摘要
[目的]构建人泛素结合酶UbcH5c的活性位点突变体S22R和F62A,同时分析这2种突变体蛋白与野生型蛋白在泛素化反应中的活性差异。[方法]通过点突变(Site-Directed Mutagenesis)的方法构建2种突变体质粒,利用0.5 mmol/L IPTG分别诱导2种突变体蛋白在大肠杆菌中进行表达;将菌体超声破碎后,依次利用阳离子交换层析法对UbcH5c突变蛋白进行分离纯化,最后利用体外泛素化酶促反应结合蛋白免疫印迹杂交的方法分析野生型UbcH5c与其活性突变体UbcH5c S22R和UbcH5c F62A在泛素化修饰上的差异。[结果]人泛素结合酶UbcH5c的2个突变体蛋白S22R、F62A的泛素化修饰程度明显弱于野生型蛋白。[结论]突变体S22R和F62A突变均不同程度的改变了人泛素结合酶UbcH5c与泛素分子之间的结合活性,即2个突变的位点中第62位苯丙氨酸残基及第22位丝氨酸残基均是UbcH5c结合泛素的关键位点,而且后者对于UbcH5c结合泛素活性的影响更大。
[ Objective] To investigate the key amino acid that contributing to the ubiquitin conjugating activity in UbcH5c catalyzed ubiquitin conjugation, we construct two site mutants including S22R and F62A, and analyze the activity difference of these two mutants in contrast with wide type UbcH5c, subsequently. [ Method] The site mutant was constructed with site-directed mutagenesis based on PCR. The overexpres- sion of these two mutants in E. coli were induced with 0.5 mmol/L IPTG. When the E. coli were homogenized with sonication, the over- pressed recombinant protein were purified with cation exchange chromatography. Finally, the ubiquitin-conjugating activities of these two mu- tants in compare with wide type UbcHSc were analyzed with in vitro ubiquitination assay. [ Result] In comparison with wide type UbcHSc, these two mutants including S22R and F62A showed obvious weaker ubiquitin conjugating activity. [ Conclusion] The displace of these two a- mino acids including S22 and F62 significantly changed the combination of UbcH5c with ubiquitin in different degree, which means that both the S22 and F62 are the key site for the combination of UbcHSc with ubiquitin. Moreover, F62 indicates more important combination capability with ubiquitin than S22.
出处
《安徽农业科学》
CAS
2014年第3期663-666,共4页
Journal of Anhui Agricultural Sciences
基金
国家自然科学基金(81101220)
天津市应用基础与前沿研究计划项目(12JCQNJC08100)
"十二五"天津市中青年骨干创新人才支持计划
