重组羧肽酶原B的复性及纯化

目的　建立重组羧肽酶原B复性方法及活性羧肽酶B的纯化方法。方法　重组大肠杆菌发酵后 ,表达的羧肽酶原B包涵体经洗涤、变性 ,采用凝胶过滤的方法对其进行复性及纯化。所得的复性液经Trypsin酶解后 ,采用DEAE FF阴离子交换层析进行羧肽酶B的分离纯化 ,并采用SDS PAGE检测纯度。结果　经凝胶过滤后得到了复性和纯化的羧肽酶原B包涵体 ,在蛋白终浓度相同的情况下 ,复性效率比稀释复性提高了 5 0 % ,经DEAE离子交换柱一步纯化 ,得到了活性羧肽酶B ,纯度达到 90 %以上。以Hippuryl -L arg为底物测活 ,得到的活性酶的比活为 15 0u mg。结论　所建立复性方法及纯化方法为进一步的研究奠定了基础。

Renaturation of Recombinant Pro-carboxypepfidase B by Gel Filtration and Purification of Active Carboxypeptidase B

Objective To develop the methods for renatura tion of recombinant pro-carboxypeptidase B and for purification of active carboxype ptidase B.Methods After recombinant E.coli was cultured by fermentation,the expressed pro-carboxypeptidase B,in a form of inclusion body,w as washed and denaturalized,then purified and re-naturalized by gel filtration. The obtained solution was digested with trypsin,from which carboxypeptidase B wa s purified by DEAE-FF anion exchange chromatography.The purity of carboxypeptid ase B was determined by SDS-PAGE.Results In case of the same f inal concentration of pro-caroxypeptidase B,the renaturation efficiency by gel filtration increased by 50% compared with that by dilution.After purification by DEAE-FF anion exchange chromatography,the purity of carboxypeptidase reached a bove 95%,and its specific activity determined using Hippuryl-L-arg as a substr ate was 150 u/mg.Conclusion The developed methods were effectiv e and laid a foundation of further study on property of caroxypeptidase. [