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A. G. Gvritishvili, K. Leung, C. J. Barnstable, J. Tombran-Tink; Codon Optimization Increases Bacterial Expression of Functionally Active Pigment Epithelium Derived Factor (pedf). Invest. Ophthalmol. Vis. Sci. 2008;49(13):4927. doi: https://doi.org/.
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PEDF is a non-inhibitory member of the serpin super family. The protein is a broad-acting neurotrophic factor and an effective antiangiogenic agent, making this a key polypeptide in developing treatments for retinal degenerative diseases. The expression of non-optimized PEDF in bacteria results in poor yields of biologically active protein. Since large amounts of PEDF are required for in vitro and in vivo studies, we used codon optimization strategies to improve the expression of PEDF in bacteria.
In this study, we converted the human PEDF nucleotide sequence to one that is codon optimized (coPEDF) for expression in bacteria using proprietary algorithm machinery. The gene was then synthesized and cloned into pET32a using Kpn I and Hind III sites and the resulting clone was verified by DNA sequencing. 5 ng of the pET32a-coPEDF plasmid was transformed into E. coli BL21(star)DE3 cells. coPEDF was purified using Ni His-binding resin and the protein cleaved from the 5’ Trx.tag coding sequence by treatment with rEK. The purity of the protein was determined by SDS-PAGE and molecular weight confirmed by western blot and MALDI-TOF analyses. coPEDF was characterized by UV spectrometry and circular diochroism (CD) and compared to non-optimized (noPEDF) PEDF. The protein was tested for neurite outgrowth in PC12 pheochromocytoma cells and the neuroblastoma cell line, SHSY5Y, as well tube formation in HUVEC cultures. CoPEDF was also used to generate polyclonal antibodies.
We showed that bacteria express approximately 61mg of coPEDF/L of LB media (26.7mg/g of wet E. coli cells). Purification using Ni His-binding resin yields approximately 35mg of coPEDF/L of LB media (15.3mg/g of wet E.coli cells). There is an ~11.8 fold greater yield of purified coPEDF protein in bacteria compared to non-codon optimized PEDF expression (1.3 mg/gram wet E.coli cells). CD experiments confirmed that coPEDF had similar far UV/CD spectra to that of commercially available noPEDF, indicating that the coPEDF is correctly folded. MALDI-TOF confirmed the precise molecular weight of the protein. In addition, coPEDF is equally as effective in stimulating outgrowth of neurites in PC12 and SHSY5Y cells and in blocking endothelial tube formation as noPEDF. coPEDF was also highly immunogenic, producing high yields of polyclonal antibodies that specifically recognize the ~50 kDa protein by both western blot analysis and immunocytochemistry.
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