Biological equivalence of natural bovine and recombinant human alpha-endothelial cell growth factors.
The cDNA encoding human alpha-endothelial cell growth factor (alpha-ECGF) has been engineered for high-level expression in Escherichia coli. Induction of bacterial cultures harboring the recombinant plasmid pMJ26 results in the appearance of a prominent 16-kDa polypeptide. This protein has been purified from bacterial lysates using a rapid, 2-step procedure employing heparin-Sepharose affinity based chromatography and reversed-phase high pressure liquid chromatography. Recombinant human alpha-ECGF was compared to bovine brain-derived alpha-ECGF in three biological assays: receptor binding on murine lung capillary endothelial cells (LE-II cells), stimulation of [3H]thymidine incorporation in LE-II cells, and stimulation of human umbilical vein endothelial cell proliferation. The results demonstrate that the recombinant human mitogen has the same biological potency as the bovine brain-derived material. Fluorescence spectroscopy was used to study the interaction between recombinant ECGF and heparin. Heparin-binding resulted in a 40% reduction in the intrinsic fluorescence of ECGF, consistent with a heparin-induced conformational change. The intrinsic fluorescence of ECGF also varied as a function of pH.
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1Rorer Biotechnology, Inc., Springfield, Virginia 22151.
Burgess
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Shaw
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Drohan
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005346:Fibroblast Growth Factors / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009113:Muramidase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D005346:Fibroblast Growth Factors; D004247:DNA; D009113:Muramidase