| 摘要: |
| 【目的】建立范式酵母（Vanderwaltozyma polyspora）Wss1（VpWss1）金属蛋白酶的原核表达纯化系统，对该蛋白酶的均一性和自切活性进行初步分析，为VpWss1的晶体制备及结构解析奠定基础。【方法】构建VpWss1基因的融合表达载体，将其转化入E.coli 2566菌株中进行诱导表达，利用Ni-NTA亲和柱和SP阳离子交换柱对融合蛋白进行纯化。通过动态激光散射仪（DLS）和Superdex 200分子筛对该蛋白酶的均一性和聚集状态进行分析，同时与不同类型和长度的DNA底物孵育，分析野生型和突变型SUMO VpWss1蛋白的自切活性。【结果】表达纯化的目的蛋白SUMO VpWss1/SUMO VpWss1E96Q为可溶性蛋白，分子质量约为50 ku，纯度>95%。野生型SUMO-VpWss1蛋白均一性较差，呈现多种状态，但突变型SUMO VpWss1E96Q蛋白的均一性较好，其主要单一状态（Mass）的含量达到93.2%。在不同类型和长度DNA下，野生型SUMO-VpWss1均可发生自切，且随DNA长度的延长呈现出先增强后减弱的趋势，而突变型SUMO VpWss1E96Q的自切活性显著减弱。【结论】成功表达纯化了金属蛋白酶SUMO VpWss1及其突变体SUMO VpWss1E96Q，明晰了两者的均一性和自切活性。 |
| 关键词: VpWss1金属蛋白酶 DNA-蛋白交联物 均一性 自切活性 |
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| 基金项目:中央高校基本科研业务费专项（2014YB032）；教育部回国人员科研启动基金项目（11304252） |
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| Prokaryotic expression and physicochemical properties of Vanderwaltozyma polyspora Wss1 |
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LUO Yixin,XI Xuguang,SHAN Liwei,,et al
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| Abstract: |
| 【Objective】This paper established a protocol for prokaryotic expression and purification of metalloproteinase VpWss1 and preliminarily analyzed its homogeneity and cleavage activity.【Method】This research constructed prokaryotic expression vector of the Vanderwaltozyma polyspora Wss1 and transformed it into E.coli 2566 bacteria for expression.The fusion protein was purified using Ni-NTA resin and SP cation exchange column by fast liquid chromatography system (AKTA primer).Then dynamic laser light scattering instrument (DLS) and Superdex 200 molecular sieve were used to detect homogeneity and aggregation state of the protein.A detailed analysis was also conducted to analyze the self cleavage activity of SUMO VpWss1 and SUMO VpWss1E96Q.【Result】The fusion protein obtained by prokaryotic expression was soluble with molecular weight of about 50 ku and high purity of >95%.SUMO VpWss1 had a poor uniformity and variety of aggregation states by dynamic laser light scattering (DLS) and Superdex 200 molecular sieve analysis.The mutant SUMO VpWss1E96Q had higher uniformity,more stable homogeneous state and the content of “Mass” was 93.2%.In the presence of different lengths and types of DNA,metalloproteinase VpWss1 had self cleavage activity and showed a trend of increasing first and then decreasing.The self cleavage activity of the mutant SUMO VpWss1E96Q was notably weaker than the wild type.【Conclusion】This study successfully expressed and purified the SUMO VpWss1 and the mutant SUMO VpWss1E96Q,and clarified the uniformity and self cleavage activity. |
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