INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 34: 321-326, 2014 Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system YONG-ZHI LUN, XUE-LEI WANG and JIE FENG Liaoning Provincial University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China Received January 21, 2014; Accepted April 24, 2014 DOI: 10.3892/ijmm.2014.1778 Abstract. In this study, Hespintor, a protein with unknown func- and basement membrane (BM). Tumor cells first secrete uroki- tion, was screened and obtained from the hepatoblastoma cell nase-type plasminogen activator (uPA), which is a serine protease line, HepG2, using suppression subtractive hybridization (SSH). that can activate plasminogen and produce plasmin (PLM). PLM Sequence analysis demonstrated that the protein is a novel then degrades the ECM, and can also activate the precursor and secreting member of the Kazal-type serine protease inhibitor produce matrix metalloproteinases, which are involved in the (serpin) family, and possesses the basic structure of serpin, hydrolysis of ECM. Finally, the ECM and BM are hydrolyzed which is highly homologous to esophageal cancer-related and the tumor cells spread (1). Therefore, controlling the levels or gene 2 (ECRG2). To further elucidate its biological functions, functions of uPA can be used for cancer treatment. In particular, the Hespintor protein was expressed and purified. The coding the inhibition of uPA activity by Kazal-type serine protease sequence of the Hespintor Kazal domain was cloned into the inhibitors is the most direct approach and has the most prospects prokaryotic expression vector, pET-40b(+), and was then trans- in clinical application (2-4). formed into host bacteria (Escherichia coli) Rosetta (DE3). The Suppression subtractive hybridization (SSH) was used optimally expressed recombinant fusion protein, Hespintor-Kazal, in the present study to investigate the transregulation of the with a molecular weight of 42 kDa was obtained by 0.25 mmol/l target genes by HBV RT/DNA polymerase, and a protein of isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 30˚C unknown function was screened and obtained from the hepa- for 5 h. Western blot analysis was performed to further confirm toblastoma cell line, HepG2. The gene was a new member of the specificity of the recombinant protein, Hespintor-Kazal. The the Kazal-type serpin family identified by RT-PCR verifica- recombinant fusion protein, Hespintor-Kazal, was expressed in tion and bioinformatics, and was named Hespintor (GenBank the host bacteria in the form of an inclusion body. Two-step metal Accession no. DQ438947). chelating affinity chromatography and anion exchange chroma- The cloned Hespintor Kazal domain encoding region tography columns were used to purify the recombinant protein. from the HepG2 cells was subcloned into the prokaryotic The preliminary activity identification results revealed that the expression vector, pET-40b(+), in the present study. Large purified recombinant fusion protein, Hespintor-Kazal, specifi- quantities of Hespintor-Kazal recombinant protein with fusion cally inhibited the hydrolysis activity of trypsin, suggesting that tag proteins were expressed, as shown by isopropyl β-D-1- Hespintor has potential value as a novel antitumor drug. thiogalactopyranoside (IPTG) induction. High-efficiency expression of the recombinant fusion protein, Hespintor-Kazal, Introduction was obtained by optimization screening under IPTG induc- tion conditions. Finally, the purified recombinant protein with The most crucial step in the progression of cancer is the invasion specific inhibition of trypsin hydrolysis activity was obtained and metastasis of tumor cells, which depends on certain tumor by a further two-step purification of the Ni2+ and Q columns cell secretions that can hydrolyze the extracellular matrix (ECM) and renaturation on columns, which provides the basis for the research into the next step. Materials and methods Correspondence to: Dr Yong-Zhi Lun, Liaoning Provincial Plasmids and strains. The cloning vector, pMD 20-T/Hespintor University Key Laboratory of Biophysics, College of Medicine, Dalian University, Dalian, Liaoning 116622, P.R. China cDNA, and Escherichia coli DH5α were obtained from our E-mail: [email protected] laboratory; Escherichia coli Rosetta (DE3) and the prokary- otic expression vector, pET-40b(+), were purchased from the Key words: serine protease inhibitor, Hespintor, Kazal domain, Physical and Chemical Analysis and Testing Center of Beijing; prokaryotic expression the pMD 19-T Simple Vector was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). 322 LUN et al: PURIFICATION AND IDENTIFICATION OF THE RECOMBINANT FUSION PROTEIN, Hespintor-Kazal Primers. The sequence-specific primers with restriction endo- Purification and identification of the recombinant fusion nuclease (BamHI and HindIII) cleavage sites of forward (5'-GGA protein, Hespintor-Kazal. Western blot analysis was used to TCCGCCTAAGCCCCG-3') and reverse (5'-GCGCAAGC determine the expression of the recombinant proteins. The TTATCACATTTTCCATATTTTTC-3') were designed harvested bacteria induced by the optimal conditions were according to the gene sequence of Hespintor (GenBank lysed by ultrasonic wave. The precipitate (i.e., inclusion) Accession no. DQ438947) and were synthesized by Takara was dissolved in 8 mol/l urea solution, and the supernatants Biotechnology Co., Ltd.). were harvested after centrifugation. Solution A (8 mol/l urea, 0.5 mol/l NaCl, 20 mmol/l imidazole; pH 8) was used to Main reagents. Restriction Enzyme Starter BOX, Permix balance the Ni2+ column, and the supernatant was applied to Ex Taq Version 2.0, λ-HindIII digest and DL2 000 DNA the column after sucking filtration and degassing. Solution A Marker were purchased from Takara Biotechnology Co., Ltd.; was used to flush continuously, and solution B (0.5 mol/l NaCl, PageRuler Prestained Protein Ladder was purchased from 20 mmol/l imidazole; pH 8) was used in protein refolding Fermentas (Waltham, MA, USA); mouse anti-human His Tag with the gradient elution program from 0% B to 100% B. The monoclonal antibody (primary antibody) was purchased from refolding time of the protein was 150 min. During the process, Kebaiao Biotech Co., Ltd. (Beijing, China); goat anti-mouse the fusion protein solution was adjusted from 8 mol/l urea to HRP-IgG (secondary antibody) was purchased from Beijing 0 mol/l urea. The denatured fusion proteins were folded again Sequoia Jinqiao Biological Technology. Co., Ltd. (Beijing, and formed a spatial structure with biological activity (column China); bovine serum albumin (BSA) and trypsin (active refolding). All processes were conducted in low temperature ≥250 NF U/mg) were purchased from Amresco Inc. (Solon, environments. Following complete urea removal, solution C OH, USA); Na-benzoyl arginine-DL-p-nitrophenyl amide (0.5 mol/l NaCl, 500 mmol/l imidazole; pH 8) was used for hydrochloride (BAPNA) was purchased from Sigma (St. Louis, elution. The eluting peak was determined by 15% SDS-PAGE. MO, USA); Ni2+ column (Ni2+-Histrap FF crude 5 ml) and Solution D (20 mmol/l Tris-HCl; pH 8) was used to balance the Q column (Histrap Q FF column 1 ml) were purchased from Q column. The samples purified with the Ni2+ columns were GE Healthcare (Piscataway, NJ, USA); Coomassie brilliant diluted with solution D and analyzed. Solution E (20 mmol/l blue G250 was purchased from Solarbio (Beijing, China). Tris-HCl, 1 mol/l NaCl; pH 8) was used for gradient elutions. The eluting peaks were determined by 15% SDS-PAGE. The Construction and identification of prokaryotic expression concentration of the recombinant protein was detected by the vector, PET-40b(+)/Hespintor-Kazal. The domain coding Bradford method. sequence of the Kazal-type Hespintor was amplified by PCR, with pMD 20-T/Hespintor cDNA as the template. The PCR Activity identification of the recombinant fusion protein, products were analyzed by 3% agarose gel electrophoresis and Hespintor-Kazal. The activity of the recombinant fusion were purified and refolded. The purified target gene fragment protein, Hespintor-Kazal, was identified according to the was incubated with the pMD 19-T Simple Vector at room Sigma method with minor modifications. Briefly, 8 tubes with temperature overnight for ligation, and the ligation product a volume of 5 ml were taken and numbered. Subsequently, was then transformed into DH5α competent cells. The plas- 200 µl 40 µg/ml trypsin followed by 0, 20, 40, 80, 120, 160, mids were extracted using alkaline lysis and further sequenced or 200 µl of the recombinant protein purification liquid were to prove that the target gene fragment was inserted into the added to 7 tubes. To the remaining centrifugation tube, 20 µl T vector correctly. The target gene fragment was then digested 1 mmol/l added, with HCl as the blank control. Tris-HCl was from the recombinant T vector with BamHI/HindIII, and used to supplement to a final volume of 2 ml, and the reaction was purified and obtained by 3% agarose gel electrophoresis. was conducted in a 37˚C water bath for 10 min. To each tube The purified target fragment was ligated to the prokaryotic was added 1 mmol/l BAPNA, and the mixture was incubated in expression vector pET-40b(+) digested with the same restric- a 37˚C water bath for 5 min. Finally, 1 ml 60% acetic acid was tion enzymes, and the ligation product was used to transform added