辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 <sup>*</sup>

doi:10.13523/j.cb.1906036

中国生物工程杂志

2020, Vol. 40

Issue (1-2): 116-123 DOI: 10.13523/j.cb.1906036

技术与方法

辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 ^*

朱彤彤¹,杨磊¹,刘应保¹,孙文秀^1,^**(

),张修国²

1 长江大学生命科学学院荆州 434000
2 山东农业大学植物保护学院泰安 271018

Purification and Crystallization of PcCRN20-C from Phytophthora capsici

ZHU Tong-tong¹,YANG Lei¹,LIU Ying-bao¹,SUN Wen-xiu^1,^**(

),ZHANG Xiu-guo²

1 College of Life Science,Yangtze University,Jingzhou 434000,China
2 College of Plant Protection,Shandong Agricultural University,Tai’an 271018,China

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摘要：

CRN(crinkling and necrosis-inducing protein)为疫霉菌在与寄主互作过程中分泌的一类特有胞质效应因子,干扰寄主细胞正常的生理代谢和功能。采用PCR法从辣椒疫霉LT1534菌株cDNA中克隆PcCRN20-C基因。该基因序列长783bp,编码261个氨基酸。构建重组表达载体,并转化大肠杆菌BL21(DE3)。在优化条件下诱导表达重组蛋白,利用Ni-NTA金属螯合层析、离子交换层析、分子筛层析和胰蛋白酶酶解技术获得高纯目的蛋白,SDS-PAGE分析表明,蛋白质分子量约为25kDa。采用座滴气相扩散法进行晶体制备和筛选,成功获得了蛋白质晶体,并通过X-射线衍射仪收集了晶体衍射花样。结合蛋白质晶体学方法,获得了有衍射的辣椒疫霉PcCRN20-C蛋白晶体,为进一步研究CRN蛋白的结构与病原菌致病机制提供参考资料。

关键词： 辣椒疫霉; 皱缩坏死蛋白; 蛋白质纯化结晶; X-射线衍射

Abstract:

Crinkling and necrosis-inducing protein is a class of special secreted cytoplasmic protein when phytophthora occurs, which interferes with the normal physiological metabolism and function of host cells. However, the relationship between its three-dimensional structure and infection mechanisms need to be further studied. The sequence of PcCRN20-C was amplified by PCR from cDNA of Phytophthora capsici LT1534, which is 783bp in length and encodes 261 amino acids. The expression vector of pET-28a-MBP-PcCRN20-C was constructed and transformed to Escherichia coli BL21(DE3). The recombinant protein was induced under optimal condition, subsequently purified by nickel chelate affinity chromatography, ion-exchange chromatography, molecular-exclusion chromatography and trypsin hydrolysis technology. The product identified as a specific band of 25kDa by SDS-PAGE, which is the same as predicted. Subsequently, crystals were grown with the sitting-drop vapour-diffusion method and diffracted by X-ray. As a result, the crystals of CRN were obtained in a way of protein crystallography, which set a foundation for further study on the connect of CRN structure with function to analyze the pathogenic mechanism of P. capsici at the molecular level.

Key words: Phytophthora capsici Crinkling and necrosis-inducing protein Purification crystallization X-ray diffraction

收稿日期: 2019-06-26 出版日期: 2020-03-27

ZTFLH:

S432.1

基金资助: * 国家自然科学基金(31701573);湖北省教育厅科学研究计划重点项目(D20181302)

通讯作者: 孙文秀 E-mail: wenxiusun@163.com

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引用本文:

朱彤彤,杨磊,刘应保,孙文秀,张修国. 辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 116-123.

ZHU Tong-tong,YANG Lei,LIU Ying-bao,SUN Wen-xiu,ZHANG Xiu-guo. Purification and Crystallization of PcCRN20-C from Phytophthora capsici. China Biotechnology, 2020, 40(1-2): 116-123.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1906036 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I1-2/116

图1 PCR扩增PcCRN20-C基因片段

图2 PcCRN20-C基因核苷酸及氨基酸序列

图3 SDS-PAGE检测PcCRN20-C蛋白亲和层析结果

图4 PcCRN20-C蛋白纯化结果

图5 PcCRN20-C纯化蛋白SDS-PAGE检测示意图

表1 PcCRN20-C质谱检测结果

图6 不同条件PcCRN20-C蛋白结晶形态

图7 PcCRN20-C优化晶体及衍射花样

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