抗栓肽Ala~(34)→Trp突变体的表达及活性研究

目的:将抗栓肽34位的丙氨酸突变为色氨酸,构建活性较高的突变体。方法:通过设计合成两对引物经PCR获得抗栓肽突变体基因,利用质粒pET32a( )作为载体,在大肠杆菌BL21(DE4)内表达。利用融合蛋白本身的组氨酸标签,以金属亲和色谱进行纯化,然后用肠激酶切除融合部分,所得蛋白用体外抗血小板凝集实验检测其活性。结果与结论:抗栓肽突变体以可溶形式高效表达,活性检测显示其抑制二磷酸腺苷诱导的血小板聚集的IC50为150 nmol/L。与原型蛋白相比,突变体活性明显提高。

Expression and bioactivity of decorsin mutant: Ala34→Trp

Aim:To obtain a decorsin mutant with higher bioactivity by replacing the Ala34 with Trp.Methods: Decorsin mutant gene was obtained and inserted into the expression vector pET32a by DNA recombinanation.The fusion protein was produced in E.coli BL21(DE4) and purified over a HisTrap chelating HP column followed by enterokinase cleavage.Its function was finally characterized.Results and Conclusion: The mutant protein was highly expressed,showing a potent inhibition of ADP-induced platelet aggregation with observed IC50 of 150 nmol/L.In comparison to decorsin,the mutant protein shows higher bioactivity.