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.