Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli

Tonglin Shi, Lichao Zhang, Zhuoyu Li (Lead / Corresponding author), Ian P. Newton, Quanbin Zhang

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    9 Citations (Scopus)

    Abstract

    Integrins are a family of transmembrane receptors and amongst 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 E. coli 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 8 M 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 per liter 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 β. 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.

    Original languageEnglish
    Pages (from-to)13-19
    Number of pages7
    JournalProtein Expression and Purification
    Volume107
    DOIs
    Publication statusPublished - Mar 2015

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