Jaye M, Burgess W H, Shaw A B, Drohan W N
1Rorer Biotechnology, Inc., Springfield, Virginia 22151.
J Biol Chem. 1987 Dec 5;262(34):16612-7.
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.
编码人α-内皮细胞生长因子（α-ECGF）的cDNA已被设计用于在大肠杆菌中进行高水平表达。携带重组质粒pMJ26的细菌培养物的诱导导致出现一条突出的16 kDa多肽。该蛋白质已使用基于肝素-琼脂糖亲和色谱和反相高压液相色谱的快速两步法从细菌裂解物中纯化出来。在三种生物学测定中，将重组人α-ECGF与牛脑来源的α-ECGF进行了比较：在鼠肺毛细血管内皮细胞（LE-II细胞）上的受体结合、LE-II细胞中[3H]胸腺嘧啶核苷掺入的刺激以及人脐静脉内皮细胞增殖的刺激。结果表明，重组人有丝分裂原与牛脑来源的物质具有相同的生物学效力。荧光光谱法用于研究重组ECGF与肝素之间的相互作用。肝素结合导致ECGF的固有荧光降低40%，这与肝素诱导的构象变化一致。ECGF的固有荧光也随pH值而变化。
