title: Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli.
abstract: Integrins are a family of transmembrane receptors and among their members, integrin β1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin β1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin β1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin β1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, PR China.
authors.forename: Tonglin
authors.initials: T
authors.lastname: Shi
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, PR China.
authors.forename: Lichao
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, PR China; College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, PR China. Electronic address: lzy@sxu.edu.cn.
authors.forename: Zhuoyu
authors.initials: Z
authors.lastname: Li
authors.affiliation: Cell and Developmental Biology, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Ian P
authors.initials: IP
authors.lastname: Newton
authors.affiliation: Central Laboratory of Taiyuan Central Hospital, No. 1, East Sandao Alley, Xinghualing District, Taiyuan, Shanxi 030009, PR China.
authors.forename: Quanbin
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019012:Integrin beta1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019012:Integrin beta1; D011994:Recombinant Proteins
keywords: Inclusion body; Integrin β1; Prokaryotic expression; pET-28a vector