Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system

  • Authors:
    • Yong-Zhi Lun
    • Xue-Lei Wang
    • Jie Feng
  • View Affiliations

  • Published online on: May 9, 2014     https://doi.org/10.3892/ijmm.2014.1778
  • Pages: 321-326
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Abstract

In this study, Hespintor, a protein with unknown function, was screened and obtained from the hepatoblastoma cell line, HepG2, using suppression subtractive hybridization (SSH). Sequence analysis demonstrated that the protein is a novel secreting member of the Kazal-type serine protease inhibitor (serpin) family, and possesses the basic structure of serpin, which is highly homologous to esophageal cancer-related gene 2 (ECRG2). To further elucidate its biological functions, the Hespintor protein was expressed and purified. The coding sequence of the Hespintor Kazal domain was cloned into the prokaryotic expression vector, pET-40b(+), and was then transformed into host bacteria (Escherichia coli) Rosetta (DE3). The optimally expressed recombinant fusion protein, Hespintor-Kazal, with a molecular weight of 42 kDa was obtained by 0.25 mmol/l isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 30˚C for 5 h. Western blot analysis was performed to further confirm the specificity of the recombinant protein, Hespintor-Kazal. The recombinant fusion protein, Hespintor‑Kazal, was expressed in the host bacteria in the form of an inclusion body. Two-step metal chelating affinity chromatography and anion exchange chromatography columns were used to purify the recombinant protein. The preliminary activity identification results revealed that the purified recombinant fusion protein, Hespintor-Kazal, specifically inhibited the hydrolysis activity of trypsin, suggesting that Hespintor has potential value as a novel antitumor drug.

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July 2014
Volume 34 Issue 1

Print ISSN: 1107-3756
Online ISSN:1791-244X

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APA
Lun, Y., Wang, X., & Feng, J. (2014). Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system. International Journal of Molecular Medicine, 34, 321-326. https://doi.org/10.3892/ijmm.2014.1778
MLA
Lun, Y., Wang, X., Feng, J."Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system". International Journal of Molecular Medicine 34.1 (2014): 321-326.
Chicago
Lun, Y., Wang, X., Feng, J."Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system". International Journal of Molecular Medicine 34, no. 1 (2014): 321-326. https://doi.org/10.3892/ijmm.2014.1778