Overexpression, purification, and characterization of a membrane-bound receptor for PEDF
Thursday, September 15, 2016 — Poster Session III
- J Bullock
- P Subramanian
- SP Becerra
Retinitis pigmentosa and macular degeneration are caused by degeneration of photoreceptor cells, which progressively leads to blindness. Our lab has identified pigment epithelium-derived factor receptor (PEDF-R) as a cell surface receptor for the retinoprotective protein PEDF. Upon binding, PEDF enhances the PEDF-R lipase activities, which are critical for the retinal survival effects mediated by PEDF. Given the interest in structure-function studies of PEDF-R, we propose to use the PNPLA2 gene to produce large amounts of biochemically active recombinant PEDF-R protein in E. coli. We overexpressed the gene from plasmids containing full-length and truncated versions of human PNPLA2 cDNA fused to His6x/Xpress peptide tags at their N-termini. Induction for expression produced the recombinant proteins as inclusion bodies with a yield of about 8mg of recombinant protein per L of culture. The recombinant proteins were solubilized with urea. Protein refolding and urea removal were achieved by buffer exchange into deoxycholate containing buffers using filtration devices. Recombinant PEDF-R proteins exhibited phospholipase and triglyceride lipase activities. Only the PEDF-R versions containing the ligand binding domain retained the ability to bind PEDF, which stimulated the triglyceride lipase activity. A high-throughput screen assay was optimized to evaluate multiple compounds for the ability to stimulate the enzymatic activity of the recombinant proteins. Our findings show the production of high levels of biochemically active PEDF-R versions, which are useful in high-throughput assays for the identification of PEDF-R enhancers, and in turn, discover potential retinal degeneration therapeutics.
Category: Molecular Biology and Biochemistry