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

Abstract

Glutamic Acid Rich 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 cGMPgated cation channel; 2) as a 590 amino acid polypeptide (f-GARP); and 3) as a 299 amino acid polypeptide (t-GARP). The function of GARP in photoreceptors is unknown. Although it is not visible by Coomassie Blue staining, t-GARP appears to be the most abundant form of GARP as seen by western blotting. Such an abundant protein must have a role in the function or structure of the photoreceptor. As a first step in examining the function of GARP, a method for the recombinant production and purification of t-GARP was developed in this study. Heterologous expression o f t-GARP was attempted in three different systems— bacterial, yeast and mammalian systems. t-GARP 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 ROS Units of t-GARP (1 ROS Unit = amount of t-GARP in 12.5 μg of ROS) per 10 cm plate. From Pichia, t-GARP was both secreted and produced intracellularly at 280 and 700 ROS units per 500 mL of culture, respectively. Considering the cost, ease of production, quality and yield of recombinant t-GARP, the intracellularly produced t-GARP was selected as the system from which to purify recombinant t-GARP. Using affinity chromatography, 9% of the t-GARP produced intracellularly was isolated from Frenchpressed, detergent solubilized yeast lysates.

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