一种高效表达多肽或小分子蛋白平台的构建

Construction of multimeric gene of peptides or small protein molecules to improve expression level in E.coli

目的 克服小分子蛋白或多肽在大肠杆菌表达系统中易降解、表达量低的问题 ,为基因工程生产此类产品提供一种简便有效的解决方案。方法 以 33个氨基酸的小分子蛋白 (factorC的S3功能区 )为例 ,用PCR方法扩增了基因 ,并克隆到载体pBBS。利用载体带有的BbsⅠ酶切位点特性和添加到引物上的特殊序列 ,使插入片段在体外定向连接成多联体 ,然后重新连接到pBBS。结果 成功构建了S3的二联体、四联体及八联体克隆。转移至表达载体pET2 2b后 ,在BL2 1细菌宿主中诱导表达。S3单体表达不明显 ,而四联体 (rS3 4mer)表达量最高。rS3 4mer经纯化后在酸性溶液中水解为rS3单体。以Westernblot证明 ,多联体及其分解单体均可与S3抗血清特异性结合。结论 应用这一系统能有效地构建串联基因 ,从而提高小分子蛋白或多肽在大肠杆菌中的表达量。

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.