静水椎螺乙酰胆碱结合蛋白在Bac-to-Bac系统中的表达、纯化与结晶

生物技术通报 ›› 2014, Vol. 0 ›› Issue (8): 126-131.

静水椎螺乙酰胆碱结合蛋白在Bac-to-Bac系统中的表达、纯化与结晶

林波, 孟海玲, 吴勇, 邴晖, 长孙东亭, 罗素兰

海南大学热带生物资源教育部重点实验室海口市海洋药物重点实验室, 海口 570228

修回日期:2013-12-17 出版日期:2014-08-15 发布日期:2014-08-01
作者简介:作者简介: 林波，男，博士研究生，研究方向：蛋白质结构；E-mail：linbo_752@163.com

Expression，Purification，Crystallization of Acetylcholine Binding Proteins from Lymnaea stagnalis in Bac-to-Bac System

Lin Bo, Meng Hailing, Wu Yong, Bing Hui, Zhangsun Dongting, Luo Sulan

Key Lab for Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou 570228

Revised:2013-12-17 Published:2014-08-15 Online:2014-08-01
Supported by:
国家自然科学基金项目(41366002);国家国际科技合作专项(2011DFR31210);海南省社会发展科技专项(SF201328);海口市重点科技计划项目(2013-16);长江学者和创新团队发展计划(IRT1123);海南省研究生创新科研课题(B201305,B201306)

摘要/Abstract

摘要：

表达、纯化静水椎螺（Lymnaea stagnalis）乙酰胆碱结合蛋白（Ls-AChBP）并得到晶体。将静水椎螺乙酰胆碱结合蛋白cDNA 序列克隆到表达载体pFastBac1上，构建了重组表达质粒pFastBac1-Ls-AChBP-His，经重组子筛选，得到重组杆状病毒质粒Bacmid，采用CellfectinⅡ脂质体，把带有外源基因的杆状病毒质粒Bacmid 转染到昆虫细胞中，经镍柱和凝胶色谱柱对重组蛋白进行纯化得到五聚体，并用机器人进行结晶筛选获得其蛋白晶体。重组蛋白Ls-AChBP在Bac-to-Bac表达系统中得到高效表达并被纯化，结晶筛选得到晶体。

关键词: 静水椎螺乙酰胆碱结合蛋白, Bac-to-Bac杆状病毒表达系统, 重组表达, 纯化, 结晶

Abstract:

It was to construct a recombinant bacmid that expresses acetylcholine binding protein from Lymnaea stagnalis（Ls-AChBP）and express the Ls-AChBP functional genes in Bac-to-Bac expression system to obtain the crystal. Synthesized Ls-AChBP cDNA was inserted into the correct position of the baculovirus expression vector pFastBac1. After the positive recombinant bacmid was screened, the recombinant bacmid was transfected into the insect cells to generate restructured baculovirus for high expression of the recombinant protein Ls-AChBP（rLs-AChBP）, which was captured by nickel-charged resin, and further purified by gel filtration chromatography. The rLs-AChBP pentamer was obtained and performed crystallization by robot screen. Recombinant Ls-AChBP expression in Bac-to-Bac system was successfully and we got Ls-AChBP crystal. Ls-AChBP is one of the AchBPs. The crystal structure of rLs-AchBP is an available template of description of nAChRs structure and function.

Key words: Ls-AChBP, Bac-to-Bac expression system, Recombinant expression, Purification, Crystallization

林波, 孟海玲, 吴勇, 邴晖, 长孙东亭, 罗素兰. 静水椎螺乙酰胆碱结合蛋白在Bac-to-Bac系统中的表达、纯化与结晶[J]. 生物技术通报, 2014, 0(8): 126-131.

Lin Bo, Meng Hailing, Wu Yong, Bing Hui, Zhangsun Dongting, Luo Sulan. Expression，Purification，Crystallization of Acetylcholine Binding Proteins from Lymnaea stagnalis in Bac-to-Bac System[J]. Biotechnology Bulletin, 2014, 0(8): 126-131.

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