原核表达hbFGF结构与功能优化的研究

野生型hbFGF蛋白可溶性与稳定性较低一直是困扰研发人员的难题。分析其氨基酸序列发现含有 4个游离巯基 ,其中第 78、96、1 0 1位半胱氨酸游离巯基可能直接影响其可溶性、稳定性及活性。对天然hbFGF进行定点突变 ,一组将Cys78、Cys96同时突变 ,另一组将Cys78、Cys96与Cys1 0 1进行联合突变 ,均改为丝氨酸密码子 ,然后克隆入原核表达载体pET 3c ,进行表达 ,结果发现两种突变蛋白的可溶性大幅度增加 ,稳定性明显上升 ,其中三点突变的效果更为显著 ,但蛋白质的活性明显下降。可以认为 78、96与 1 0 1位半胱氨酸的游离巯基同时参与了天然hbFGF多聚体的形成 ,是导致野生型hbFGF可溶性与稳定性较低的主要原因 ,Cys1 0 1还与维持hbFGF的活性直接有关

Study on Optimizing The Structure and Function of hbFGF Expressed in Escherichia coli

The poor stability and solubility of human basic Fibroblast Growth Factor expressed in Escherichia coli is the major problem which prevent the protein to be widely used. The free thiol groups of cysteine residues in the amino acid sequence of hbFGF may play an important role. Two kinds of mutations had been used to investigate the possible reasons:2 points mutation(in which both Cys78 and Cys96 were replaced by serines)and 3 points mutation( Cys78,Cys96,Cys101 were all changed into serines). Both the mutants were cloned into pET 3c, and transducted into E.coli strain BL21(DE3) plysS, induced for expression with IPTG. Solubility and stability were assayed by SDS PAGE, bioactivity was observed with MTT. The solubility and stability of the two mutants increased significantly, but the mutation of Cys101 destroyed the bioactivity of the mutated protein. It may be concluded that Cys78, Cys96 and Cys101 all contribute to the formation of disulphide bonds among the protein molecules, and as a result, it influences the structure and function of hbFGF directly.