Construction of multimeric gene of peptides or small protein molecules to improve expression level in E.coli
Objective To inhibit the disstability and enhance the expression level of peptides or small proteins, a special prokaryotic expression system was applied. Methods The Sushi3 domain (S3) gene of factor C was amplified by PCR from plasmid and cloned into pBBS vector. This vector contains the BbsⅠ restriction site, which can cut the DNA into two bases adjacent to the recognition site. The resultant cohensive terminals can direct the self-ligation of digested insert in vitro, hence producing multimers of S3 genes. The multimers can be recloned into pBBS vector. Results We successfully constructed pBBSS3-2mer, -4mer and -8mer guided by this strategy. All these multimers were transferred to expression vector pET22b and induced by IPTG to expression in bacterial strain BL21. The rS3-4mer has achieved the highest expression level among these constructs. rS3-4mer was purified by anion exchange and followed by gel filtration. The additional linker between the repetitive units, Aspartic acid (D) and proline (P), can be cleaved in mild acid solution. So rS3-4mer was digested into rS3 monomers in vitro by 70% formic acid treatment. Tricine-PAGE analysis shows that all recombinant proteins and the digested products have the correct sizes, and all of them can react with the S3 anti-serum in Western blot test. Conclusion Tandem repeats of genes can be effectively achieved through this system and enhance the expression level of smaller proteins and peptides in E.coli.