title: 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.affiliation: Liaoning Provincial University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China.
authors.forename: Yong-Zhi
authors.initials: YZ
authors.lastname: Lun
authors.affiliation: Liaoning Provincial University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China.
authors.forename: Xue-Lei
authors.initials: XL
authors.lastname: Wang
authors.affiliation: Liaoning Provincial University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China.
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056945:Hep G2 Cells; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D053491:Proteinase Inhibitory Proteins, Secretory / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000074162:Serine Peptidase Inhibitors, Kazal Type; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D066251:Subtractive Hybridization Techniques; D014357:Trypsin / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D053491:Proteinase Inhibitory Proteins, Secretory; D011993:Recombinant Fusion Proteins; C473799:SPINK7 protein, human; D000074162:Serine Peptidase Inhibitors, Kazal Type; D015843:Serpins; D014361:Trypsin Inhibitors; D007544:Isopropyl Thiogalactoside; D014357:Trypsin