Site-directed Mutagenesis of Human Endostatin Gene and Expression of Mutant in E. coli

Objective To increase the soluble expression level of human endiostatin (hES)gene in E. coli by site-directed mutagenesis. Methods The mutation sites were designed according to the information on structure as well as relationship between structure and function of hES from internet sites. The rationality of the designed mutation sites was confirmed by predicting the three-dimensional structure of hES mutant in relevant internet sites of bioinformatics. The site-directed mutagenesis of hES was achieved by overlapping extension PCR, and the mutated and original hES genes were subcloned into vector pGEX-4T-3 respectively for fusion expression in E. coli . The soluble expression levels of target protein before and after mutation were compared. Results The prediction of three-dimensional structure of hES showed rational design of mutation sites. Sequencing result proved the site-directed mutation of hES gene. Of the expressed product of hES gene mutant, 40% existed in a soluble form. However, almost all the expressed product of original hES gene were in forms of inclusion bodies. Conclusion The site-directed mutagenesis of hES gene was successfully achieved, which increased the soluble expression level of target protein.