摘要
目的原核表达代谢型谷氨酸受体4胞内段(metabolic glutamate receptor 4 intracellular,GRM4 intracellular,GRM4 intra),并用Ni柱进行纯化。方法采用PAS(PCR-based accurate synthesis)法合成GRM4 intra基因序列,将目的片段和质粒p Czn1双酶切后,经连接,构建重组质粒pCzn1-GRM4 intra;将重组质粒转化至Rosseta菌株,IPTG诱导GRM4 intra蛋白的表达,表达的蛋白利用Ni柱亲和层析法纯化。结果经测序和双酶切鉴定证明质粒pCzn1-GRM4 intra构建正确;在IPTG诱导下,质粒能够在Rosseta菌株中高效表达,表达的蛋白相对分子质量约18 400,主要以可溶性性形式存在,纯度> 90%。结论成功获得了原核表达的GRM4 intra蛋白,为利用体外pull down方法鉴定GRM4的相互作用蛋白提供了参考。
Objective To express metabolic glutamate receptor 4 intracellular(GRM4 intra) in prokaryotic cells and purify the expressed product by nickel ion affinity chromatography. Methods GRM4 intra gene was synthesized by PCRbased accurate synthesis method. The target gene and plasmid p Czn1 were digested with NdeⅠand XbaⅠand linked,and the constructed recombinant plasmid pCzn1-GRM4 intra was transformed to E. coli Rosseta and induced with IPTG. The expressed protein was purified by nickel ion affinity chromatography. Results Recombinant plasmid pCzn1-GRM4 was constructed correctly as proved by restriction analysis and sequencing,and was highly expressed in E. coli Rosseta. The expressed protein,with a relative molecular mass of about 18 400,mainly existed in a soluble form,of which the purity reached more than 90%. Conclusion GRM4 intra was successfully expressed in prokaryotic cells,which provided a reference for identification of GRM4 interectome in pull down assays in vitro.
作者
李建勋
肖斌
孙朝晖
王露霞
陈丽丹
曹玲
王丽志
李林海
LI Jian-xun;XIAO Bin;SUN Zhao-hui;WANG Lu-xia;CHEN Li-dan;CAO Ling;WANG Li-zhi;LI Lin-hai(Department of Laboratory Medicine,Guangzhou General Hospital of PLA,Guangzhou 510010,Guangdong Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第2期134-138,共5页
Chinese Journal of Biologicals
基金
2015年广州市科创委项目(201604040003)
关键词
代谢型谷氨酸受体4
相互作用
原核表达
纯化
Metabotropic glutamate receptor 4(GRM4)
Interaction
Prokaryotic expression
Purification
