一种促分裂活性降低的hbFGF突变体的表达及纯化(英文)

为了降低由人碱性成纤维细胞生长因子(hbFGF)的广谱促分裂活性引起的潜在副作用,用中性氨基酸丙氨酸取代了hbFGF第108位的丝氨酸,构建了促分裂活性降低的hbFGF突变体(mhbFGF)。IPTG诱导突变体在大肠杆菌BL21(DE3)中高效表达。mhbFGF的表达量约为全菌体蛋白的30％。通过离子交换和肝素亲和层析从菌体上清中纯化目标蛋白。MTT法检测促分裂活性表明,mhbFGF的促分裂活性显著低于野生型hbFGF。纯化的:mhbFGF可用于进一步的药效和安全性研究。

Expression and Purification of Basic Fibroblast Growth Factor Mutant with Reduced Mitogenic Activity

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broad-spectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.