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目的:探讨聚乙二醇(PEG)修饰重组溶葡球菌酶(lysostaphin)的反应条件以及修饰后产物的纯化方法。方法:采用超声波细胞粉碎机进行菌体破碎,阳离子交换层析、疏水层析进行蛋白纯化;在不同条件下,将活化的单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)与纯化后的lysostaphin反应,以单个PEG-Lysostaphin的比例为指标,用SDS-PAGE、MALDI-TOF-MS方法确定其在修饰产物中的所占比例;采用Sephacryl S-200分子筛凝胶层析法对修饰产物进行分离纯化。结果:mPEG-SPA修饰lysostaphin的反应条件为pH 8.0,温度4℃,lysostaphin与mPEG-SPA的质量比为1∶5,反应时间2.0h;反应产物经一步Sephacryl S-200分子筛凝胶层析纯化后,初步实现分离。结论:初步确定了聚乙二醇修饰lysostaphin的反应条件及修饰产物的纯化方法。
OBJECTIVE: To investigate the reaction conditions of PEG-modified lysostaphin and the purification method of the modified product. Methods: The cells were disrupted by cation exchange cell disintegration, purified by cation exchange chromatography and hydrophobic chromatography. Under different conditions, the activated monomethoxy polyethylene glycol succinimidyl propionate (mPEG- SPA) with purified lysostaphin. The proportion of individual PEG-Lysostaphin was determined by SDS-PAGE and MALDI-TOF-MS. The content of modified PEG-Lysostaphin was determined by using Sephacryl S-200 molecular sieve gel layer Analysis of the modified products were isolated and purified. Results: The reaction conditions of lysostaphin modified by mPEG-SPA were pH 8.0, temperature 4 ℃, the mass ratio of lysostaphin to mPEG-SPA was 1: 5 and the reaction time was 2.0 h. The reaction product was purified by one-step Sephacryl S-200 molecular sieve gel chromatography After the initial separation. Conclusion: The reaction conditions of PEGylation of lysostaphin and the purification of the modified product were preliminarily determined.