您的位置：山东大学 -> 科技期刊社 -> 《山东大学学报（理学版）》

《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

•   •    下一篇

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)

RichHTML

63

PDF (PC)

790

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。"

1 LARSEN H , OMANG S , STEENSLAND H . On the gas vacuoles of the halobacteria[J]. Archives of Microbiology, 1967, 59 (1/2/3): 197- 203.
2 PFENNIG N , COHEN-BAZIRE G . Some properties of the green bacterium Pelodictyonclathratiforme[J]. Archives of Microbiology, 1967, 59 (1): 226- 236.
3 STOECKENIUS W , KUNAU W H . Further characterization of particulate fractions from lysed cell envelopes of Halobacteriumhalobium and isolation of gas vacuole membranes[J]. The Journal of Cell Biology, 1968, 38 (2): 336- 357.
4 WALSBY A E . Gas vesicles[J]. Microbiological Reviews, 1994, 58 (1): 94- 144.
5 BOWEN C C , JENSEN T E . Blue-green algae: fine structure of the gas vacuoles[J]. Science, 1965, 147 (3664): 1460- 1462.
doi: 10.1126/science.147.3664.1460
6 JOST M . Die Ultrastruktur von Oscillatoriarubescens D. C[J]. Archives of Microbiology, 1965, 50 (3): 211- 245.
7 WALSBY A E , BUCKLAND B . Isolation and purification of intact gas vesicles from a blue-green alga[J]. Nature, 1969, 224 (1): 716- 717.
8 JONES D D , JOST M . Characterization of the protein from gas-vacuole membranes of the blue-green alga, Microcystis aeruginosa[J]. Planta, 1971, 100 (4): 277- 287.
doi: 10.1007/BF00385192
9 WALSBY A E . The permeability of blue-green algal gas-vacuole membranes to gas[J]. Proceedings of the Royal Society B: Biological Sciences, 1969, 173 (1031): 235- 255.
doi: 10.1098/rspb.1969.0049
10 WALSBY A E . Permeability of gas vesicles to perfluorocyclobutane[J]. Microbiology, 1982, 128 (8): 1679- 1684.
doi: 10.1099/00221287-128-8-1679
11 HAYES P K , WALSBY A E . The inverse correlation between width and strength of gas vesicles in cyanobacteria[J]. European Journal of Phycology, 1986, 21 (2): 191- 197.
12 DUNTON P G , WALSBY A E . The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length[J]. FEMS Microbiology Letters, 2005, 247 (1): 37- 43.
doi: 10.1016/j.femsle.2005.04.026
13 WALSBY A E . Structure and function of gas vacuoles[J]. Bacteriological Reviews, 1972, 36 (1): 1- 32.
14 PFEIFER F . Distribution, formation and regulation of gas vesicles[J]. Nature Reviews Microbiology, 2012, 10 (10): 705- 715.
doi: 10.1038/nrmicro2834
15 MLOUKA A , COMTE K , CASTETS A M , et al. The gas vesicle gene cluster from Microcystis aeruginosa and DNA rearrangements that lead to loss of cell buoyancy[J]. Journal of Bacteriology, 2004, 186 (8): 2355- 2365.
doi: 10.1128/JB.186.8.2355-2365.2004
16 XU B Y , DAI YN , ZHOU K , et al. Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa[J]. Actacrystallographica. Section D, Biological crystallography, 2014, 70 (11): 3013- 22.
doi: 10.1107/S1399004714021312
17 许柏英, 张甜甜. 铜绿微囊藻气囊蛋白GvpW的异源表达纯化与晶体生长研究[J]. 山东大学学报(理学版), 2017, 52 (9): 103- 110.
XU Boying , ZHANG Tiantian . Heterelogous expression, purification andcrystal growth of gas vesicle protein GvpW from the cyanobacterial Microcystis aeruginosa[J]. Journal of Shandong University(Natural Science), 2017, 52 (9): 103- 110.
18 HAYES P K , WALSBY A E , WALKER J E . Complete amino acid sequence of cyanobacterial gas-vesicle protein indicates a 70-residue molecule that corresponds in size to the crystallographic unit cell[J]. Biochemical Journal, 1986, 236 (1): 31- 36.
19 BUCHHOLZ B E , HAYES P K , WALSBY A E . The distribution of the outer gas vesicle protein, GvpC, on the Anabaena gas vesicle, and its ratio to GvpA[J]. Journal of General Microbiology, 1993, 139 (10): 2353- 2363.
doi: 10.1099/00221287-139-10-2353
20 HAYES P K , LAZARUS C M , BEES A , et al. The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcystissp[J]. Molecular Microbiology, 1988, 2 (5): 545- 552.
21 WALSBY A E , HAYES P K . The minor cyanobacterial gas vesicle protein, GvpC, is attached to the outer surface of the gas vesicle[J]. Journal of General Microbiology, 1988, 134 (10): 2647- 2657.
22 ENGLERT C , PFEIFER F . Analysis of gas vesicle gene expression in Haloferaxmediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins[J]. Journal of Biological Chemistry, 1993, 268 (13): 9329- 9336.
23 DUNTON P G , MAWBY W J , SHAW V A , et al. Analysis of tryptic digests indicates regions of GvpC that bind to gas vesicles of Anabaena flos-aquae[J]. Microbiology, 2006, 152 (6): 1661- 1669.
doi: 10.1099/mic.0.28755-0
24 BELENKY M , MEYERS R , HERZFELD J . Subunit structure of gas vesicles:a MALDI-TOF mass spectrometry study[J]. Biophysical Journal, 2004, 86 (1): 499- 505.
25 KINSMAN R , WALSBY A E , HAYES P K . GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles[J]. Molecular Microbiology, 1995, 17 (1): 147- 154.
[1] 许柏英,张甜甜. 铜绿微囊藻气囊蛋白GvpW的异源表达纯化与晶体生长研究[J]. 山东大学学报（理学版）, 2017, 52(9): 103-110.
[2] 巨颖琳,李小明*. 南四湖3种沉水植物对铜绿微囊藻化感作用研究[J]. J4, 2011, 46(3): 1-8.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] 杨军. 金属基纳米材料表征和纳米结构调控[J]. 山东大学学报（理学版）, 2013, 48(1): 1 -22 .
[2] 孙小婷1,靳岚2*. DOSY在寡糖混合物分析中的应用[J]. J4, 2013, 48(1): 43 -45 .
[3] 罗斯特,卢丽倩,崔若飞,周伟伟,李增勇*. Monte-Carlo仿真酒精特征波长光子在皮肤中的传输规律及光纤探头设计[J]. J4, 2013, 48(1): 46 -50 .
[4] 杨伦，徐正刚，王慧*，陈其美，陈伟，胡艳霞，石元，祝洪磊，曾勇庆*. RNA干扰沉默PID1基因在C2C12细胞中表达的研究[J]. J4, 2013, 48(1): 36 -42 .
[5] 冒爱琴1, 2, 杨明君2, 3, 俞海云2, 张品1, 潘仁明1*. 五氟乙烷灭火剂高温热解机理研究[J]. J4, 2013, 48(1): 51 -55 .
[6] 李永明1, 丁立旺2. PA误差下半参数回归模型估计的r-阶矩相合[J]. J4, 2013, 48(1): 83 -88 .
[7] 赵同欣1,刘林德1*,张莉1,潘成臣2,贾兴军1. 紫藤传粉昆虫与花粉多型性研究[J]. 山东大学学报（理学版）, 2014, 49(03): 1 -5 .
[8] 汤晓宏1，胡文效2*，魏彦锋2，蒋锡龙2，张晶莹2，. 葡萄酒野生酿酒酵母的筛选及其生物特性的研究[J]. 山东大学学报（理学版）, 2014, 49(03): 12 -17 .
[9] 王碧玉,曹小红*. 算子矩阵的Browder定理的摄动[J]. 山东大学学报（理学版）, 2014, 49(03): 90 -95 .
[10] 郭兰兰1,2,耿介1,石硕1,3,苑飞1,雷丽1,杜广生1*. 基于UDF方法的阀门变速关闭过程中的#br# 水击压强计算研究[J]. 山东大学学报（理学版）, 2014, 49(03): 27 -30 .
版权所有 © 2018 《山东大学学报(理学版)》编辑部　
地址: 山东省济南市山大南路27号山东大学中心校区（250100）　电话: +86-0531-88366917　E-mail: xblxb@sdu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发