改良的幽门螺杆菌噬菌体裂解酶在大肠杆菌和毕赤酵母中表达的活性差异

Difference of Activity of Improved Helicobacter pylori Phage Lysins Expressed in Escherichia coli and Pichia pastoris

目的:改良型幽门螺杆菌噬菌体裂解酶由具有穿透外膜作用的疏水多肽和幽门螺杆菌噬菌体编码的裂解酶和穴蛋白构成,比较其在大肠杆菌和毕赤酵母中表达活性的差异。方法:构建的重组质粒分别转化大肠杆菌BL21(DE3)和毕赤酵母X33,经过表达纯化得到改良型裂解酶,体外通过滤纸片法和液体法比较不同表达系统所表达的改良型裂解酶的活性差异。结果:体外抑菌实验表明,大肠杆菌表达的改良型裂解酶的溶菌效果明显强于毕赤酵母表达的改良型裂解酶。结论:2种表达系统各有优势,就活性而言,大肠杆菌表达系统更佳,毕赤酵母表达系统对外源蛋白的糖基化修饰影响改良型裂解酶活性。

Objective:The improved Helicobacter pylori phage lysins involved in this paper were composed of hy⁃drophobic polypeptide with the function of penetrating outer membrane and lyase and holin encoded by H.pylori phage,and the difference activity expressed in Escherichia coli and Pichia pastoris was compared.Methods:The constructed recombinant plasmids were transformed into E.coli BL21(DE3)and P.pastoris X33,respectively.After expression and purification,the improved lysins were obtained.The difference in the activity of the improved ly⁃sins expressed by different expression systems was compared in vitro by Kirby-Bauer test and liquid method.Re⁃sults:In vitro antibacterial experiments showed that improved lysin expressed by E.coli BL21(DE3)was more ef⁃fective than that expressed by P.pastoris X33.Conclusion:The two expression systems had their own advantages.In terms of activity,the expression system of E.coli BL21(DE3)was better,while the expression system of P.pasto⁃ris X33 reduced the activity of improved lysin due to its glycosylation of exogenous proteins.