title: Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.
abstract: In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3-1mg of pure and highly active rαBtx per liter of E. coli culture.
authors.affiliation: Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China. Electronic address: foxjuly@gmail.com.
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Neurology, No. 323 Hospital of PLA, Xi'An, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Microbiology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Xingan
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Neurology, General Hospital of Airforce, Beijing, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Youxiao
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Microbiology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.affiliation: Department of Neurology, General Hospital of Navy, Beijing, China.
authors.forename: Chenjing
authors.initials: C
authors.lastname: Sun
authors.affiliation: Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Xianni
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pathophysiology, Fourth Military Medical University, Xi'An, China.
authors.forename: ZhiChao
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Pathophysiology, Fourth Military Medical University, Xi'An, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Nuclear Medicine, Tangdu Hospital, Fourth Military Medical University, Xi'An, China.
authors.forename: Longxiao
authors.initials: L
authors.lastname: Wei
authors.affiliation: Department of Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China. Electronic address: lizhuyi@fmmu.edu.cn.
authors.forename: Zhuyi
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Microbiology, Tangdu Hospital, Fourth Military Medical University, Xi'An, China. Electronic address: zhikaixu@fmmu.edu.cn.
authors.forename: Zhikai
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000818:Animals; D001483:Base Sequence; D002038:Bungarotoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012911:Snakes / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D003062:Codon; D011993:Recombinant Fusion Proteins
keywords: Protein refolding; Recombinant protein expression; nAChR; α-Bungarotoxin