c mice which had been injected with 4B1 hybridoma cells. The ascites was diluted 1:1 with P B S . The antibody was precipitated by adding an equal amount of saturated ammonium sulfate to the diluted ascites with stirring at 4°C. The precipitate was collected by a 20 min spin at 8000 rpm and resuspended in binding buffer (10 m M N a 2 H P 0 4 p H 7, 150 m M N a C l , 10 m M E D T A ) . Ammonium sulfate was dialysed out in three changes o f binding buffer. The G A R 4 B 1 antibody was purified on GammaBind Plus Protein G beads (Genex Corp). Protein G Agarose beads (2 mL) were washed five times in binding buffer and poured into a glass chromatography column (Kontes Glass Co.). Concentrated, dialysed ascites was passed through the column, and the flow through was saved. The column was washed ten times in binding buffer. Antibody was eluted in 2 m L aliquots of 0.5 M acetic acid p H 3.0 into a tube containing 1.3 m L of 1 M Tris HC1 p H 9 to quickly neutralize the acid. Eluted fractions were run on a 10 % S D S - P A G E gel. Bands were visualized by Coomassie Blue staining (Section 2.3.1). 23 2.3.3 Preparation of GAR4B1 coupled Sepharose beads. Purified antibody was coupled to Sepharose 2B beads (Pharmacia). Sepharose beads were activated by C N B r as described by Cuatrecasas (1970). Antibody was dialysed in three changes of 20 m M borate p H 8.4, 0.1 M N a C l . Dialysed antibody was incubated with activated beads at a concentration of 1 mg antibody per 1 m L of beads. After 4 h of incubation on a slowly rotating wheel at 4°C, GAR4B1-Sepharose beads were washed in 20 m M Tris HC1 p H 8, 0.15 M N a C l , 0.05 M glycine to block any free reactive sites. Beads were then stored at 4°C in 20 m M Tris p H 8, 0.15 M N a C l , 10 m M sodium azide. 2.4 Expression of t-GARP in COS Cells For transient expression of t - G A R P in mammalian cells, the expression vector p c D N A l / A m p (Invitrogen) was used. In this vector, expression of recombinant protein was controlled by a human cytomegalovirus promoter for high level constitutive expression. 2.4.1 Maintenance of COS cells. A l l tissue culture media and antibiotics were obtained from Gibco B R L . Cells were grown in Dulbecco Modi f ied Eagle Medium ( D M E M ) supplemented with 10 % fetal calf serum, 100 U / m L pennicil l in, 0.1 mg/mL streptomycin, and 0.625 pg/mL Fungizone®. Media was filter sterilized and stored at 4°C. Cells were incubated in a 5 % CO2, 37°C incubator until nearly confluent. The cells were then trypsinized from the plate by adding trypsin to 0.05 %, incubating 3 minutes at 37°C, and tapping the plate to loosen the cells. As soon as the cells were released from the plate, 10 m L 24 of D M E M was added to stop the trypsinization. The cells were re-plated at a dilution of 1:10 and grown as before. 2.4.2 Transfection of COS cells. Constructs containing the c D N A of t - G A R P and f - G A R P were previously made (Colvi l le and Molday, 1996). Constructs containing peripherin and rhodopsin were generated by Chris Loewen and Dr. Andy F .X. Goldberg, respectively. The expression plasmids were transformed into COS cells using the calcium phosphate precipitation method o f Chen and Okayama (1987). COS cells were plated at a concentration of 2 X 10 5 cells per 3 m L plate or 6 X 105 cells per 10 m L plate. Plates were incubated for 24 h at 37°C in 5% CO2. The following amounts are for a 3 m L / 1 0 mL plate. The next day, 8 - 1 2 pg/ 24 - 36 pg expression vector was mixed in a total of 124/ 372 uL sterile water. Then 41/ 123 uL of 1 M C a C l 2 was added to the diluted D N A . Next, 165/ 495 uL of B B S (50 m M N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) , 280 m M N a C l , 1.4 m M N a 2 H P 0 4 , p H to 6.95) was added dropwise to the D N A - C a C l 2 mixture while gently vortexing. After the mixture was incubated 20 min at room temperature, it was slowly added dropwise to a plate o f COS cells while gently swirl ing the plate. The plates were incubated at 35°C in 3% C 0 2 overnight. The following morning, the plates were washed in P B S and 3/ 9 m L of fresh medium was added. The cells were further incubated for 24 h at 37°C in 5% co2. 2.4.3 Analysis of total cell lysates. Cells were washed twice in P B S before solubilization buffer (PBS, 1% Triton-X-100, 0.1 mg/mL P M S F ) was added. The plates were incubated at 4°C for 20 min and the cells were then scraped off the plates and centrifuged 30 min at 30000 25 g in a Beckman Optima™ T L Ultracentrifuge. Samples of both the detergent soluble supernatant and the detergent insoluble pellet were prepared for S D S - P A G E . Sample was added to an equal amount of 2 X S D S - P A G E loading buffer and 20 uL of each sample was loaded onto a 10 % acrylamide gel. Insoluble fractions were boiled 5 min prior to loading. The gel was transferred to Immobilon-P membrane as described in Section 2.3.1. Specific proteins were visualized using antibodies against the protein of interest and the E C L detection method. 2.4.4 Immunofluorescence and immunoperoxidase labelling. To determine the distribution of recombinant t - G A R P in the COS cel l , the cells were labeled by immunofluorescence or immunoperoxidase methods. Transfection was performed as described in Section 2.4.1. The morning after incubating at 35°C in 3% CO2, the cells were washed two times in P B S and then trypsinized as in Section 2.4.1. To visualize the cells under the microscope, the cells were grown on sterile glass disks. A sterile glass disk was placed in a well of a 24-well tissue culture plate. The disks were wetted with a drop o f media, and then 0.5 m L of the trypsinized cells was pipetted into the well . Cells were then incubated 24 h at 37°C in 5% CO2. The cells on the glass disks were washed in two times in P B S , 5 min each wash. To fix the cells, each well was incubated in 40% paraformaldehyde made in 100 m M sodium phosphate, p H 7.4, for 10 min at room temperature. Wells were then washed in P B S two times, 10 min per wash. Next, the cells were blocked 1 5 - 3 0 min in 100 m M sodium phosphate buffer p H 7.4 containing 10% normal goat serum and 0.5% Triton-X-100. Blocking buffer was aspirated away and then diluted primary antibody in 100 m M sodium phosphate buffer p H 7.4 containing 5% normal 26 goat serum and 0.5% Triton-X-100 was added. For t - G A R P transfected cells, ant i -GARP rabbit polyclonal antibody was diluted 1:1000, and G A R 4 B 1 mouse monoclonal was diluted 1:5. For rhodopsin-transfected cells, R h o l D 4 mouse monoclonal antibody was diluted 1:500 or 1:100. For peripherin-transfected cells, Per2B6 mouse monoclonal was diluted 1:5. After l h incubation in primary antibody, cells were washed two times in P B S for 10 min each and incubated 30 min in secondary antibody. For immunochemical staining with 3,3'-diaminobenzidine tetrahydrochloride (DAB) , anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase was diluted to 1:50 in a total of 150 pL. For immunofluorescence labelling, Cy3™ -conjugated Aff iniPure Goat Anti-Rabbit or Ant i-Mouse IgG Antibody (Jackson ImmunoResearch Laboratories, Inc.) was diluted to 1:700 in a total volume of 150 uL. Fol lowing secondary antibody incubation, the cells were washed two times in P B S , 10 min each. A t this point, the immunofluorescence labelled glass disks were fixed with media to glass slides. The immunochemical labelled disks required staining to visualize labelling. Stain solution was prepared by diluting 0.5 m L of 10 X DAB/Me ta l Chelating Concentrate (Pierce) in 4.5 m L of I X peroxidase buffer (Pierce). About 200 uL of stain solution was added to each wel l , and the reaction was monitored at 10X magnification under a microscope. When the cells turned to a dark brown to black color, the reaction was stopped by addition of P B S . The glass disks were then fixed to glass slides. Slides were viewed and photographed using a Zeiss Axioplan 2 microscope. 27 2.5 Expression of t-GARP in E. coli The pET system for bacterial expression was used to express t - G A R P in E. coli. In this system, the target protein is produced under the control of bacteriophage T7 transcription and translation signals. T7 R N A polymerase, which is present in the host BL (DE3 ) , is tightly controlled by the lac UV5 promoter. Upon addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), T7 R N A polymerase from the host is expressed, and the target protein is produced. 2.5.1 Cloning of bacterial expression construct. Because the t - G A R P c D N A did not contain convenient sites for cloning, P C R primers G l , G2 , and G 3 A (Table 3) were designed to amplify out the coding region of t - G A R P and to provide convenient sites for cloning into pET24b (Novagen), as well as cloning into intracellular or extracellular Pichia expression vectors (Invitrogen). The template for the reaction was p K S 6 C l + , the original c D N A clone of t - G A R P isolated from a bovine retinal c D N A library (Colvi l le and Molday, 1996). The coding region was amplified by P C R . The reaction was carried out by 1 uL of a 10"6 dilution o f D N A template, 5 uL 10X P C R buffer (Gibco B R L ) , 2 uL 50mM M g C l 2 (Gibco B R L ) , 1 uL 12.5 u M Primer G l , 1 uL 12.5 u M Primer G2 or G3 , 0.5 uL 20 m M dNTP (Pharmacia) and 1 uL Taq polymerase (Pharmacia or Gibco B R L ) in a total volume of 50 uL. A typical P C R program used was 4 min at 94°C, then 25 cycles of denaturation at 94°C for 15 s, annealing at 52-56°C for 20 s and extension at 72°C for 90 s. P C R products purified from 1% agarose gels were digested with Nde l/Xho I for cloning into PET24b. Due to the high error rate of Taq polymerase, errors were expected in the P C R product. To reduce the 28 amount of D N A sequencing to be done, a portion in the middle of the P C R product was replaced by cloned D N A , which was presumed to be error-free. The construct was transformed into JM110, a bacterial strain which was deficient in D N A cytosine methylase. Plasmids isolated from this strain enabled the methylation sensitive restriction enzyme Stu I to cut. A 390 bp fragment between Stu I and Bam HI in the middle of the P C R product was then cut out and replaced by a cloned fragment (Figure 8). Flanking sequence which arose from P C R was then sequenced as previously described in Section 2.2.4. Final constructs were named p E T 6 C l . Table 3. Primers Designed for P C R Amplif ication of t - G A R P Name Direction Sequence Description G l Forward 5'-G G A A T T C C A T A T G C T G G G C T G G G T C C-3' This 26-mer contains an Nde I site for cloning into bacterial expression vector pET24b after the ribosome binding site and before the T7 tag. The Eco R l site can be used for intracellular yeast expression vector and is in-frame for yeast secretion vectors. G2 Reverse 5 ' -GGA A T T C T C G A G C G G T C A G A G G C T T C C A G C T G C C-3' This 34-mer contains a stop codon for non-expression of C-terminal tags and an Xho I site for cloning into pET24b. There is also an Eco R l site for cloning into the yeast intra/extracellular expression vectors. G 3 A Reverse 5'-C C A C C G T C G A C C T T C G G A G T G A G C T C G T TGT-3 ' This 31-mer lacks a stop codon for expression of C-terminal tags and contains an in-frame Xho I site for fusion to His tag of pET24b vector. 2.5.2 Induction of t-GARP in E. coli. Final expression constructs were transformed into the E. coli strain BL21(DE3) . For expression studies, protein induction was carried out based on 29 T7 CAC3 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 kb -\ h ATG Stu I Bam HI H 1 h TGA 521501 T7tt Figure 8. Map of truncated GARP in pET. Truncated G A R P was cloned into pET24b as described in text. Shaded rectangle denotes cloned D N A fragment. Open boxes show D N A amplified by P C R . Single line indicates pET24b sequence. Arrows above and below map show positions of forward and reverse primers used for D N A sequencing. Sequence of primers is described in Table 2. 30 Novagen manual. A single colony was used to inoculate 2 m L LB/kanamycin. The culture was incubated at 37°C with shaking until the ODgoo w a s between 0.6 - 1.0. This preculture was stored at 4°C overnight. The next day, the cells were resuspended in fresh LB/kanamycin and used to inoculate 50 m L of LB/kanamycin. The culture was grown for about 2 h to an OD500 of 0.6. A t this point, three 500 uL aliquots were removed for samples of uninduced total protein. The inducer IPTG was added to the 50 m L culture to a final concentration of 1 m M . The culture was incubated at 37°C or 30°C with shaking. A t various time points (9 min, 1 h, 2 h, 3 h, 21 h), 500 uL samples were removed for analysis. Cells were harvested by centrifuging the culture at 5000 g for 5 min at 4°C. 2.5.3 Analysis of expression. To examine total cell protein, the cell pellet was resusupended in 0.1 culture volumes (50 uL) of buffer (10 m M Tris HC1, p H 8.0). A n equal volume of 2 X SDS sample buffer was added and the sample was heated 70°C 5 min prior to loading on duplicate S D S - P A G E gels. Volumes of sample loaded were adjusted so that each lane would contain about the same amount of protein. Thus 10 uL of the zero time point and 5 uL of the subsequent time points were loaded per lane. One gel was stained by Coomassie Blue, while the other was blotted onto Immobilon-P. Western blots were probed by G A R 4 B 1 mouse monoclonal antibody as described in Section 2.4.2. 2.6 Intracellular Expression of t-GARP in Pichia pastoris The p G A P Z series of expression vectors from Invitrogen intracellularly produce target protein under the constitutive promoter for glyceraldehyde-3-phosphate dehydrogenase 31 ( G A P D H ) . The gene of interest is cloned directly after the promoter. In the absence of a secretion signal, the protein is produced intracellularly. Selection of transformants containing inserts is facilitated by the marker Zeocin™, which can be used in both E. coli and Pichia. 2.6.1 Cloning of t-GARP into expression vectors. For the intracellular expression in yeast, two constructs were made. First, the entire c D N A was cloned from p K S 6 C l into the Eco Rl/Xho I sites of p G A P Z B using techniques described in Section 2.2. This construct, named p G A P Z B 6 C l , contained 5' and 3' non-coding sequence, as wel l as start and stop codons within the coding sequence. The vector was electroporated into yeast as in Section 2.2.5. The second construct was made by only amplifying out the coding region. VentR® D N A polymerase from New England Biolabs was used instead of Taq polymerase since VentR® had a proofreading subunit whereas Taq polymerase did not. Conditions for Vent R® D N A polymerase were optimized by adding formamide to the reaction and by varying the M g S 0 4 concentration, the annealing temperature and the amount of enzyme. The final reaction mixture for VentR® P C R included 1 uL of D N A template (10"°" dilution of p S K 6 C l ) , 5 uL of 10X ThermoPol Reaction buffer (NEB) , 1 uL of 12.5 u M stock of primer G l and G2 , 0.5 uL of 20 m M dNTP, 2.5 uL of 100 % formamide, 0.25 uL of Vent R® D N A polymerase (2 U/mL) and sterile water to 50 uL. The P C R program was as follows: 4 min at 94°C, then 19 cycles of 15 s at 94°C, 20 s at 65 - 56°C at a decreasing rate of 0.5°C per cycle, and 1 min at 72°C. Amplif ication was completed by 20 cycles of 15 s at 94°C, 20 s at 56°C, and 1 min at 72°C. P C R products were digested with Eco RI andXho I and cloned into the Eco RVXho I sites of p G A P Z B using methods described in Section 2.2. Two clones 32 were isolated and named p G A P Z B 6 C l - 1 0 and p G A P Z B 6 C l - l 1. The c D N A portion was D N A sequenced by methods in Section 2.2.4 using primers shown in Figure 9. Both clones were transformed into yeast by electroporation as described in Section 2.2.5. 2.6.2 Growth and analysis of transformants. Transformants which grew wel l on 1000 ug/mL Zeocin™ were patched onto a fresh Y P D plate containing 1000 pg/mL Zeocin™ and incubated 3 days at 30°C. Using a sterile pipet tip, the patched yeast cells were transferred to a microfuge tube containing 100 uL 2 X SDS sample buffer. The cells were well mixed in the buffer and then boiled 5 min prior to loading on an S D S - P A G E gel. Yeast clones were analysed for t - G A R P expression by western blot and E C L detection (Section 2.3.1). One clone named #10-SMD1168-67 was chosen for further analysis. 2.7 Secreted Expression of t-GARP in Pichia pastoris The p G A P Z a series of Pichia expression vectors is identical to the p G A P Z vectors described in Section 2.6, except an a-factor secretion signal is provided between the promoter and the multicloning site. For secreted expression, the target protein must be cloned in-frame with the secretion signal. 2.7.1 Cloning of t-GARP into extracellular yeast expression vector. For extracellular expression in yeast, the expression vector p G A P Z a A was used. This vector was identical to p G A P Z B except that p G A P Z a A had a few different cloning sites and contained an a-factor signal sequence which had to be ligated in-frame with the coding sequence of the t - G A R P 33 A pGAPF 153176 328346 501523 CAC3 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 kb ' 1 1 | 1 1 1 1 1 1 ' ' 1 plrnH 1— ATG TGA 282266 521501 CAC2 887867 myc B pGAPF 153176 328346 501523 CAC3 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 kb , • • • • • * * ' ' ' ' II—Ir ATG TGA 282266 521501 CAC2 887867 myc Figure 9. Map of truncated GARP in Pichia pGAPZ vectors. (A) Truncated G A R P was cloned into the Eco R l / X h o I sites o f p G A P Z B . (B) Truncated G A R P was cloned in-frame with a secretion factor of p G A P Z a as described in text. Shaded rectangle shows P C R amplified product. Open boxes show vector elements surrounding the insert: 1. G A P D H promoter; 2. myc epitope; 3. polyHis tag; 4. a secretion factor. Single line indicates p G A P Z sequence. Arrows above and below map show positions of forward and reverse primers used for D N A . sequencing. Sequence of primers is described in Table 3. 34 insert. t - G A R P insert was cut out of p G A P Z B 6 C l - l 1 using Eco R l andXba I, and inserted into the Eco Rl/Xba I sites of p G A P Z a A . The resulting expression construct was named p G A P Z a A 6 C l . 2.7.2 Growth and screening of transformants. P G A P Z a 6 C l was electroporated into Pichia strain SMD1168 as described in Section 2.2.5. Selected transformants which grew wel l upon re-streaking on fresh plates were used to inoculate 10 m L of Y P D in a 50 m L sterile conical tube. The cultures were grown at 30°C with shaking (250 rpm). Samples of 100 uL were removed each day for 5 days. To analyse for t - G A R P expression, cells were pelleted by centrifugation. The cell pellet was resuspended in 100 uL 2 X SDS Sample Buffer, while 25 uL 2 X SDS was added to 75 uL of supernatant. Pellet and supernatant samples were run on S D S - P A G E gels, western blotted and detected by E C L as in Section 2.3.1. For larger scale expression, 100 uL of an overnight preculture was used to inoculate 100 m L Y P D for incubation at 30°C for 24 h. The culture was centrifuged 10 min at 3000 g and the supernatant was collected. To concentrate t - G A R P present in the supernatant, solid ammonium sulfate was added to 30% saturation and stirred at 4°C for 1 h. Precipitated protein was collected by centrifuging 30 min at 4900 g. The pellet was resuspended in 3 m L of buffer ( 20 m M Tris p H 7.4, 0.1 M NaCl ) . The supernatant was precipitated two more times at 50 % and 70 % saturation. Re-dissolved pellets were dialysed at 4°C in three changes of buffer (20 m M Tris p H 7.4, 0.1 M NaCl) using dialysis tubing with a molecular cutoff o f 10-12 000 M W (Spectrum Medical Industries, Inc.). Proteins obtained at 30, 50 and 70 % saturation of ammonium sulfate were analysed for t - G A R P by Western blotting as described in Section 2.3.1. 35 2.7.3 Enzymatic analysis of secreted t-GARP. Secreted t - G A R P was digested with neuraminidase and O-glycosidase to remove any glycosylation by the yeast cells. The buffer in which secreted t - G A R P was originally resuspended was changed to 20 m M sodium cacodylate p H 6.0 by dialysis. A n aliquot (99 uL) of t - G A R P was then treated with 0.01 U of neuraminidase (Boeringher) and/or 0.5 m U O-glycosidase (Boeringher) at 37°C for 4 h. Digested samples were analysed by western blotting and E C L detection as previously described. 2.8 Characterization of GAR4B1 Antibody Epitope The epitope of G A R 4 B 1 antibody was determined using a SPOTs kit (Genosys Biotechnologies, Inc.) according to kit instructions. Basically, a series of overlapping peptides was synthesized on a SPOTs membrane, and then the membrane was probed with G A R 4 B 1 antibody to determine which peptides contained the epitope. In this instance, a series o f 8-amino acid peptides with an off-set of one amino acid (Figure 10) was used to scan the original 16 amino acid sequence used to generate G A R 4 B l ( C o l v i l l e and Molday, 1996). K i t protocols were followed except for the final SPOTs analysis, where the kit protocol did not work. Instead, E C L detection (Section 2.3.1) was used to detect which peptides contained the epitope. 36 Original Peptide M L G W V Q R V L P Q P P G T P Scanning Peptides 1 M L G W V Q R V 2 L G W V Q R V L 3 G W V Q R V L P 4 W V Q R V L P Q 5 V Q R V L P Q P 6 Q R V L P Q P P 7 R V L P Q P P G 8 V L P Q P P G T 9 L P Q P P G T Figure 10. Overlapping series of peptides generated by SPOTs kit. 37 2.9 Synthesis of Peptide Two peptides, both containing the epitope of G A R 4 B 1 antibody, were used. The first peptide, named "4B1 Peptide" had the sequence: N - t e r m — G W V Q R L P Q P G — C - t e r m . This peptide was manually synthesized on a L K B B io lynx 4175 Peptide Synthesizer (Pharmacia). Synthesis was completed on a Fmoc-gly Novasyn P A 500 resin (Novabiochem) using solid phase synthesis (Merrif ield, 1963) with Fmoc polyamide active ester chemistry (Atherton et al, 1978). Addit ional peptide, named " P e p G A R P # l " , had the sequence: N-term— L G W V Q R V L P Q P — C - t e r m and was commercially synthesized by Genemed Synthesis, Inc. 2.10 Isolation of Bovine Rod Outer Segments. Rod outer segments (ROS) were isolated from frozen bovine retinas obtained from Schenk Packing Co. Inc. Retinas were thawed overnight at -20°C and processed under dim light as follows. To 50 retinas, 20 m L of homogenization buffer (20 m M Tris H A c p H 7.4, 10 m M taurine, 0.25 m M MgCl2, 10 m M glucose, and 20 % sucrose) was added. The rods were broken off from the retina by shaking for one min. The retinas were then filtered through a Teflon membrane. Material retained by the filter was subject to another round o f homogenization and filtering. The resulting filtrate from both rounds was layered on a 28 -50 % sucrose gradient with 20 m M Tris H A c p H 7.4, 10 m M taurine, 0.25 m M M g C l 2 , and 10 m M glucose. The gradients were centrifuged at 26 000 rpm for 1 h at 4°C on a Beckman L8-70 Ultracentrifuge. A major pink band containing R O S was removed from the gradient and washed in 40 m L of homogenization buffer. R O S was pelleted by centrifuging at 10 000 38 rpm, 10 min at 4 °C. R O S pellets were resuspended in 5 m L homogenization buffer containing 1 m M Pefabloc®SC (Boehringer Mannheim). R O S was then aliquoted into microfuge tubes, wrapped in foi l and stored at -80°C. The protein concentration of R O S was determined by B C A Protein Assay (Pierce) according to manufacturer's instructions. 2.11 Purification of GARP from ROS R O S were solubilized in Triton-X-100 as follows. R O S (1 mg) was pelleted from sucrose by centrifugation at 10 000 g for 10 min at 4°C. The R O S pellet was resuspended in 1 m L of 10 m M Tris HC1 p H 7.4 and added dropwise with stirring to a solution of 10 m M Tris H C L p H 7.4, 2 % Triton-X-100, and 0.1 M N a C l . After 5 min at room temperature, the solution was stirred for 1 h at 4°C. Detergent insoluble material was removed by centrifugation for 15 min at 98 000 g. The supernatant was saved for loading on a G A R 4 B 1 affinity column. G A R P was isolated from R O S by affinity chromatography. Solubil ized R O S (2 mL) was diluted in an equal amount of column buffer (10 m M Tris HC1 p H 7.4, 0.1 M N a C l , and 0.1 % Triton-X-100) to reduce the concentration of Triton-X-100 to 0.5 %. The R O S was then added to 100 pL of G A R 4 B 1 beads washed in column buffer. Binding was allowed to occur during incubation for 30 - 60 min on a rotating wheel at 4°C. The unbound material was collected by centrifugation through a 0.45 um spin filter unit (Mil l ipore). The beads were washed in column buffer and then eluted in 2 X SDS sample buffer without 0-mercaptoethanol or eluted with 1 mg/mL 4B1 Peptide. Samples including solubilized R O S , R O S pellet, G A R 4 B 1 column flow through and eluted R O S proteins were loaded on 39 duplicate S D S - P A G E gels for analysis. One gel was stained with Coomassie Blue or silver stained, while the other gel was subject to Western blotting and E C L detection. 2.12 Purification of t-GARP from Pichia pastoris 2.12.1 Mechanical disruption of yeast cells. To produce t - G A R P for purification, 500 m L of Y P D was inoculated with 0.1 m L of overnight pre-culture, and incubated with shaking at 30°C for 22 h, until OD600 = 1.3 - 1.6. The cells were pelleted by centrifuging 5 min at 3000 g, flash frozen in liquid nitrogen, and stored at -80°C. Two different methods were employed to disrupt the yeast cells. In the first method, an ultrasonicator (Misonix) was used to break open the cells. Defrosted cells were washed in 10 m M Tris HC1 p H 7.4, 0.1 M N a C l and resuspended in the same buffer including protease inhibitor tablets (Boehringer Mannheim). A n equal amount of 425 - 600 micron glass beads (Sigma) was added. The cells were then sonicated at power level 7 for 20 min in a dry ice-ethanol bath to dissipate heat produced by sonication. Glass beads were removed by passing the homogenate through a 10 m L syringe plugged with fish filter floss. The homogenate was then solublized by adding the solution dropwise to an equal volume of 10 m M Tris HC1, 0.1 M N a C l , 2 % Triton-X-100. The solubilized homogenate was then homogenized further by five passes in a glass hand homogenizer (Kontes). Large cell debris was removed by centrifugation for 20 min at 10 000 g in a Beckman T L Ultracentrifuge. Clarifed supernatant was spun 440 000 g for 1 h. Large cell debris from the low speed spin and samples from the high speed spin pellet and supernatant were analysed for t - G A R P by western blotting. 40 In the second method, the cells were disrupted by French press based on a procedure used by Beaudet et al. (1998). A thawed cell pellet was resuspended in 25 m L of cold S M + glycerol (1.2 M sorbitol, 20 m M Tris HC1 p H 7.0, 5 m M E D T A , 20 % glycerol). Cells were then frozen in liquid nitrogen and rapidly thawed in a room temperature water bath. The cells were centrifuged and the cell pellet was resuspended in cold buffer (20 m M Tris HC1 p H 7.4, 0.15 M N a C l , 1 m M E G T A , 1 m M E D T A , 1 m M D T T , protease inhibitor tablets) at a volume of 5.3 m L per g wet cell weight. The cell suspension was passed through a French press (Aminco) three times at a pressure of 20 000 psi. Triton -X-100 (to a final concentration o f 1%) was then added dropwise to the lysate with stirring at 4°C. After 10 min of mixing, the lysate was spun two times in a chilled centrifuge for 20 min at 14000 g. Cel l samples collected throughout the procedure, as wel l as the final clarified lysate, were analysed for G A R P by methods in Section 2.3.1. 2.12.2 Affinity purification of t-GARP. t - G A R P was purified from detergent solubilized high speed supernatants (Section 2.12.1) from sonicated yeast or from clarified, detergent solublized French press lysates. Basically, the yeast lysate was diluted with an equal volume of column buffer (10 m M Tris HC1 p H 7.4, 0.1 M N a C l , 0.2 % Triton-X-100). Recombinant t - G A R P was isolated from the diluted lysates by affinity chromatography as described in Section 2.11, except that the final washes were done in detergent-free column buffer. 2.12.3 Quantitative analysis of recombinant t-GARP. Yie lds of recombinant t - G A R P from Pichia were compared to amounts o f t - G A R P present in R O S . A standard amount o f R O S (12.5 pg) was run on each western blot containing recombinant t - G A R P samples. The blots 41 were probed with G A R 4 B 1 antibody and signal was detected as described in Section 2.3.1. To compare the amounts of R O S t - G A R P and recombinant t - G A R P , the density of the t-G A R P bands at 60 kDa were quantitated using an L K B Ultroscan X L Laser Densitometer ( L K B Productker A B ) . The bands were scanned multiple times to find the maximum signal for each band. Rather than using the instrument's integrator function, peaks corresponding to the maximum signal of each band on the f i lm were cut out and weighed. The weight of each of the cut-out peaks was assumed to be directly proportional to the amount of t - G A R P on the blot. Peak weights of recombinant t - G A R P were compared to the peak weights o f the 12.5 pg of R O S . One R O S unit of t - G A R P was defined to be the amount of t - G A R P in 12.5 pg of R O S . The amounts of recombinant t - G A R P were expressed in R O S units. 42 RESULTS The goal of this study is to express t - G A R P at a high level in a heterologous system, as a first step to understanding its structure and function. To this end, expression of t - G A R P was tried in three different expression systems. 3.1 Expression of t-GARP in E. coli Six different independent clones of the expression construct p E T 2 4 6 C l were transformed into the expression strain BL21(DE3) and induced at 37°C and 30°C. For a negative control, an empty pET24b plasmid was used. For a positive control, a pET expression construct which expressed a 50 kDa protein (generously provided by X i n Miao) was used. Total cel l extracts of the induced cells at different time points were subject to S D S - P A G E (Figure 11) and western blot analysis (Figure 12). None of the cultures went clear during induction, indicating that the cells did not lyse due to toxicity of the expressed product. In the Coomassie stained gel of the samples grown at 37°C, no protein was visibly over-expressed in any o f the samples containing pET246C l plasmids or p E T vector alone, whereas the positive control showed strong over-expression of the 50 kDa protein after 1 h of induction. Because t - G A R P does not stain well by Coomassie Blue, duplicate blots of the gels were probed with G A R 4 B 1 mouse monoclonal antibody. A one minute exposure of film to the E C L treated blot detected a number of faint bands in the protein extract lanes, and a strong signal from G A R P in R O S , which was loaded as a positive control (not shown). A 43 1 3 5 pET24b kDa m 0 9 1 2 3 0 9 1 2 3 0 9 1 2 21 0 9 1 2 2 0 5 -116— 9 7 . 4 -6 6 -4 5 -2 9 - <M* - i t mm v n rttf , .. .... ... ......... A 6(7) 6(8) 6(9) 2-11 kDa m 0 9 1 2 3 0 9 1 2 3 0 9 1 2 3 0 9 1 21 2 0 5 -116— 9 7 . 4 -6 6 -4 5 -2 9 -Figure 11. Coomassie stained SDS-PAGE gel of selected clones containing bacterial expression vector pET246Cl. Cultures were grown to O.D.=0.6. Six different transformants (1, 3, 5, 6(7), 6(8), 6(9)) were induced at 3 7 C in 1 m M IPTG. Empty pET24b vector and positive control 2-11 were also induced. Samples were withdrawn at 0 and 9 min, 1, 2, 3 and 21 h. Twice as much sample (10 uL) was loaded in the zero time point lane compared to other time points. Most lanes contained about 1 pg of protein, except sample 5, which did not grow as fast as the other cultures. No induced bands at the molecular weight of t - G A R P (60 kDa) were visible in the stained gel. Negative control (pET24b) displayed no induced bands, while positive control (2-11) contained a strong induced band at 50 kDa. Lane 'm ' contains standard protein markers from Sigma. 44 kDa 1 3 5 pET24b 0 9 1 2 3 0 9 1 2 0 9 1 2 21 0 9 1 2 116— 97.4- A M 66-45- m m 29- m ^ Hi •j||§jp HI 6(71 6(81 6(91 2-11 kDa RosO 9 1 2 3 0 9 1 2 21 0 9 1 2 3 0 1 2 116-97.4-66-45-29-f • it • « t • Figure 12. Western blot of total cell extracts of selected clones containing bacter ial expression vector p E T 2 4 6 C l . These blots correspond exactly to the Coomassie stained gels in Figure 11. A lane of R O S was loaded for the second blot to show the size of t-G A R P from R O S . Blots were probed with G A R 4 B 1 antibody. After a ten minute exposure, some clones showed that bands were induced at 60, 55, 30, and 25 kDa fol lowing one hour of induction. No bands were strongly labeled in the positive (2-11) or negative (pET24b) control. 45 10 minute exposure of the same blot revealed that the antibody labeled a number of bands. A signal at about 30 kDa seemed to be induced after 1 and 2 h of induction. However, this band was smaller than the apparent molecular weight o f R O S t - G A R P , 60 kDa (Figure 12). A fainter band at about 60 kDa also seemed to be induced after 1 h of induction. However, both of these induced bands were only visible after a long exposure to the f i lm. Induction of the bacteria at 30°C produced no detectable induction. There was a high background and no signal was induced over time (not shown). Because the expression constructs were created using fragments generated by P C R using Taq polymerase, it was possible that errors had been introduced in the D N A sequence. D N A sequencing of two of the expressed clones (#5, 6(8)), showed that no stop codons had been introduced, but a single base pair change was found in each of two clones. These changes resulted in amino acid substitutions o f Arg l05Trp in one clone and Met265Val in another. 3.2 Expression of t-GARP in COS cells To evaluate mammalian COS cells as a high-level expression system, COS cells were transiently transfected with the mammalian expression construct p c D N A - 6 C l . The COS cells were solubilized in Triton-X-100 and samples were centrifuged to separate the detergent-soluble and insoluble proteins. COS cell extracts were subjected to S D S - P A G E and western blotting (Figure 13). A s a transfection control, p c D N A containing the disk membrane protein peripherin was also transfected and expressed in COS cells. A s seen in the western blot of the COS cell extracts, G A R 4 B 1 antibody strongly labeled a band 46 1 t-garp i 15 ug 20 ug 30 ug kDa m ROS sol insol sol insol sol insol 205-116 -97.4-66 -45 -29 -peripherin 30 MS kDa m ROS sol insol 205-1 116-97.4 -66-45 H 29 A B t-garp kDa I I 15 ug 20 ug 30 jag m ROS sol insol sol insol sol insol peripherin , i 30 ug kDa m sol insol Figure 13. Detergent solublized COS cell extracts containing t - G A R P . (A) Coomassie stained S D S - P A G E gel of COS cell extracts. Cells were transfected with 15, 20 or 30 ug of a p C D N A containing t -GARP. P C D N A containing peripherin (30 pg) was transfected as a positive control for the transfection procedure. Out of 500 p L of solubilized cells from one 10 cm plate, 10 uL of a detergent soluble (Lane 'sol') and a detergent insoluble sample (Lane 'insol') were loaded on the gel. Also loaded were 12.5 pg R O S (Lane 'ROS') and 10 pg of marker (Lane'm'). About 4-5 pg of COS cell protein was loaded per lane. (B) Western blots o f duplicate S D S - P A G E gels from A . Blots were probed with ant i -GARP rabbit polyclonal antibody (for t-GARP-transfected cells) or Per2B6 mouse monoclonal antibody (for peripherin-transfected cells). After a one minute exposure to film, t - G A R P was detected in detergent soluble COS cell extracts. Peripherin was also expressed as a positive control for the transfection. 47 corresponding to the molecular weight of t - G A R P after a one minute exposure. t - G A R P was produced by COS cells after transfection of 15-30 pg of plasmid construct. Virtual ly all o f the recombinant t - G A R P was solublized in Triton-X-100. From densitometry of the t - G A R P signal on the blot, 500 uL of a COS cell protein extract from a 10 cm plate yielded an average of 23 R O S units of t - G A R P (1 R O S unit=amount of t - G A R P in 12.5 pg ROS) . This is equivalent to the amount of t - G A R P in 288 pg of R O S . The transfection was repeated to examine the transfection efficiency and localization of the expressed protein within the COS cell using immunoperoxidase labeling and immunofluorescence microscopy. To compare expression patterns of t - G A R P , peripherin and rhodopsin were also transfected into COS cells and labeled with their respective primary antibodies. A s a negative control, untransformed cells were labeled with rabbit polyclonal antibody against t - G A R P (rPC6N). Immunoperoxidase labeling with D A B (Figure 14) showed that COS cells expressing rhodopsin labeled evenly throughout the cel l , l ikely distributed all over the plasma membrane since it is an integral membrane protein (Oprian et al., 1987). Peripherin appears to be concentrated around the nucleus of the COS cel l , perhaps the result of being stalled in the endoplasmic reticulum or golgi apparatus as previously reported (Goldberg et al., 1995). t - G A R P , predicted to be a soluble protein, also appears to be expressed throughout the cell. Immunofluorescence labeling revealed the same result (Figure 15). Most o f the cells observed on the slide were not labeled, suggesting that the transfection efficiency was low, only about 10%. 48 Figure 1 4 . Immunoperoxidase labeling of COS cells transfected with various expression constructs. Cells were fixed to glass cover slides and permeablized with Triton-X-100 prior to labeling with primary antibody. Untransfected cells (Panel A ) and cells transfected with t - G A R P (Panel D) were labeled with ant i -GARP rabbit polyclonal antibody, while peripherin (Panel C) and rhodopsin (Panel B) transfected cells were labeled with Per2B6 and R h o l D 4 mouse monoclonal antibody, respectively. For staining with D A B , the cells were incubated with anti-mouse or anti-rabbit conjugated to horseradish peroxidase and stained with D A B to yield a dark precipitate. Untransfected cells (panel A ) did not stain, while staining was visible in cells transfected with rhodopsin (panel B ) , peripherin (panel C) and t - G A R P (panel D). Cells were magnified 1000 X . 49 50 ( Figure 15. Immunofluorescence labeling of transfected COS cells. Untransfected COS cells were labeled with ant i -GARP rabbit polyclonal antibody and anti-rabbit secondary antibody and viewed by normal phase contrast (Panel A ) and by fluorescence microscopy (Panel B) . COS cells transfected with rhodopsin were labeled with R h o l D 4 mouse monoclonal antibody and anti-mouse antibody conjugated to Cy3 dye, and viewed by normal phase contrast (Panel C) and by fluorescence microscopy (Panel D). Cells transfected with peripherin were labeled with Per2B6 monoclonal antibody and anti-mouse antibody conjugated to Cy3 dye, and viewed by normal phase contrast (Panel E) and by fluorescence microscopy (Panel F). Finally, cells transfected with t - G A R P were labeled with ant i -GARP rabbit polyclonal and anti-rabbit antibody conjugated to Cy3 dye and viewed by normal phase contrast (Panel G) and fluorescence microscopy (Panel H). 51 B 52 53 3.3 Expression of t-GARP in Pichia pastoris The Pichia expression system offers advantages over bacterial and mammalian cells. Yeast cells are eukaryotic, and are more similar to a mammalian cell than a prokaryotic bacterial cell . In addition, yeast can be grown like bacteria in flasks, whereas mammalian cells require meticulous care in tissue culture. Furthermore, the growth rate o f yeast is much faster than mammalian tissue culture, so a large cell mass can be grown faster and more cost effectively. This makes scale-up much simpler for yeast cells than for mammalian cell culture. 3.3.1 Extracellular expression of t-GARP in Pichia. Two different methods of Pichia expression were tried. In one method, Pichia was engineered to secrete t - G A R P into the culture media. The extracellular expression construct p G A P Z a 6 C l was electroporated into yeast. A total of 54 colonies grew on Y P D plates containing 100 pg/mL Zeocin. To screen for expression, selected transformants were grown in 10 m L of Y P D at 30°C for 2-5 days. Ce l l pellets and supernatants were assayed for t - G A R P production by western blot. t - G A R P was secreted from most clones (Figure 16). After a one minute exposure, G A R 4 B 1 monoclonal antibody strongly labeled different proteins (33, 70, and 80 kDa) in the cell pellet (not shown). These may represent differentially glycosylated or degraded forms of t - G A R P . After a 10 minute exposure of the f i lm, two secreted proteins at about 70 kDa and 33 k D a became visible. The 70 kDa proteins probably represent a processed form of t - G A R P , while the 33 kDa protein could be a degraded form of t - G A R P . 54 23 24 25 26 49 50 kDa pell 1 2 pell 1 2 pell 1 2 pell 1 2 pell 1 2 pell 1 2 B 51 52 53 54 kDa pell 1 2 pell 1 2 pell 1 2 pell 1 2 pell 1 2 3 4 5 Figure 16. Screening of Pichia transformants for extracellular expression of t-GARP. Selected transformants (23, 24, 25, 26, 49, 50, 51, 52, 53, 54, 2) were grown in 10 m L of Y P D for 1-5 days at 30°C. Ce l l pellets (pell) from day 2 and unconcentrated culture supernatants from days 1 and 2 or 1 through 5 were loaded onto S D S - P A G E gels and western blotted (shown). After a 10 min exposure, G A R 4 B 1 antibody labels a major band at about 70 k D a and a minor band at 33 kDa in the culture supernatants. Many proteins strongly label within the pellet. These proteins may represent degraded or post-translationally modified forms within the yeast cell. 55 Although this system was able to secrete t - G A R P , its molecular weight was higher than that of t - G A R P from R O S (60 kDa). A signal sequence was added onto the N-terminal of t - G A R P to facilitate secretion of t - G A R P , but the signal sequence should be cleaved off by Pichia. It is possible that the increase in the molecular weight of t - G A R P was caused by glycosylation. Since t - G A R P contains no N-glycosylation sites, the secreted t - G A R P may be O-glycosylated on serine or threonine residues for a total of 36 potential sites. To remove these undesirable post-translational modifications, two selected supernatants (#2, 26) were concentrated and then enzymatically digested with neuraminidase and O-glycosidase. The supernatants were differentially precipitated with ammonium sulfate at 30, 50, and 70 % concentrations. A l l of the t - G A R P precipitated in the 30% ammonium sulfate fraction (Figure 17). Sample #2 was chosen for enzymatic analysis since the G A R 4 B 1 antibody only labeled one protein in this sample, whereas the supernatant for #26 contained multiple bands. Digestion with neuraminidase, which cleaves terminal sialic acid residues, yielded two products at 70 kDa and 66 kDa. Further digestion with O-glycosidase did not further reduce the apparent molecular weight (Figure 18). Densitometry of the bands of #2 reyealed that 56 R O S units of t - G A R P were produced from a 100 m L Pichia culture. This is equivalent to the amount o f t - G A R P in 700 pg of R O S . The construct was D N A sequenced since the entire coding region had been amplified by P C R . Despite using a polymerase with a proofreading function, a single base pair change was found (A->T). This resulted in an amino acid change (Ala295Thr). 3.3.2 Intracellular expression in Pichia. Two different constructs containing t - G A R P were expressed intracellularly in Pichia. First, a construct o f p G A P Z containing the entire c D N A 56 26 m ROS 30 50 70 30 50 70 Figure 17. A m m o n i u m sulfate fractionation of culture supernatants containing secreted t - G A R P . Selected clones (2, 26) were grown in 100 m L Y P D 24 h at 30°C. The culture supernatant was precipitated at 30%, 50% and 70% ammonium sulfate and resuspended in 3 m L of buffer (33 times concentration). Dialysed fractions (20 p L ) were loaded onto S D S - P A G E and stained with Coomassie Blue (Panel A ) . For reference, R O S (12.5 pg) was also loaded on the gel. A duplicate gel was blotted and probed with G A R 4 B 1 antibody (Panel B) . After a one minute exposure, secreted t - G A R P was detected in the 30% ammonium sulfate precipitate fraction. Its molecular weight (-70 K D a ) was higher than that of t - G A R P from R O S (60 KDa) . 57 A kDa 205-116— 97.4— 6 6 — 4 5 -2 9 -m 1 2 3 4 5 B kDa 1 2 3 4 5 Figure 18. Enzymat ic digestion of t - G A R P f rom Pichia culture supernatant #2. Supernatant from clone #2 (Lane 2) was concentrated 33 times by ammonium sulfate precipitation and dialysed in sodium cacodylate (Lane 3). Supernatant was then subject to enzymatic digestion by neuraminidase (Lane 4) and both O-glycosidase and neuraminidase (Lane 5). For reference, Lane 1 contains 12.5 p g of R O S . Samples were run on an SDS-P A G E gel and Coomassie stained (Panel A ) . A duplicate gel was western blotted and probed with G A R 4 B 1 antibody (Panel B) . Digestion with neuraminidase yielded a doublet at 70 and 66 kDa, but additional digestion with O-glycosidase did not reduce the molecular weight of t - G A R P any further. 58 of t - G A R P ( p G A P Z B 6 C l ) was transformed and expressed in Pichia. To test i f intact t-G A R P had been successfully integrated into yeast without gross rearrangement, the coding region of the c D N A insert was amplified by P C R using the yeast cells as the template. Out of 19 transformants screened by P C R , 17 amplified the expected product. However, upon screening these transformants for t - G A R P expression, none showed detectable expression of t - G A R P (not shown). Further expression screening of transformants indicated that only 26 out of a total 68 colonies screened showed a detectable, but very low level of expression from the yeast cells which was only visible after a 10 minute exposure (Figure 19). A n estimated 50 pg of cell protein was loaded on the gel, so the weak signal was not due to lack o f protein. Thus the some of the constructs did not express, even when the c D N A was shown to be present in the recombinant yeast. In a second approach, two expression constructs containing only the coding region ( p G A P Z B 6 C l - 1 0 , p G A P Z B 6 C l - l 1) were transformed and expressed in two different Pichia strains. Selected transformants were screened for expression by western blots (Figure 20). Results of different electroporations are tabulated in Table 4. Overall, each electroporation yielded varying numbers of t - G A R P expressing transformants, from 0 to 70 % of the colonies screened. Generally, a higher proportion of transformants expressed t - G A R P as the antibiotic concentration increased. Apparent differences in the level of expression between strains were due to the amounts of protein loaded on the gel. The amido black stained blot showed that there was less protein in the SMD1168 lanes (not shown). Although there was no obvious difference in product degradation between using the wi ld type or protease deficient stain, further expression studies were done using the protease deficient strain. 59 X33 + kDa 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 ROS X33 kDa ROS 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 1 1 6 -9 7 . 4 - 1 66 - 1 45 -M 29 -Figure 19. Intracel lular expression of t - G A R P in Pichia using expression vector p G A P Z B 6 C l . Selected transformants containing p G A P Z B 6 C l were replica plated to Y P D plates containing 100 mg/mL Zeocin. Cells were harvested off the replica plates, lysed by boil ing in 2 X sample buffer, and run on an S D S - P A G E gel and western blotted. A n estimated 50 pg of yeast proteins were loaded in each lane. For reference, R O S (12.5 pg) was loaded on the gels. A s a negative control for the western blot, untransforrned X33 cells were also included on the blot. Blots were probed with G A R 4 B 1 antibody (shown). After a 10 min exposure, a weak signal at 60 kDa was detected in most samples. This band corresponded to the molecular weight of t - G A R P in R O S . 60 A kDa 61 6 2 6 3 6 4 6 5 6 6 4 5 -2 9 -B kDa R O S 5 7 5 8 5 9 6 3 6 4 6 6 6 7 6 8 6 9 Figure 20. Intracel lular expression of t - G A R P in Pichia using p G A P Z B 6 C l - 1 0 . Expression construct p G A P Z B 6 C l - 1 0 was electroporated into wi ld type strain X 3 3 and mutant strain SMD1168. Selected transformants were replica plated on Y P D plates containing 100-200 pg/mL Zeocin. To analyse for expression, cells were run on S D S - P A G E gels and western blotted. After labeling of the blot with G A R 4 B 1 antibody, a one minute exposure showed that a band corresponding to t - G A R P (60 kDa) was detected in the wi ld type (Panel A ) and protease-deficient strains (Panel B) . 61 Clone #67 (Figure 20) was chosen for scale-up. This clone was able to produce about 200 R O S units of t - G A R P per g of wet yeast cells. Table 4. Results of Electroporation of Pichia with Intracellular Construct p G A P Z B 6 C l Electroporation Plasmid Pichia Host [Zeocin] on Y P D plates (pg/mL) Transformants Obtained Transformants expressing t - G A R P per total screened 1 p G A P Z B 6 C l X33 100 160 26/68 2 p G A P Z B 6 C l -11 X33 100 518 2/5 500 316 3/5 1000 46 6/7 2000 0 -3 p G A P Z B 6 C l -11 X33 100 276 0/4 500 241 0/5 1000 35 3/5 2000 0 -4 p G A P Z B 6 C l -10 S M D l l 68 100 19 0/19 500 2 0/2 1000 1 0/1 2000 0 -5 p G A P Z B 6 C l -10 S M D l l 68 100 72 29/52 1000 17 5/11 Since the inserts of p G A P B 6 C l - 1 0 and p G A P B 6 C l - l 1 were derived from a P C R product, both inserts were D N A sequenced. A single base-pair change was found in p G A P B 6 C l - l 1, changing A l a 295 to Thr. P G A P B 6 C 1 - 1 0 was error-free and thus selected for all further expression studies. 62 3.4 Development of a Purification Protocol 3.4.1 Preparation of an affinity column. Mouse monoclonal G A R 4 B 1 antibody was purified from ascites using Protein G Agarose beads. About 7 mg of G A R 4 B 1 antibody was isolated from 10 m L of mouse ascites (Figure 21). The elutions containing the light (26 kDa) and the heavy chains (55 kDa) showed little trace of other contaminating proteins. This purified antibody preparation was coupled to 2B Sepharose beads and subsequently used to purify G A R P from R O S and recombinant Pichia extracts. 3.4.2 Characterization of GAR4B1 epitope. To determine the epitope o f the G A R 4 B 1 antibody, a commercial kit was used to generate a series o f overlapping peptides spanning the original peptide sequence which was used to generate the antibody. A s seen by E C L detection , the G A R 4 B 1 monoclonal antibody bound to the first four spots (Figure 22). The epitope was narrowed down to the five amino acids common to all of these s p o t s — W V Q R V . Based on this information, peptides '4B1 Peptide' and 'PepGARP#l ' were designed to contain this sequence. The ability of the peptide to bind the G A R 4 B 1 antibody was determined qualitatively by looking at the competitive inhibition of G A R 4 B 1 antibody binding to R O S . Incubating 0.13 mg/mL of 4B1 Peptide with a 1:30 dilution of G A R 4 B 1 monoclonal antibody inhibited the binding of the antibody to G A R P proteins in R O S (Figure 23). 63 k D a m 1 2 3 4 5 6 7 8 9 Figure 21. Pur i f icat ion of G A R 4 B 1 mouse monoclonal antibody by Protein G column chromatography. Samples were run on an S D S - P A G E gel which was stained by Coomassie Blue. Mouse ascites (10 mL) containing G A R 4 B 1 antibody (Lane 1, 10 pL ) were loaded onto 2 m L of Protein G Agarose beads. Ascites flow-through was collected in lanes 2 (10 pL) and 3 (10 pL) . The column was washed in binding buffer 10 times. Lane 4 contains a sample (10 pL) of the first 4 mL of that wash. Antibody was eluted four times with 0.5 M acetic acid p H 3 (Lanes 5-8, 10 pL each). Finally, the column was washed with 1 M acetic acid p H 2.5 (Lane 9,10 pL) . Most of the G A R 4 B 1 antibody eluted in lanes 6 and 7. 64 Original Peptide: M L G W V Q R V L P Q P P G T P Scanning Peptides Immunoblot of SPOTs Membrane l 2 3 4 5 6 7 8 9 M L G W V Q R V L G W V Q R V L G W V Q R V L P WVQRVJLPQ V Q R V L P Q P Q R V L P Q P P R V L P Q P P G V L P Q P P G T L P Q P P G T P Figure 2 2 . Character izat ion of G A R 4 B 1 epitope by scanning peptide analysis. A commercial kit was used to synthesize a series of 8 residue peptides which scanned the original amino-acid sequence used to generate G A R 4 B 1 antibody. Labeling of the peptides by G A R 4 B 1 antibody showed that the epitope was contained in the first four peptides. A box indicates the five residues common to the first four peptides. 65 1 2 - p-subunit of CNG channel \ - f-GARP • - t-GARP Figure 23. Compet i t ion of 4B1 Peptide for binding to G A R 4 B 1 antibody. G A R 4 B 1 antibody (1:30) was used to probe a strip of Immobilon containing R O S proteins. Incubation of G A R 4 B 1 with 0.13 mg/mL peptide (Strip 1) resulted in no signal after a 3 min exposure. Wtihout peptide (Strip 2), G A R 4 B 1 strongly labeled G A R P proteins in R O S . 66 3.5 Purification of t-GARP from ROS Native G A R P was purifed from R O S using G A R 4 B 1 affinity chromatography. Detergent solubilized R O S was bound to G A R 4 B 1 beads and bound proteins were eluted with 1 mg/mL 4B1 Peptide (Figure 24). From densitometry, it was determined that 16% of the t - G A R P in R O S was recovered by G A R 4 B 1 affinity chromatography. Coomassie staining of the S D S - P A G E gel of the elutions indicates that many different proteins are present in the elution. A long with the three G A R P forms, the disk membrane proteins peripherin and rom-1 were positively identified by western blot using primary antibodies specifically against peripherin and rom-1 (not shown). Other proteins in the stained gel most l ikely correspond to proteins which participate in phototransduction. These include the a and p subunits o f phosphodiesterase (88, 84 kDa), the a subunit o f the c G M P gated channel (63 kDa), the a and P subunits of transducin (37, 39 kDa), and rhodopsin (38 kDa). 3.6 Purification of t-GARP from Pichia 3.6.1 Disruption of the yeast cell. Unl ike bacterial cells, yeast have a tough cell wall which requires more rigorous methods to rupture. In order to assess the best way to disrupt the yeast cell and purify recombinant t - G A R P , two different methods of yeast cell disruption were tried. The better method should be quick, simple to perform, reproducible, have few manipulations, and yield a reasonable amount of intact protein. 67 A B kDa FT 1 2 3 - p-subunit of CNG channel - f-GARP - t-GARP Figure 24. Pur i f icat ion of G A R P f rom R O S using G A R 4 B 1 affinity chromatography. R O S (1.6 mg) was solubilized in 1% Triton-X-100 and incubated with 200 u L of Sepharose beads covalently linked to G A R 4 B 1 antibody. Flow-through was saved for analysis (Lane FT). Proteins which bound to the beads were eluted three times with 80 p L of 1 mg/mL Pep G A R P peptide (Lanesl-3). 20 p L of each elution was loaded on the Coomassie stained SDS-P A G E gel (Panel A ) and the corresponding western blot (Panel B) . Coomassie staining showed that many proteins were eluted with peptide. After 5 min of exposure the blot to f i lm, labeling by G A R 4 B 1 antibody showed that all three forms of G A R P were eluted by peptide. 68 A yeast clone (#10-SMD1168-67) expressing t - G A R P intracellularly was chosen for large-scale expression. A 500 mL culture incubated 22 h at 30°C typically yielded 4-6 g of wet cells. Two different mechanical methods were used to break open the yeast cells. In the first method, a sonicator was used to disrupt the yeast cells. The sonicator breaks up cells by creating transient microbubbles, which implode and send powerful sonic shock waves through the liquid sample, thus shearing the cells (Hopkins, 1991). Observation under a phase-contrast microscope showed that 70% of the cells were lysed after a 20 minute sonication. A f low chart of the steps following sonication is shown in Figure 25. After sonication and hand homogenization, a low speed centrifugation showed that most of the G A R P was associated with the cell debris (Figure 26, Lanes 3 and 4). A trace amount of t-G A R P in the untreated homogenate was soluble after a high speed spin (Figure 26, Lane 5), but most o f the t - G A R P was in the insoluble pellet (Lane 6). Subsequent solubilization o f the low-speed spin pellet (Lane 4) with CHAPS/Tr i ton in the absence/presence of salt was able to extract t - G A R P from the pellet into the detergent soluble fraction (Figure 26, Lanes 8-10, 12-14, 16-18, 20-22). The addition of 0.1 M salt to either detergent did not have any effect on the yield of t - G A R P . A lso, both Triton-X-100 and C H A P S both solubilized t-G A R P equally wel l . The only appreciable difference in using the detergent/salt conditions was that in the Triton-X-100 solubilized lysate with 0.1 M N a C l , some of the t - G A R P appeared to be running smaller than the native t - G A R P . These smaller bands most l ikely represent degraded protein. The second method of disrupting the yeast cells was the French press. Cells were broken by forcing the cell suspension through a small orfice under high pressure (20 000 psi). After three passes of the yeast cell suspension through the French press, 90 % of the cells 69 cell pellet (T) i sonicated cells 4 hand homogenize i homogenate (2 spin 10000g pel l e t ® supernatant (3) 440 000 g spin pegt supernatant CHAPS Triton-X-100 -KCI +KCI spin 10000 g NaCl +NaCi spin 10000 g pellet super-(7) natant (8) pellet super-(ij) natant (12) pellet super-(15) natant (16) pellet super-(19) natant 440 000 g spin 440 000 g spin pellet (9) super- pellet super- pellet natant (To) natant ® super- pellet super-natant (n^ natant m 2 J @ Figure 25. Flowchart of steps involved in purification of t-GARP from sonicated Pichia cells. Circled numbers indicated samples analysed for t - G A R P (Figure 26). 70 B CHAPS Triton -KCI +KCI -NaCl +NaCI kDa ROS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 ROS19 20 21 22 Figure 26. Ext ract ion of recombinant t - G A R P f rom sonicated Pichia extracts. Samples from various steps (see Figure 25) following the sonication of cells were analysed by SDS-P A G E (Panel A ) and western blotting and probing with G A R 4 B 1 antibody (Panel B) . R O S was loaded onto the gels as a reference. Lane numbers correspond to numbers in Figure 25. Lanes 2-5, 7, 11, 15, 19 contain samples at the same dilution. Four times more sample was loaded in lanes 8-10, 12-14, 16-18, 20-22. After sonication and homogenization, most of the t - G A R P remained associated with the solid debris (Lane 4). Solubilization of the pellet with detergent in the presence or absence of salt allowed extraction of t - G A R P into a detergent soluble fraction. 71 were lysed. The lysate was detergent solubilized in 1% Triton-X-100. Ce l l debris and unbroken cells were removed by centrifugation. Samples were collected from before (Figure 27, Lane 1) and after the French press procedure (Lanes 2,3). Since the signal in the resulting clarified supernatant (Lane 2) was only slightly diminished compared to the pre-press t - G A R P signal (Lane 1), most of the t - G A R P must have remained in the supernatant. The t - G A R P within the lysate migrates anomalously at 50 kDa on the gel when the sample is not boiled (Lane 3). Boi l ing or dilution of the sample appears to alleviate this effect (Lanes 2, 4). Curiously, t - G A R P isolated from these lysates by G A R 4 B 1 beads yields none of the 50 kDa bands that were present in the lysate (Lane 6). 3.6.2 Affinity purification of t-GARP from Pichia. Detergent solubilized ( C H A P S or Triton-X-100) Pichia extracts from sonication were loaded onto a G A R 4 B 1 affinity column. Bound proteins were specifically eluted with peptide, and then all proteins were non-specifically eluted with 4% SDS (Figure 28). From the silver stained gel, the elution lanes have a light background of protein bands ranging from 30 to 60 kDa. It is not obvious i f one of these bands is t - G A R P because t - G A R P does not stain well . Most l ikely, these background proteins are yeast proteins which have errantly contaminated the samples. B y measuring the density of the bands in Figure 28 (Panel B) , the yield of recombinant t - G A R P was estimated. Solubilization in either C H A P S or Triton-X-100 gave approximately the same amount o f t - G A R P . Three elutions with 4B1 peptide ( lmg/mL) were sufficient to remove about 70% of the total t - G A R P bound by the beads. Overal l , using sonication and detergent solubilization to isolate t - G A R P yielded about 76 R O S units of t-72 B kDa ROS 1 3 4 5 6 116-9 7 . 5 " 6 6 -4 5 -29H Figure 27. Ext ract ion of recombinant t - G A R P f rom Pichia cells by French press. Pichia cells (5.27 g) expressing t - G A R P (Lane 1, pre-press cells, 5 p L ) were lysed by three passes through a French press. Cel l lysates were solubilized in 1% Triton-X-100. The lysate was clarified by low speed centrifugation and the resulting supernatants were analysed for t-G A R P (Lanes 2 and 3, 5 p L each). Lanes 1 and 2 were boiled prior to loading. To examine i f t - G A R P could be purified by G A R 4 B 1 Sepharose beads, clarified supernatant (200 p L ) was diluted in an equal volume of buffer (Lane 4, 5 p L ) and incubated with 100 p L of G A R 4 B 1 Sepharose beads. Flow-through (5 pL) was saved for analysis (Lane 5). Bound proteins were eluted in 100 p L of 4% SDS and 10 p L of the SDS elution was loaded in Lane 6. Samples were run on S D S - P A G E gels for silver staining (Panel A ) and western blot (Panel B) . R O S (12.5 pg) was also run on the gels for comparison. A s seen in a one minute exposure of the blot probed with G A R 4 B 1 antibody, t - G A R P ran as a major band at 60 kDa. A band was also seen at about 50 kDa. This band diminishes upon boil ing the sample, and may represent a form of t - G A R P which migrates anomalously. t - G A R P was successfully isolated from French press lysate (Lane 1), without any minor bands as seen in the column pre-load (Lane 4). 73 A CHAPS Triton pre- pre-kDa R 0 S l o a d F T w a s h E 1 E 2 E 3 S D S l o a d F T w a s h E 1 E 2 E 3 S D S B CHAPS Triton pre- pre-kDa R 0 S l o a d F T w a s h E 1 E 2 E 3 S D S l o a d F T w a s h E 1 5 2 E 3 S D S Figure 28. Af f in i ty pur i f icat ion of t - G A R P f rom Pichia extracts produced by sonication. Cel l lysates solubilized in 1% C H A P S (3 mL) or 1% Triton-X-100 (3 mL) were diluted in an equal amount of buffer (Lane pre-load, 20 p L loaded) prior to binding to 250 p L o f G A R 4 B 1 beads. F low through was collected for analysis (Lane FT, 20 p L loaded). Beads were washed six times, and 25 p L of the first wash was loaded. Bound proteins were eluted three times in 125 p L of 1 mg/mL 4B1 Peptide (Lanes E1-E3 , 12.5 p L loaded).To determine the portion which did not elute off the beads, beads were eluted in 125 p L of 4% SDS (Lane SDS, 12.5 p L loaded). For reference, R O S (12.5 pg) was also loaded. Samples were run on an S D S - P A G E gel (Panel A ) and blotted (Panel B) . After a 10 s exposure, G A R 4 B 1 antibody labeled a single strong band of recombinant t - G A R P in all elutions. 74 G A R P from 6.5 g of wet cells (12 R O S units/g wet cells). This is equivalent to the amount o f t - G A R P in 1 mg of R O S . In the purification of t - G A R P from French press cell lysates, an additional step was added to try to reduce the amount of yeast protein run through the G A R 4 B 1 affinity column. Triton-X-100 solubilized French pressed lysates were precipitated at increasing concentrations of ammonium sulfate and subsequently analysed for t - G A R P . For comparison, a lysate without detergent was also fractionated by ammonium sulfate precipitation (Figure 29). t - G A R P precipitated over a wider range in the non-detergent sample (25-40%) while in the detergent solubilized lysate, all the t - G A R P was detected in the 27.5% fraction. A lso, there appeared to be more t - G A R P in the detergent solubilized lysate than in the non-detergent lysate. For some reason, the proteins ran anomalously in a few of the lanes. It appeared that t - G A R P ran at its expected molecular weight at 60 kDa, but G A R 4 B 1 antibody also detected bands at about 50 kDa, 40 kDa, and 20 kDa. These smaller bands were previously observed in the sonicated yeast lysate (Figure 26, Lanes 20, 22). Boi l ing the sample appears to alleviate this effect (Figure 29, 1% Triton, Lanes SI and S2). The 27.5 % precipitated fraction of the detergent solubilized lysate was selected for further purification, despite containing anomalous bands. To determine i f t - G A R P could be purified without the use of detergent, the clarified 27.5% ammonium sulfate fraction was bound to G A R 4 B 1 beads. The beads were washed extensively in buffer with/without 1%CHAPS. Bound t - G A R P was eluted in 1 mg/mL peptide containing 1% C H A P S or lmg/mL 4B1 Peptide in buffer. Extensive over-staining of the corresponding S D S - P A G E gel with silver stain reveals a prominent band at 60 KDA (Figure 30, Panel A ) . This band is l ikely purified recombinant t - G A R P . Other bands are 75 A no detergent 1% Triton kDa R O S s1 s2 25 40 50 60 80 100 s1 s2 27.5 40 50 60 80 100 B no detergent 1% Triton kDa R O S s1 s2 25 40 50 60 80 100 s1 s2 27.5 40 50 60 80 100 Figure 29. Ammonium sulfate fractionation of French pressed Pichia lysates. French pressed lysates (7 mL) were solubilized in 1% Triton or untreated. Clarified lysates were not boiled (Lane SI) or boiled (Lane S2) prior to loading onto duplicate SDS-gels for Coomassie staining (Panel A) or western blotting (Panel B). ROS (12.5 pg) was included on both gels as a reference. Lysates were differentially precipitated with ammonium sulfate at 25/27.5, 40, 50, 60, 80, and 100% concentrations. Ammonium sulfate pellets were resuspended in 1.5 mL of buffer and dialysed before loading onto the gels. The fractions on the gel represent a 4.7 times concentration compared to the clarified lysates (Lanes SI, S2). After a one minute exposure, labeling by GAR4B1 antibody showed that intact t-GARP precipitated to the 27.5% fraction of the 1% Triton-X-100 solubilized lysate. The lysate which was not detergent solubilized yielded much lower levels of t-GARP. 76 B +CHAPS -CHAPS kDa R O S pre FT E1 E2 FT E1 E2 Figure 3 0 . Purification of recombinant t-GARP from French pressed detergent solubilized Pichia lysates using GAR4B1 affinity chromatography. The 27.5% ammonium sulfate fraction (900pL) was diluted with an equal amount of buffer (Lane pre, 10 pL) . The diluted lysate was bound to 150 p L of beads, and the flow through was saved (Lane FT, 10 pL) . The beads were washed in buffer with/without C H A P S and eluted two times in 75 p L o f 1 mg/mL 4B1 Peptide with/without C H A P S (Lanes E l , E2, 10 p L loaded) R O S (12.5 pg) was loaded as a reference. Samples were run on duplicate S D S - P A G E gels. One gel was deliberately over-stained with silver to visualize t - G A R P (Panel A , Lanes E l , E2). A one minute exposure of the western blot probed with G A R 4 B 1 showed that t - G A R P was isolated whether detergent was present or not. 77 also present in the elution lanes, but they are more faint than the 60 kDa band. A s seen in the western blot, t - G A R P was successfully eluted from the beads without using detergent (Figure 30, Panel B) . Densitometry analysis of the eluted t - G A R P bands showed that about 10 R O S units o f t - G A R P were isolated regardless of whether detergent was used or not. Using this method, 68 R O S units of t - G A R P were isolated from 4.38 g of wet yeast cells (16 R O S units/g wet cells). This is equivalent to the amount of t - G A R P in 850 pg of R O S . 78 DISCUSSION 4.1 Purification of t-GARP from ROS The use of the G A R 4 B 1 affinity column to purify t - G A R P from R O S is complicated by the fact that not only are all G A R P variants expected to bind, but many other proteins bind as well . The interaction between these proteins and the G A R 4 B 1 column is not due to a non-specific interaction with the column, since the peptide binds specifically to G A R 4 B 1 antibody. Therefore, only specifically bound proteins should be eluted by peptide. The peptide was designed to contain the five amino acids responsible for the epitope recognition of G A R 4 B 1 antibody, and the peptide was shown to inhibit G A R 4 B 1 binding to R O S (Section 3.4.2). These other proteins in the elution were probably interacting with the G A R P variants on the column, rather than the column itself. Further steps would be required to separate t - G A R P from these proteins and this would complicate the purification procedure. It would be more efficient and cost effective to produce t - G A R P in a recombinant system. This would eliminate the need to obtain additional antibodies for the separation o f t - G A R P from the other two G A R P forms and bovine eyes would not need to be processed. 4.2 Heterologous Expression of t-GARP in E. coli Cells and in COS Cells Heterologous expression of t - G A R P in E. coli yielded low levels o f induced proteins at 60, 55, 30, and 25 kDa. The expression levels o f these proteins were very low compared to that seen in the positive control on the Coomassie Blue stained gel. These products could 79 be t - G A R P and a degraded t - G A R P form, but it is difficult to be certain since there was such a high background on the blot. If t - G A R P had truly been expressed at a high level, one would expect that it would have given a strong western blot signal after one minute of exposure, despite the high background signals. Induction of the bacteria at a lower temperature (30°C) has been used to optimize the expression of the target protein (Schein et al, 1989). In the case of t - G A R P , lowering the induction temperature did not improve expression; rather, no induced expression of any proteins was seen in the test samples. Temperature did not affect over-expression of the positive control. Two of the expressed constructs were D N A sequenced. The start codons were intact and no frameshifts or premature stop codons were introduced in the P C R amplified segments of these two constructs. This suggests that gross errors in the D N A sequence were not the cause of non-expression of t - G A R P . However, two mutations leading to amino acid substitutions were detected. In one clone, the change Arg l05Trp was introduced by a T->A base change, and in another clone a single base pair change from A - > G produced Met265Val . It is possible that a single amino acid substitution out of 299 residues could cause non- or low expression of t - G A R P in bacteria. However, it is unlikely that two separate clones with different single amino acid substitutions in opposite parts of the polypeptide would both lead to the non-expression of t - G A R P . Furthermore, the four additional clones, which were not sequenced, did not show significant differences in expression. Overall , expression in E. coli was not successful. Though a 60 kDa protein was induced in some bacterial clones, it was not expressed at the high level that this bacterial 80 system has shown to produce for other proteins (25-30% of protein in E.coli). There are two major reasons as to why t - G A R P may not be stably expressed in E. coli. One possibility is that t - G A R P is being degraded according to the 'N-end Rule' in bacteria (Tobias etal., 1991). This rule states that the half-life of a protein is dependent on the identity of the N-terminal residue. Proteins whose N-terminal amino acids are R, K, L, F, V , and W have shorter half lives than those proteins which start with other amino acids. Furthermore, the efficiency of the methionyl aminopeptidase, which cleaves the N-terminal methionine, increases as the size of the penultimate residue decreases (Hirel, 1989). Proteins containing G , A , P, S, T, V , C , N , D, L or I as the second amino acid are more likely to undergo N-terminal methionine removal than those proteins containing all other amino acids. t - G A R P is particularly vulnerable to degradation since its penultimate residue is Leu. According to these rules, t-G A R P would l ikely undergo N-terminal Met processing to expose Leu as the N-terminal amino acid, and then t - G A R P would be targeted for degradation. Another explanation as to why t - G A R P does not express well is that the sequence of t - G A R P contains too many codons that are rarely used by E. coli. The abundance of rare codons in the gene may hinder translation because there are relatively fewer amino acyl-t R N A s available for translation of the gene. According to a list of low usage codons compiled by Makrides (1996), 31% (94/299) of the codons in t - G A R P are rarely used in E. coli. Replacement of these rare codons with more cognate codons may improve expression. This strategy has worked for the expression of a transcription factor, c G A T A - 1 , a 305-residue protein which contained a total of 62 low-frequency codons from Pro and G l y (Pikaart and Felsenfeld, 1996). Upon replacing 28 of the rare codons, good expression in E. coli was obtained. 81 In contrast, only 4% (11/299) o f the codons in t - G A R P are low-usage codons in primates, as calculated by Zhang et al. (1991). This may be responsible for the successful expression of t - G A R P in COS cells. Truncated G A R P was readily detectable in COS cells by western blotting of the detergent solubilized cells. It was also detected throughout the entire COS cell by immunolabeling of whole cells. This finding is consistent with the belief that t-G A R P is a soluble or membrane associated protein. The total amount o f recombinant t-G A R P produced in one 10 cm plate was 23 R O S units, or roughly 30 times less than the amount of t - G A R P produced from a 500 mL culture of Pichia cells. Therefore, to produce t-G A R P in COS cells much greater expense and labor would be required to make the same amount of t - G A R P as in Pichia.. This system was not chosen as the system for routine production of recombinant t - G A R P , since it was less amenable to scale-up compared to the bacterial or yeast system. 4.3 Heterologous Expression of t-GARP in Pichia 4.3.1 Secreted Expression of t-GARP. Pichia was able to secrete 56 R O S units o f recombinant t - G A R P from a 100 m L culture. This success may be due to the presence of fewer rare codons in the sequence of t - G A R P when it is expressed in yeast rather than in E. coli. In fact, there is only 9% (26/299) rare codons, according to the 8 lowest codon frequencies as tablulated by a Pichia pastoris codon usage table (www.dna.affrc.go.jp) using data from Genbank Release 108. However, the molecular weight of the secreted t - G A R P (-70 kDa) was higher than that in R O S (60 kDa). The N-terminal of t - G A R P is fused to the a-factor signal sequence for secretion into the media, but the increased molecular weight is 82 unlikely to be due to this, since the signal sequence should be cleaved for secretion. This unprocessed form of t - G A R P (a-factor-t-GARP) is present in the pellets as a ~80-90 kDa band, larger than the secreted form at - 7 0 kDa (Figure 16). More l ikely, the increased size o f t - G A R P is due to glycosylation of t - G A R P by Pichia. Since t - G A R P contains no N -glycosylation sites, t - G A R P could be O-glycosylated. Two enzymes were used to try to remove the glycosylation. Use of neuraminidase reduced the molecular weight of some the t-G A R P by 4 kDa (Figure 18), but O-glycosidase was ineffective in reducing the molecular weight further. O-glycosylation in Pichia has not been well-characterized, but as the Pichia expression system becomes more popular, more attention is being paid to post-translational processing in Pichia. Although studies have not detected sialic acid in O-linked carbohydrates, mannose has been detected (Duman et al., 1999). It is l ikely that sugars other than sialic acid are present in the carbohydrate chain and as a result, O-glycosidase is prevented from cutting. Further digestion with a variety of other glycosidases may be able to completely remove the carbohydrates. O f all systems attempted, this one is the simplest to purify t - G A R P from since it is secreted into the media, where Pichia secretes very few other proteins (Figure 18 A and Cregg, 1993). Unfortunately, secreted t - G A R P is not the same as t - G A R P from R O S , since its apparent molecular weight is about 10 kDa higher than expected. Without solving the problem of the extra molecular weight, it would be meaningless to use this form for further studies, since the undesirable post-translational modifications could interfere with the real native structure and functional analysis of t - G A R P . Hence, secreted expression was not developed any further. 83 4.3.2 Intracellular expression of t-GARP. For intracellular expression of t - G A R P in Pichia, two constructs were made. The first construct, p G A P Z B 6 C l , contained the entire t-G A R P c D N A clone. This c D N A included 56 bp of 5' untranslated sequence, the coding region, and 532 bp of 3' untranslated sequence. A very low level of expression of t - G A R P was observed using this construct. In contrast, the second expression vector ( p G A P B Z 6 C l -10), which only contained the coding region of t - G A R P , yielded Pichia transformants which produced much more t - G A R P . The difference in expression is probably due to the 5' or 3' untranslated regions in the c D N A which contained elements that interfered with translation or transcription of t - G A R P in Pichia. Expression of the second construct resulted in the production of 177 R O S units of t - G A R P per gram of wet cells. O f all expression systems tried, the intracellular expression of Pichia was the most promising from which to purify recombinant t - G A R P . This system produced recombinant t-G A R P which had the same apparent molecular weight as t - G A R P from R O S , unlike the Pichia secretion system, which produced a recombinant protein with higher apparent molecular weight. Presumably, non-native-like post-translational modifications such as glycosylation occurred as secreted t - G A R P passed through the endoplasmic reticulum and Golg i apparatus of Pichia. The best yield of recombinant t - G A R P was obtained from intracellular expression in Pichia, taking in consideration the ease and cost of production. From a 500 m L culture, intracellular Pichia expression of t - G A R P yielded about 700 R O S units, and secreted expression yielded about 280 R O S units. In comparison, it would take more than 30-10 cm plates of COS cells to obtain 700 R O S units of t - G A R P . Since the intracellular system easily produces the most t - G A R P which is more like the native t - G A R P from R O S , it was chosen as the source from which to purify t - G A R P . 84 4.4 Purification of t-GARP from Pichia The purification scheme for recombinant t - G A R P basically involved two steps. First, the yeast cells were broken open and solubilized with detergent. Second, t - G A R P was isolated from the yeast lysate by affinity chromatography. Various methods have been used to disrupt yeast cells (reviewed by Hopkins, 1991). there are chemical methods (autolysis by solvent, heat-shock, and enzymatic digestion) and mechanical methods (ultrasonication, crushing in a bead mi l l , and high pressure homogenization). For this study, two methods which were readily available to the lab were used—disruption by ultrasonication and homogenization by French press. In both methods, the cells were disrupted and solubilized in detergent. The lysate was clarified by centrifugation before isolating t - G A R P by affinity chromatography. Both methods yielded comparable amounts of recombinant t - G A R P (12 and 16 R O S Units/g wet cells for sonication and French press, respectively). However, the French press was the better instrument to use. It was more efficient at breaking the yeast cells, required less manipulation of the sample, and was more reliable to use. Losses of t - G A R P occurred at different steps. Detergent was required to solubilize and stabilize t - G A R P . A s seen in sonicated lysates, almost all of the t - G A R P isolated with the crude membrane fraction in the absence of detergent (Figure 26, Lane 6). This suggests that recombinant t - G A R P is a membrane-associated protein in yeast. Upon solubilization with 1% Triton-X-100, most, but not all of the t - G A R P , remained in the detergent soluble fraction (Figure 26, Lane 21), rather than in the insoluble pellet (Lane 22). Thus, some of the t - G A R P could not be solubilized. French press lysates were precipitated using 25% 85 arnmoniurri sulfate fractionation as an initial crude purification step. Without solubil izing the lysate in Triton-X-100, a trace amount o f t - G A R P was detected in the 25% ammonium sulfate fraction (Figure 29B), whereas, solubilizing the lysate in detergent yielded much more t - G A R P in the 25% ammonium sulfate fraction. This indicates that detergent helped to stabilize t - G A R P during the precipitation step. However, upon dialysing out the ammonium sulfate from the 25% fraction, some of the t - G A R P precipitated or aggregated out of solution. t - G A R P was not recovered from these insolubles. Finally, a loss in yield occurred in the affinity chromatography of the lysates. Despite using a molar peptide concentration 100-fold in excess of the theoretical molar binding capacity of the G A R 4 B 1 affinity column, only 70% of the t - G A R P was eluted by peptide. The lack of sufficient secondary structure in the 11-amino acid peptide may cause inefficient exchange of the t - G A R P for peptide, since t-G A R P retains secondary structure and thus may have a higher affinity to the G A R 4 B 1 antibody than the peptide. In the end, only 9% of the recombinant t - G A R P produced by Pichia was isolated by G A R 4 B 1 affinity chromatography. The concentration of t - G A R P eluted from the G A R 4 B 1 affinity column was not determined. Conventional methods of quantitating proteins through B C A protein assay or by staining with dyes such as Coomassie Blue or with silver were not useful since t - G A R P does not stain well with these reagents. One reason that t - G A R P does not stain wel l is due to its unusual amino acid composition. In comparison to bovine serum albumin (BSA) , a common protein standard, t - G A R P contains fewer amino acids which have been suggested to be important for detection (Table 5). Other factors such as hydrophobic interactions and interactions with the peptide bond are also thought to be responsible for good staining. 86 Table 5. Comparison of the Amino Acids Responsible for Determining Protein Concentration in B S A and t - G A R P Method of Detection* Amino Acids Reponsible for Detection Percent of Amino Acids Responsible for Detection Within Each Protein B S A t - G A R P B C A Cys, Tyr, Trp 9.7 3.3 Coomassie Bril l iant Blue A rg , Lys 14.1 7.0 Silver Stain Asp, Cys, G lu , His, Lys , Met 36.5 22.4 * methods examined are the Bicinchoninic Ac i d Protein Assay ( B C A ) (Wiechelman et al, 1988), Coomassie Bril l iant Blue staining (Tai et al, 1985), and silver staining (Syrovy and Hodny, 1991) Efforts to definitively determine the concentration of t - G A R P by amino acid analysis were not completed. t - G A R P was prepared for amino acid analysis by dialysing a sample o f peptide-eluted t - G A R P in 50 m M N a C l . However, western blot analysis o f the post-dialysed fractions indicated that most of the t - G A R P was lost. Possibly, t - G A R P degraded or aggregated during dialysis. A s a result, only relative comparisons between recombinant t-G A R P and t - G A R P from R O S could be made. A l l yields of t - G A R P were expressed in R O S Units, defined as the amount of t - G A R P in 12.5 pg of R O S . 4.5 Summary and Future Directions Bovine rod photoreceptor truncated G A R P has no known function. A s a first step in examining its structure and function, a method of producing and purifying recombinant t-G A R P was developed. t - G A R P was heterologously expressed in two out of three systems attempted. Expression in bacteria was unsuccessful, possibly due to degradation of t - G A R P by the host or the presence of rare codons in the D N A sequence of t - G A R P . Expression was 87 achieved in mammalian COS cells at a level of 23 R O S Units per 10 cm plate. However, t-G A R P could be expressed extracellularly or intracellularly in Pichia at 280 and 700 R O S Units per 500 m L culture, respectively. The relative ease and cost of production of t - G A R P in Pichia as compared to its production in COS cells made Pichia the more desirable system for routine large-scale expression of t - G A R P . t - G A R P was secreted from Pichia, but the recombinant protein had a molecular weight about 10 kDa higher than t - G A R P from native R O S . This extra molecular weight was l ikely due to O-glycosylation of secreted t - G A R P by Pichia. In contrast, t - G A R P produced intracellularly had a molecular weight identical to native t - G A R P from R O S . A s it seemed more like native t - G A R P and was produced in higher amounts, intracellularly expressed t - G A R P was chosen as the source from which to purify recombinant t - G A R P . Yeast cells expressing t - G A R P were lysed using a French press and t - G A R P was isolated from the detergent solubilized lysate by affinity chromatography. Overall , 9% of the t-G A R P produced intracellularly was purified from the lysate. The methods developed in this study can be used to generate an inexhaustible source of recombinant t - G A R P for future experiments. These may include investigations into the function of t - G A R P by identifying R O S proteins with which t - G A R P interacts. Recombinant t - G A R P may be labeled and used as a probe in a far-western blot of R O S proteins, or t-G A R P may be covalently bound to Sepharose beads to make a t - G A R P affinity column. Recombinant t - G A R P produced by Pichia can also provide a source of pure protein for X-ray crystallography studies. Once more information is known about t - G A R P , site-directed mutagenesis can be performed and the mutant proteins can be produced using methods in the 88 study. 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(1988) Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal. Biochem. 175, 231-237. Wiesner, B., Weiner, J . , Middendorff, R., Hage, V . , Kaupp, U.B. , and Weyand, I. (1998) Cycl ic nucleotide-gated channels on the flagellum control C a 2 + entry into sperm. J. Cell. Biol. Ul, 473-484. Wray, W., Boulikas, T., Wray, V . P . and Hancock, R. (1981) Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197-203. Yau , K . -W. (1994) Phototransduction mechanism in retinal rods and cones. Invest. Ophthalmol. Visual Sci. 35 (1), 9-32. Zhang, S., Zubay, G. , and Goldman, E. (1991) Low-usage codons in E. col i , yeast, fruit fly, and primates. Gene. 105, 61-72. 94 Appendix 1. Nucleic acid and protein sequence of bovine rod photoreceptor truncated G A R P 1 a t g c t g g g c t g g g t c c a g a g g g t g c t g c c t c a g c c c c c a g g g a c c c c t c a g a a g a c c a a g 1 M L G W V Q R V L P Q P P G T P Q K T K 61 c a a g a g g a g g a g g g g a c a g a a c c a g a g c c a g a g t t g g a g c c g a a g c c t g a a a c a g c t c c c 21 Q E E E G T E P E P E L E P K P E T A P 121 g a g g a g a c t g a g c t a g a g g a a g t g t c c c t g c c a c c a g a g g a g c c c t g c g t g g g g a a g g a a 41 E E T E L E E V S L P P E E P C V G K E 181 g t g g c t g c g g t t a c t c t g g g t c c t c a g g g a a c c c a g g a g a c t g c t c t c a c t c c g c c c a c a 61 V A A V T L G P Q G T Q E T A L T . P P T 241 t c c c t c c a g g c t c a g g t c t c t g t g g c t c c c g a a g c t c a c a g c a g t c c c a g a g g c t g g g t g 81 S L Q A Q V S V A P E A H S S P R G W V 301 c t g a c c t g g c t c a g g a a g g g t g t g g a g a a g g t t g t c c c g c a g c c c g c c c a c a g c a g c a g g 101 L T W L R K G V E K V V P Q P A H S S R 361 c c c t c c c a g a a c a t t g c t g c a g g c c t g g a g a g t c c a g a t c a g c a g g c a g g a g c a c a g a t c 121 P S Q N I A A G L E S P D Q Q A G A Q I 421 c t t g g g c a g t g c g g c a c t g g g g g c t c a g a t g a a c c c a g t g a a c c c t c c a g g g c c g a a g a c 141 L G Q C G T G G S D E P S E P S R A E D 481 c c c g g g c c t g g g c c c t g g c t c c t c c g g t g g t t c g a a c a g a a t c t g g a g a a a a t g c t g c c t 161 P G P G P W L L R W F E Q N L E K M L P 541 c a g c c c c c g a a a a t c t c t g a g g g c t g g a g a g a t g a g c c t a c a g a t g c t g c c t t g g g t c c a 181 Q P P K I S E G W R D E P T D A A L G P 601 g a g c c c c c a g g a c c c g c c t t g g a g a t c a a g c c c a t g c t g c a g g c c c a g g a g a g c c c c t c c 201 E P P G P A L E I K P M L Q A Q E S P S 661 c t g c c a g c t c c t g g c c c c c c g g a g c c t g a g g a g g a a c c g a t c c c a g a g c c c c a g c c c a c t 221 L P A P G P P E P E E E P I P E P Q P T 721 a t t c a a g c c t c c t c c c t g c c g c c c c c g c a g g a c t c t g c c a g g t t g a t g g c a t g g a t c c t g 241 I Q A S S L P P P Q D S A R L M A W I L 781 c a c a g g c t g g a g a t g g c c c t g c c a c a g c c a g t g a t c c g t g g g a a g g g t g g g g a g c a g g a g 261 H R L E M A L P Q P V I R G K G G E Q E 8 41 t c t g a t g c c c c a g t g a c a t g t g a c g t g c a g a c c a g g g t g g t g g c a g c t g g a a g c c t c t g a 281 S D A P , V T C D V Q T R V V A A G S L -95 Appendix 2. Codon usage in rod bovine truncated G A R P Amino A c i d Codons Number Total % of Total Number Amino Acids Pro C C T 9 C C C 21 51 17 C C A 13 C C G 8 G lu C A A 10 38 12.7 G A G 28 Leu T T A 0 T T G 4 26 8.7 C T T 1 A l a G C T 10 G C C 9 25 8.4 G C A 5 G C G 1 G in C A A 2 24 8.0 C A G 22 G ly G G T 4 G G C 7 24 8.0 G G A 4 G G G 9 Ser T C T 4 T C C 7 T C A 1 20 6.7 T C G 0 A G T 3 A G C 5 V a l G T T 2 G T C 3 17 5.7 G T A 0 G T G 12 Thr A C T 6 A C C 5 16 5.4 A C A 5 A C G 0 A r g C G T 1 C G C 0 C G A 0 11 3.7 C G G 1 A G A 2 A G G 7 Lys A A A 2 10 3.3 A A G 8 Asp G A T 5 8 2.7 96 G A C 3 He A T T A T C A T A 2 6 0 8 2.7 Trp T G G 7 7 2.3 Met A T G 5 5 1.7 Cys T G T T G C 1 2 3 1.0 His C A T C A C 0 3 3 1.0 Asn A A T A A C 1 1 2 0.67 Phe T T T T T C 0 1 1 •0.33 Tyr T A T T A C 0 0 0 0 97 Appendix 3. Clustal alignment of f - G A R P and t - G A R P from bovine and human photoreceptor and rat pineal gland bF-GARP bT-GARP hT-GARP rT-GARP MLGWVQRVLPQPPGTPQKTKQ MLGWVQRVLPQPPGTPQKTKQ MLGWVQRVLPQPPGTP^ KTK MLGWVQRVLPQPPGTPQKTf E|GAGPQPETESKPEANPQP||PEV| bF- GARP 4 0 PEE bT- GARP 4 0 PEE hT- GARP 4 3 PEE rT- GARP 61 PEE SHR bF-GARP bT-GARP hT-GARP rT-GARP 1 0 0 1 0 0 1 0 2 1 1 8 V L T W L R K G V E K V V P Q P V L T W L R K G V E K V V P Q P V L T W L B K G V E K V | P Q P L I W M K G I E K W P Q P GSDEPSEPS GSDEPSEPS |N@AL D P G S E H G S D K T I bF-GARP bT-GARP hT-GARP rT-GARP 14 8 ^ .fi^ gf 177 |TQ|TEJSL| bF-GARP bT-GARP hT-GARP rT-GARP K E A P bF-GARP bT-GARP hT-GARP rT-GARP EQEELHLILEEVDPHWEEDEHQEGSTSTSP sE bF-GARP bT-GARP hT-GARP rT-GARP 3 2 8 RTSEAAPADEEKGKVVEQTPRELPRIQEEKEDEEEEKEDGEEEEEEGREKEEEEGEEKEE bF-GARP bT-GARP hT-GARP rT-GARP 3 8 8 EEGREKEEEEGEKKEEEGREKEEEEGGEKEDEEGREKEEEEGRGKEEEEGGEKEEEEGRG bF-GARP bT-GARP hT-GARP rT-GARP 4 4 8 KEEVEGREEEEDEEEEQDHSVLLDSYLVPQSEEDQSEESETQDQSEVGGAQAQGEVGGAQ 98 bF-GARP 508 ALSEESETQDQSEVGGAQDQSEVGGAQAQGEVGGAQEQDGVGGAQDQSTSHQELQEEALA bT-GARP hT-GARP rT-GARP bF-GARP 568 DSSGGSFQMSPFEALQECEALKR bT-GARP hT-GARP rT-GARP Bovine rod photoreceptor f - G A R P (bF-GARP) sequence is as described by Sugiomoto et al. (1991). Bovine rod photoreceptor t - G A R P (bT-GARP) sequence was determined by Dr. Carol Colvi l le. Human rod photoreceptor t - G A R P (hT-GARP) sequence is as described by Ardel l et al. (1995). Rat pineal gland t - G A R P ( rT-GARP) sequence was determined by Sautter et al. (1997). 99 Append ix 4. List of Suppliers Amersham Cleveland, O H Aminco Silver Spring, M D B D H Inc. Toronto, O N B i o l O l Vista, C A Boehringer Mannheim Mannheim, Germany B io-Rad Hercules, C A Di fco Laboratories Detroit, M I Fisher Scientific Nepean, O N Genemed Synthesis Inc. South San Francisco, C A Genex Corp. Gaithersburg, M D Genosys Biotechnologies Inc. The Woodlands, T X Gibco B R L Gaithersburg, M D Hoefer San Francisco, C A Invitrogen Carlsbad, C A Jackson ImmunoResearch Laboratories Inc. Westgrove, P A Kontes Vinland, N J L K B Productker A B Bromma, Sweden Mi l l ipore Bedford, M A Misonix Farmingdale, N Y New England Biolabs Beverly, M A Novabiochem L a Jolla, C A Novagen Madison, WI Qiagen Chatsworth, C A Pharmacia L a Jolla, C A Pierce Rockford, IL Schenk Packing Co. Inc. Seattle, W A Sigma St. Louis, M O Spectrum Medical Industries, Inc. Los Angeles, C A Stratagene L a Jolla, C A U V P Inc. Upland, C A Zeiss Jena, Germany 100 

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