OVEREXPRESSION OF FETAL HUMAN PIGMENT EPITHELIUM-DERIVED FACTOR IN ESCHERICHIA-COLI - A FUNCTIONALLY ACTIVE NEUROTROPHIC FACTOR

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein pre sent in low amounts in conditioned medium of cultured fetal human reti nal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence i dentified it as a member of the serine protease inhibitor (serpin) sup ergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombr an-Tink, J. (1993) Proc. Natl. Acad, Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human P EDF cDNA and obtained milligram amounts of biologically active PEDF fr om Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44- Pro41) were used in our constructs. Induc tion from a vector containing the truncated PEDF version, named pEV-BH , produced a protein (BH) of expected size (M(r) 42,800) associated wi th inclusion bodies, which contained 25-40% of expressed protein. Afte r solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified pro tein matched that of the pEV-BH construct. We have conducted neurite o utgrowth assays in a human retinoblastoma Y-79 cell culture system. Re combinant PEDF (BH) demonstrated neurotrophic activity, as reported fo r the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibiti on assays were performed. Recombinant PEDF did not have an effect on t rypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, end oproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neurotrophic activity.