NMDAR1蛋白膜外抗原结构域的重组表达, 纯化和免疫反应原性鉴定

Translated title of the contribution: Expression, purification and immunoreactivity characterization of extracellular antigenic domains of NMDAR1 protein

Ding Bao, Wei Li, Leming Shi, Quanzhen Li

Research output: Contribution to journalArticle

Abstract

This study aimed to construct prokaryotic recombinant plasmids for expression of the extracellular domains of NMDAR1 protein, purify and characterize the immunoreactivity of the recombinant proteins. Based on the mRNA sequence of human NMDAR1 gene, we predicted the structure of the antigenic domains in the extracellular part of the protein using the "phyre2" software. Primers were designed to amplify the nucleic acid fragments encoding the NMDAR1 extracellular antigenic domains by RT-PCR. The amplified gene fragments were cloned into pCold-SUMO vector to construct the recombinant plasmids which were transformed into Escherichia coli DH5α. The positive colonies harboring the recombinant plasmids were picked and verified by PCR and DNA sequencing. Then, the recombinant plasmids were transformed into E. coli BL21(DE3) strain and induced by IPTG for protein expression. The recombinant proteins were purified by Ni-NTA affinity chromatography. The target proteins were further purified by removing the 6 His-SUMO tag using enzyme excision followed by gel filtration chromatography using AKTA purifier. The purity of the recombinant proteins were evaluated by SDS-PAGE and the immunoreactivity were characterized by Western blotting. Three DNA fragments encoding the extracellular domains of NMDAR1 protein, including NR1-M1 (encoding 19-393 aa), NR1-S1 (encoding 394-544 aa) and NR1-S2 (encoding 663-800 aa), were amplified by RT-PCR. The NR1-S1 and NR1-S2 were linked with G (arginine) and T (threonine) amino acid as a combined fragment. The NR1-M1 and NR1-S1-GT-S2 fragments were cloned into pCold-SUMO vector and two recombinant plasmids, pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2, were generated and expressed in E. coli. SDS-PAGE analysis showed that the recombinant plasmids expressed soluble NR1-M1 and NR1-S1-GT-S2 proteins in bacterial. After affinity chromatography and gel filtration chromatography, we obtained high purity target proteins. Western blotting assay showed that the recombinant proteins NR1-M1 and NR1-S1-GT-S2 can bind specially with their corresponding antibodies, suggesting the recombinant proteins retained antigenic reactivity. We constructed a prokaryotic expression system for expressing the NMDAR1 protein extracellular parts that had immunoreactivity successfully, and the purified proteins can be used for studying NMDAR1 function and testing the autoantibodies.

Translated title of the contributionExpression, purification and immunoreactivity characterization of extracellular antigenic domains of NMDAR1 protein
Original languageChinese
Pages (from-to)1979-1988
Number of pages10
JournalShengwu Gongcheng Xuebao/Chinese Journal of Biotechnology
Volume33
Issue number12
DOIs
StatePublished - Dec 25 2017
Externally publishedYes

Keywords

  • Extracellular protein
  • NMDAR1
  • Prokaryotic expression
  • Purification

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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