Volume 32 Issue 4
Jul.  2011
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yu guo-Yu, XIANG Yang, ZHANG Hong-Yun, JIANG Ping, Lee Wen-Hui, ZHANG Yun, ZHANG. Expression of Bm-TFF2 mutants in Escherichia coli andtheir cell migration-promoting activity. Zoological Research, 2011, 32(4): 379-385. doi: 10.3724/SP.J.1141.2011.04379
Citation: yu guo-Yu, XIANG Yang, ZHANG Hong-Yun, JIANG Ping, Lee Wen-Hui, ZHANG Yun, ZHANG. Expression of Bm-TFF2 mutants in Escherichia coli andtheir cell migration-promoting activity. Zoological Research, 2011, 32(4): 379-385. doi: 10.3724/SP.J.1141.2011.04379

Expression of Bm-TFF2 mutants in Escherichia coli andtheir cell migration-promoting activity

doi: 10.3724/SP.J.1141.2011.04379
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  • Author Bio:

    yu guo-Yu,XIANG Yang

    yu guo-Yu,XIANG Yang

  • Corresponding author: ZHANG Yong
  • Received Date: 2011-01-05
  • Rev Recd Date: 2011-05-18
  • Publish Date: 2011-08-22
  • Bm-TFF2, a trefoil factor from the large-webbed bell toad (Bombina maxima), can stimulate cell migration and inhibit cell apoptosis. To study the structure-function relationship of Bm-TFF2, we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E. coli. The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR, while the N-terminal, C-terminal and two arginine mutated Bm-TFF2 clones were constructed, and ligated into pET-32a(+) expression vectors. The fusion proteins were induced by IPTG at 37°C. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni2+-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant. The fusion proteins were analyzed by SDS-PAGE and Western blotting. The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L. All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.
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  • [1]
     Baus-Loncar M, Kayademir T, Takaishi S, Wang T, 2005. Trefoil factor family 2 deficiency and immune response [J]. Cell Mol Life Sci, 62(24): 2947-2955.
     Canas M, Moran Y, Rivero MB, Bohorquez A, Villegas V, Rendon Y, Ramirez E, Valderrama E, Briceno Z, Chiurillo MA, 2009. Interleukin-1 genetic polymorphism: association with gastric cancer in the high-risk Central-Western population of Venezuela [J]. Rev Med Chil, 137(1): 63-70.
     Chatterjee DK, Gnanasammandhan MK, Zhang Y, 2010. Small upconverting fluorescent nanoparticles for biomedical applications [J]. Small, 6(24): 2781-2795.
     Chwieralski CE, Schnurra I, Thim L, Hoffmann W, 2004. Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades [J]. Am J Respir Cell Mol Biol, 31(5): 528-537.
     Cook GA, Familari M, Thim L, Giraud AS, 1999. The trefoil peptides TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response
    [J]. FEBS Lett, 456(1): 155-159.
     Dhar DK, Wang TC, Maruyama R, Udagawa J, Kubota H, Fuji T, Tachibana M, Ono T, Otani H, Nagasue N, 2003. Expression of cytoplasmic TFF2 is a marker of tumor metastasis and negative prognostic factor in gastric cancer [J]. Lab Invest, 83(9): 1343-1352.
     Dignass A, Lynch-Devaney K, Kindon H, Thim L, Podolsky DK, 1994. Trefoil peptides promote epithelial migration through a transforming growth factor β-independent pathway [J]. J Clin Invest, 94(1): 376-383.
     Emami S, Rodrigues S, Rodrigue CM, Le Floch N, Rivat C, Attoub S, Bruyneel E, Gespach C, 2004. Trefoil factor family (TFF) peptides and cancer progression [J]. Peptides, 25(5): 885-898.
     Fox JG, Rogers AB, Whary MT, Ge Z, Ohtani M, Jones EK, Wang TC, 2007. Accelerated progression of gastritis to dysplasia in the pyloric antrum of TFF2 -/- C57BL6 x Sv129 Helicobacter pylori-infected mice [J]. Am J Pathol, 171(5): 1520-1528.
     He YY, Liu SB, Lee WH, Zhang Y, 2008. Melanoma cell growth inhibition by βγ-CAT, which is a novel non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin [J]. Toxicon, 52(2): 341-347.
     Katoh M, 2003. Trefoil factors and human gastric cancer (review) [J]. Int J Mol Med, 12(1): 3-9.
     Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA, 1997. Regulation of cell motility by mitogen-activated protein kinase [J]. J Cell Biol, 137(2): 481-492.
     Lalani EN, Williams R, Jayaram Y, Gilbert C, Chaudhary KS, Siu LS, Koumarianou A, Playford R, Stamp GW, 1999. Trefoil factor-2, human spasmolytic polypeptide, promotes branching morphogenesis in MCF-7 cells [J]. Lab Invest, 79(5): 537-546.
     LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM, 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm [J]. Biotechnology: N Y, 11(2): 187-193.
     Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y, 2008. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications
    [J]. PLoS One, 3(3): e1770.
     Oertel M, Graness A, Thim L, Buhling F, Kalbacher H, Hoffmann W, 2001. Trefoil factor family-peptides promote migration of human bronchial epithelial cells: synergistic effect with epidermal growth factor [J]. Am J Respir Cell Mol Biol, 25(4): 418-424.
     Polshakov VI, Williams MA, Gargaro AR, Frenkiel TA, Westley BR, Chadwick MP, May FE, Feeney J, 1997. High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein [J]. J Mol Biol, 267(2): 418-432.
     Siu LS, Romanska H, Abel PD, Baus-Loncar M, Kayademir T, Stamp GW, Lalani el N, 2004. TFF2 (trefoil family factor2) inhibits apoptosis in breast and colorectal cancer cell lines [J]. Peptides, 25(5): 855-863.
     Stewart EJ, Aslund F, Beckwith J, 1998. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins [J]. EMBO J, 17(19): 5543-5550.
     Taupin D, Podolsky DK, 2003. Trefoil factors: initiators of mucosal healing [J]. Nat Rev Mol Cell Biol, 4(9): 721-732.
     Taupin DR, Kinoshita K, Podolsky DK, 2000. Intestinal trefoil factor confers colonic epithelial resistance to apoptosis [J]. Proc Natl Acad Sci USA, 97(2): 799-804.
     Wong WM, Poulsom R, Wright NA, 1999. Trefoil peptides [J]. Gut, 44(6): 890-895.
     Yu G, Zhang Y, Xiang Y, Jiang P, Chen Z, Lee W, 2010. Cell migration- promoting and apoptosis-inhibiting activities of Bm-TFF2 require distinct structure basis [J]. Biochem Biophys Res Commun, 400(4): 724-728.
     Zhang J, Zhang Y, Wan SG, Wei SS, Lee WH, 2005. Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions [J]. Biochem Biophys Res Commun, 330(4): 1027-1033.
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