溶葡球菌酶的定点突变与PEG巯基定点修饰

Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin

摘要:

为了建立聚乙二醇 (PEG) 巯基定点修饰溶葡球菌酶的方法，并检验假定连接区的突变与修饰对酶活的影响，对溶葡球菌酶的假定连接区进行了巯基聚乙二醇定点修饰研究。通过分析溶葡球菌酶的结构特征，选择两个结构域之间的氨基酸 (133-154aa) 进行定点突变引入半胱氨酸残基。使用单甲氧基聚乙二醇马来酰亚胺 (mPEG-MAL) 进行定点修饰，对修饰后的酶进行纯化并测定酶活性。结果表明定点突变的半胱氨酸残基PEG修饰效率高、产物单一，运用简便的Ni2+-NTA柱亲和层析法实现了一步分离，获得了高纯度的目标蛋白，但在连接区进行定点突变及PEG定点修饰后的酶活有不同程度的降低，表明假定连接区部分位点的PEG修饰会对溶葡球菌酶的催化活性产生一定影响。

Abstract:

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133?154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further determined. The results show that the modification method for lysostaphin was highly efficient, resulting in the single uniform PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni2+-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.