title: Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor.
abstract: Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: S P
authors.initials: SP
authors.lastname: Becerra
authors.forename: I
authors.initials: I
authors.lastname: Palmer
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: F
authors.initials: F
authors.lastname: Steele
authors.forename: J
authors.initials: J
authors.lastname: Shiloach
authors.forename: V
authors.initials: V
authors.lastname: Notario
authors.forename: G J
authors.initials: GJ
authors.lastname: Chader
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005136:Eye Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000737:chemistry; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000235:genetics*; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D018076:DNA, Complementary; D005136:Eye Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor