Study on Optimizing The Structure and Function of hbFGF Expressed in Escherichia coli
原核表达hbFGF结构与功能优化的研究

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.