重组羧肽酶B及其包涵体溶解性的研究

Research on Recombinant Carboxypeptidase B and Solubility of Its Inclusion Body

目的构建重组羧肽酶B(rCPB)的表达质粒和表达菌株,表达羧肽酶B(CPB),并对表达CPB包涵体的溶解性进行研究。方法构建CPB重组质粒pET-21a-CPB,将其导入表达菌株BL21(DE3)中,分别在25℃和37℃进行诱导表达;所得包涵体分别用不同浓度尿素添加不同还原剂溶解,以溶液中的蛋白质浓度判定其溶解性,利用非还原SDS-PAGE分析其溶解性提高的原因。结果诱导后生长温度不同对rCPB包涵体的纯度及后期处理均有影响;rCPB包涵体在10mol/L尿素溶液中的溶解度比在8mol/L尿素溶液中提高2￣3倍;添加0.75%β-巯基乙醇能显著改善rCPB包涵体的溶解效果。经非还原SDS-PAGE分析,添加β-巯基乙醇后,溶解rCPB聚体的含量减少。结论成功地表达了rCPB,并通过实验提高了rCPB包涵体的溶解度。

Objective The expression plasmid and strain of recombinant carboxypeptidase B （rCPB） were constructed. The solubility of inclusion body of the expressed carboxypeptidase （CPB） was also studied. Methods The recombinant plasmid of CPB, pET-21a-CPB, was constructed and transformed into E. coli BL21（DE3）. The expression was induced in 25 ℃ and 37℃, respectively. The obtained inclusion bodies were dissolved in different denaturing solution systems. The protein concentration was determined in order to indicate the solubility of CPB inclusion bodies in the denaturing solution. And probable mechanism for the solubility differences was illuminated by the analysis of unreduced SDS-PAGE. Results The purity and following treatment of inchision bodies of CPB were influenced by the culturing temperature after the inducement with IPTG. The solubility of inchision bodies of rCPB in 10mol/L urea was 2-3 times higher than in 8 mol/L urea, and the renature solution with 0.75% β -ME in 10mol/L urea could increase the solubility of inclusion bodies of rCPB, comparing with that in 10mol/L urea simply. As a result of the analysis of unreduced SDS-PAGE, the content of large molecular rCPB polymers was decreased in β-ME solutions as well. Conclusion The rCPB can be expressed successfully and the solubility of its inclusion bodies can be increased by experiment.