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《山东大学学报(理学版)》 ›› 2018, Vol. 53 ›› Issue (11): 1-8.doi: 10.6040/j.issn.1671-9352.0.2018.200

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铜绿微囊藻气囊结构蛋白GvpC的表达纯化与晶体生长研究

许柏英1,2,*(),张甜甜1   

  1. 1. 重庆师范大学生命科学学院, 重庆 401331
    2. 中国科学技术大学生命科学学院, 安徽 合肥 230026
  • 收稿日期:2018-04-17 出版日期:2018-11-01 发布日期:2018-11-14
  • 通讯作者: 许柏英 E-mail:xbywh@126.com
  • 作者简介:许柏英(1987—)，女，副教授，博士，研究方向为蛋白质的结构与功能. E-mail:xbywh@126.com
  • 基金资助:
    重庆市前沿与应用基础研究计划资助项目(cstc2015jcyjBX0142);重庆市前沿与应用基础研究计划资助项目(cstc2016jcyjA0375)

Expression, purification and crystallization of gas vesicle structure protein GvpC from the cyanobacterium Microcystis aeruginosa

Bo-ying XU1,2,*(),Tian-tian ZHANG1   

  1. 1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
    2. School of Life Sciences, University of Science and Technology of China, Heifei 230026, Anhui, China
  • Received:2018-04-17 Online:2018-11-01 Published:2018-11-14
  • Contact: Bo-ying XU E-mail:xbywh@126.com
  • Supported by:
    重庆市前沿与应用基础研究计划资助项目(cstc2015jcyjBX0142);重庆市前沿与应用基础研究计划资助项目(cstc2016jcyjA0375)

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摘要：

对GvpC进行了初步晶体学研究。首先对GvpC进行分子克隆，利用原核表达系统在体外进行异源表达；结合变复性和凝胶过滤层析的方法纯化GvpC；通过圆二色谱的方法鉴定GvpC的复性效果；运用坐滴蒸汽扩散法进行晶体初筛。结果表明：GvpC在大肠杆菌表达系统中表达为包涵体；通过变复性和凝胶过滤层析能纯化出纯度较高的蛋白；圆二色谱证实复性后的GvpC形成了正确的二级结构，复性效果较佳；通过晶体初筛获得了GvpC的晶体，后续即可通过解析GvpC的晶体结构而获得其三维结构。

关键词: 铜绿微囊藻, 气囊, GvpC, 变复性, 蛋白质晶体

Abstract:

Preliminary crystallographic study on the gas vesicle structure protein GvpC from the cyanobacterium Microcystis aeruginosa was performed. Firstly, gene gvpC was cloned and expressed; then, the recombinant protein was purified by denaturation and renaturation and gel filtration chromatography; the result of renaturation was detected by circular dichroism. Crystal screening was performed by sitting drop vapor diffusion techniques. GvpC was overexpressed as inclusion body in E. coli, and then the protein has been purified through denaturation and renaturation. The result of circular dichroism indicated that GvpC was refolded successfully in vitro. Crystals of GvpC were also obtained by crystal screening. Hence, the three-dimensional structure of GvpC could be determinated by solving its crystal structure.

Key words: Microcystis aeruginosa, gas vesicle, GvpC, denaturation and renaturation, protein crystal

中图分类号: 

  • Q816

图1

GvpC蛋白一级结构示意图(A)和一级序列比对(B) A. N-ter：GvpC蛋白的氨基端序列；RRⅠ/RRⅡ/RRⅢ/RRⅣ：GvpC蛋白4个高度保守的33RR重复序列单位；C-ter：GvpC蛋白的羧基端序列；B. 33RR consensus：GvpC保守的33RR重复序列单位的氨基酸序列。"

图2

PSIPREDv3.3软件预测GvpC的二级结构"

图3

GvpC 33RR保守重复区的三级结构预测模型 RRⅠ/RRⅡ/RRⅢ/RRⅣ：GvpC蛋白4个高度保守的33RR重复序列单位。 "

图4

gvpC在E.coli中的表达结果 BL21(DE3)和Rosetta(DE3)分别表示质粒在这2种表达菌株中进行表达；M：Marker；B：诱导前本底蛋白对照；S：总蛋白经超声裂解后的上清；P：总蛋白经超声裂解后的沉淀。 "

图5

GvpC分子筛层析图(A)和分子筛层析后电泳图(B) A.横轴表示层析体积(mL)，纵轴表示280 nm紫外吸收值(mAU). GvpC只有一个主峰，峰尖位于82.6 mL处; B. SDS-PAGE胶图标注从左到右依次表示蛋白标准分子质量和GvpC分子筛层析纯化出峰对应的54-66管蛋白样品。"

图6

GvpC的CD结果图"

表1

GvpC圆二色谱结果"

Secondary elements Ratio/%
Helix 30.0
Beta 0.0
Turn 31.2
Random 38.8
Total 100.0
RMS 35.479

图7

GvpC的晶体照片"

图8

不同种具气囊蓝藻GvpC的多序列比对 多序列比对是运用Multalin (http://multalin.toulouse.inra.fr/multalin/multalin.html)和ESPript 3.0 (http://espript.ibcp.fr/ESPrint/cgibin/ESPript.cgi)完成的。所有的蛋白质序列均下载自NCBI数据库(http://www.ncbi.nlm.nih.gov)。分别来自物种Microcystis aeruginosa PCC 7806 (CAE11901.1)，Microcystissp. FACHB-854 (AAS78467.1)，Planktothrixagardhii NIVA-CYA 137 (CAB59520.1)，Dolichospermumlemmermannii (AAX37399.1)，Nodulariaspumigena (AAP46544.1)，Tolypothrix sp. PCC 7601 (EKF01074.1)，Microchaetediplosiphon (P08041.1)，Raphidiopsisbrookii D9 (EFA73421.1)，Nostoc sp. PCC 7120 (BAB73951.1)，Anabaena variabilis ATCC 29413 (ABA19703.1)；高度保守的氨基酸残基显示为红色。"

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