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

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.

• Authors: Yong-Zhi Lun, Xue-Lei Wang, Jie Feng
• Journal: International journal of molecular medicine (Int J Mol Med) Vol. 34 Issue 1 Pg. 321-6 (Jul 2014) ISSN: 1791-244X [Electronic] Greece
• PMID: 24821237 (Publication Type: Journal Article)

Chemical References

• Proteinase Inhibitory Proteins, Secretory
• Recombinant Fusion Proteins
• SPINK7 protein, human
• Serine Peptidase Inhibitors, Kazal Type
• Serpins
• Trypsin Inhibitors
• Isopropyl Thiogalactoside
• Trypsin

Topics

• Amino Acid Sequence
• Base Sequence
• Cloning, Molecular
• Escherichia coli (drug effects, genetics, metabolism)
• Gene Expression
• Hep G2 Cells
• Humans
• Inclusion Bodies (chemistry)
• Isopropyl Thiogalactoside (pharmacology)
• Models, Molecular
• Molecular Sequence Data
• Molecular Weight
• Open Reading Frames
• Proteinase Inhibitory Proteins, Secretory (chemistry, genetics, metabolism)
• Recombinant Fusion Proteins (chemistry, genetics, isolation & purification, metabolism)
• Sequence Homology, Amino Acid
• Serine Peptidase Inhibitors, Kazal Type
• Serpins (chemistry, genetics, isolation & purification, metabolism)
• Subtractive Hybridization Techniques
• Trypsin (chemistry)
• Trypsin Inhibitors (chemistry, isolation & purification, metabolism)