摘要
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 study the reaction conditions for the chemical modification of recombinant lysostaphin （Lysostaphin） with activated polyethlene glycol （PEG） and the methods for the purification of PEG- Lysostaphin. Methods： Disrupt the cell wall of bacteria by ultrasonic cell disruptor and purify the lysostaphin by Cation exchange chromatography （SP） and Hydrophobic chromatography （HIC）; Lysostaphin was chemical modified with activated mono-methoxy polyethlene glycol succinimide propionate （mPEG - SPA） under various conditions, the ration of mono-mPEG-Lysostaphin was determined by SDS-PAGE and EMALDI-TOF-MS; The reaction mixture was purified by Sephacryl S-200 molecular size exclusion chromatography. Results： The optimal reaction was pH 8.0,temperature 4℃, 1：5 for the mass ratio of lysostaphin and mPEG, two hours for reaction time was selected. The reaction mixture was preliminarily separated by one step Sephacryl S-200 molecular size exclusion chromatography. Conclusion： It＇s preliminarily determined that the optimal reaction conditions for the chemical modification of lysostaphin with activated polyethlene glycol and the methods for the purification of PEG- Lysostaphin.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第6期12-17,共6页
China Biotechnology
