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


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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