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UBC Theses and Dissertations

Expression, purification, and characterization of RD3, the protein associated with Leber's congenital amaurosis type 12 Jefferies, Thomas

Abstract

RD3 is a highly conserved 23 kDa soluble protein expressed in the retinal and testicular tissues required for the trafficking of GC1 (Guanylate Cyclase 1). A lack of expression of this protein, or of GC1, results in a rapid loss of photoreceptor cells after retinal development, indicating their importance in photoreceptor function and survival. Previous work has demonstrated that RD3 shows disperse and membrane-associated distribution in HEK293T cells. When expressed in E. coli, RD3 forms inclusion bodies exclusively. RD3 is non-amenable to vigorous centrifugation, resistant to purification by way of size exclusion, and binds to and inhibits GC1. The current investigation demonstrates that RD3 is highly conserved throughout sight capable vertebrates, forms large proteolipid macro-molecular structures of 10nm size or greater and that E. coli expressed protein conforms to the predicted secondary structure of RD3. Additionally, methods of isolating RD3 used in previous studies are carried out here and found to result in the formation of the expected structures. Multiple alignment and secondary structure prediction software, circular dichroism, dynamic light scattering, electron microscopy, and standard protein purification and visualization techniques are utilized in these endeavors.

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