生物学杂志

生物学杂志

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HaV01 Pol内部半胱氨酸对其剪接活性的影响

  

  1. 东华大学 生物科学与技术研究所，上海 201620
  • 出版日期:2018-10-18 发布日期:2018-10-18
  • 通讯作者: 孟清，教授，博士生导师，研究方向为蛋白质内含子和仿生蛛丝， E-mail: mengqing@dhu.edu.cn
  • 作者简介:李佳，博士研究生，研究方向为蛋白质内含子和相关分子生物学研究，E-mail: lixuanjiajia@foxmail.com
  • 基金资助:
    国家自然科学基金（31570721）；美国国家卫生研究院项目NIH（R01GM114429）；国家留学基金委（201306630039）；上海市科委基础研究重点项目（14521100700和14520720200）；东华大学博士研究生创新基金（102552015094）

Effect of HaV01 Pol internal cysteines to its splicing activity

  1. Insititute of Biological Science and Biotechnology, Donghua University, Shanghai 201620, China
  • Online:2018-10-18 Published:2018-10-18

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摘要： 希望获得一个内部不含有半胱氨酸的蛋白质内含子作为标记和修饰的工具。通过定点突变和搭桥PCR技术对HaV01 Pol内部4个半胱氨酸Cys进行突变和替换，然后通过Western Blot检测对应的蛋白质内含子活性。结果发现，位于112位的半胱氨酸突变可以影响HaV01 Pol的剪接活性，其余3个对其剪接活性则没有任何影响，用甘氨酸Gly和苏氨酸Thr替换112Cys后，可以挽救HaV01 Pol的剪接活性。第一次发现一个非保守区的半胱氨酸可以决定蛋白质内含子的活性。通过Gly和Thr替换成功获得了2个没有半胱氨酸的HaV01 Pol蛋白质内含子，为HaV01 Pol进一步改造和应用奠定了基础。

关键词: 半胱氨酸, 蛋白剪接, 定点突变

Abstract: The purpose of this study is to obtain a Cysteine-free intein as a biological tool for labeling or modification. Four Cysteines in HaV01 Pol were mutated and replaced by site-directed mutation and over-lap PCR, and their splicing activity was detected by Western Blot. The results showed that the 112th Cysteine negatively regulates the splicing activity of HaV01 Pol but not other three Cysteines. In addition, replacement of the 112th Cysteine with Glycine and Threonine can rescue the splicing efficiency up to 50%. Surprisingly, this is the first time to report one Cysteine localized at the unconserved block which can determine the intein′s splicing activity. In the end, two Cysteine free HaV01 Pol mutants were constructed which are facilitated for HaV01 Pol′s further transformation as well as application.

Key words: cysteine, protein splicing, site-directed mutation