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Microbial Cell Biology
Expression, Purification, and Biological Characterization of The Amino-Terminal Fragment of Urokinase in Pichia pastoris
1The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093 Jiangsu, P.R. China, 2Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc., Changzhou 213164, Jiangsu, P. R. China
Received: May 3, 2013; Accepted: June 3, 2013
J. Microbiol. Biotechnol. 2013; 23(9): 1197-1205
Published September 28, 2013 https://doi.org/10.4014/jmb.1305.05004
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and
metastasis. Targeting the excessive activation of this system as well as the proliferation of the
tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt
the tumor development. In this regard, the amino-terminal fragment (ATF) of urokinase has
been confirmed as effective to inhibit the proliferation, migration, and invasiveness of cancer
cells via interrupting the interaction of uPA and uPAR. Previous studies indicated that ATF
expressed in Escherichia coli was mainly contained in inclusion bodies and also lacked
posttranslational modifications. In this study, the biologically active and soluble ATF was
cloned and expressed in Pichia pastoris. The recombinant protein was purified to be
homogenous and confirmed to be biologically active. The yield of the active ATF was about
30 mg/l of the P. pastoris culture medium. The recombinant ATF (rATF) could efficiently
inhibit angiogenesis, endothelial cell migration, and tumor cell invasion in vitro. Furthermore,
it could inhibit in vivo xenograft tumor growth and prolong the survival of tumor-bearing
mice significantly by competing with uPA for binding to cell surfaces. Therefore, P. pastoris is
a highly efficient and cost-effective expression system for large-scale production of
biologically active rATFs for potential therapeutic application.
Keywords
the amino-terminal fragment of urokinase, Pichia pastoris, recombinant protein production, anti-angiogenesis, tumor therapy
References
- Andreasen PA, Kjoller L, Christensen L, Duffy MJ. 1997. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72: 1-22.
- Behrendt N, Ronne E, Ploug M, Petri T, Lober D, Nielsen LS, et al. 1990. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J. Biol. Chem. 265: 6453-6460.
- Binder BR, Mihaly J, Prager GW. 2007. uPAR-uPA-PAI-1 interactions and signaling: a vascular biologist's view. Thromb. Haemost. 97: 336-342.
- Brondyk WH. 2009. Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol. 463: 131147.
- Cereghino JL, Cregg JM. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45-66.
- Choong PF, Nadesapillai AP. 2003. Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin. Orthop. Relat. Res. S46-S58.
- Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. 2009. Expression in the yeast Pichia pastoris. Methods Enzymol. 463: 169-189.
- Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. 1985. Plasminogen activators, tissue degradation, and cancer. Adv. Cancer Res. 44: 139-266.
- Dass K, Ahmad A, Azmi AS, Sarkar SH, Sarkar FH. 2008. Evolving role of uPA/uPAR system in human cancers. Cancer Treat. Rev. 34: 122-136.
- Demain AL, Vaishnav P. 2009. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27: 297-306.
- Foekens JA, Peters HA, Look MP, Portengen H, Schmitt M, Kramer MD, et al. 2000. The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res. 60: 636-643.
- Goldstein D, Gofrit O, Nyska A, Benita S. 2007. Anti-HER2 cationic immunoemulsion as a potential targeted drug delivery system for the treatment of prostate cancer. Cancer Res. 67:269-275.
- Guo Y, Higazi AA, Arakelian A, Sachais BS, Cines D, Goldfarb RH, et al. 2000. A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 14: 1400-1410.
- Kim TD, Song KS, Li G, Choi H, Park HD, Lim K, et al. 2006. Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer. BMC Cancer 6: 211.
- Kobayashi H, Gotoh J, Shinohara H, Moniwa N, Terao T. 1994. Inhibition of the metastasis of Lewis lung carcinoma by antibody against urokinase-type plasminogen activator in the experimental and spontaneous metastasis model. Thromb. Haemost. 71: 474-480.
- Koolwijk P, van Erck MG, de Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, et al. 1996. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J. Cell Biol. 132:1177-1188.
- Laufs S, Schumacher J, Allgayer H. 2006. Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease. Cell Cycle 5: 1760-1771.
- Li H, Griscelli F, Lindenmeyer F, Opolon P, Sun LQ, Connault E, et al. 1999. Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum. Gene Ther. 10: 30453053.
- Li H, Soria C, Griscelli F, Opolon P, Soria J, Yeh P, et al. 2005. Amino-terminal fragment of urokinase inhibits tumor cell invasion in vitro and in vivo: respective contribution of the urokinase plasminogen activator receptor-dependent or -independent pathway. Hum. Gene Ther. 16: 1157-1167.
- Mars WM, Jo M, Gonias SL. 2005. Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent. Biochem. J. 390: 311-315.
- Mohanam S, Chandrasekar N, Yanamandra N, Khawar S, Mirza F, Dinh DH, et al. 2002. Modulation of invasive properties of human glioblastoma cells stably expressing amino-terminal fragment of urokinase-type plasminogen activator. Oncogene 21: 7824-7830.
- Moreau P, Voillat L, Benboukher L, Mathiot C, Dumontet C, Robillard N, et al. 2007. Rituximab in CD20 positive multiple myeloma. Leukemia 21: 835-836.
- Odekon LE, Sato Y, Rifkin DB. 1992. Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity. J. Cell Physiol 150: 258-263.
- Pollanen J, Stephens RW, Vaheri A. 1991. Directed plasminogen activation at the surface of normal and malignant cells. Adv. Cancer Res. 57: 273-328.
- Rabbani SA, Gladu J. 2002. Urokinase receptor antibody can reduce tumor volume and detect the presence of occult tumor metastases in vivo. Cancer Res. 62: 2390-2397.
- Shih T, Lindley C. 2006. Bevacizumab: an angiogenesis inhibitor for the treatment of solid malignancies. Clin. Ther. 28: 1779-1802.
- Sidenius N, Blasi F. 2003. The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metastasis Rev. 22: 205222.
- Sun Q, Xu Q, Dong X, Cao L, Huang X, Hu Q, et al. 2008. A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy. Int. J. Cancer 123: 942-950.
- Sun QM, Cao L, Fang L, Chen C, Dai J, Chen LL, et al. 2005. Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization. Protein Expr. Purif. 39: 288-295.
- Wang X, Hou M, Tan L, Sun X, Zhang Y, Li P, et al. 2005. A hybrid protein of the amino-terminal fragment of urokinase and mutant plasminogen activator inhibitor-2 efficiently inhibits tumor cell invasion and metastasis. J. Cancer Res. Clin. Oncol. 131: 129-136.
- Zhao G, Yuan C, Bian C, Hou X, Shi X, Ye X, et al. 2006. Protein expression and preliminary crystallographic analysis of amino-terminal fragment of urokinase-type plasminogen activator. Protein Expr. Purif. 49: 71-77.
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Research article
J. Microbiol. Biotechnol. 2013; 23(9): 1197-1205
Published online September 28, 2013 https://doi.org/10.4014/jmb.1305.05004
Copyright © The Korean Society for Microbiology and Biotechnology.
Expression, Purification, and Biological Characterization of The Amino-Terminal Fragment of Urokinase in Pichia pastoris
Jianping Li 1, Yuli Lin 1, Hongqin Zhuang 1, 2 and Zi-Chun Hua 1, 2*
1The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093 Jiangsu, P.R. China, 2Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories Inc., Changzhou 213164, Jiangsu, P. R. China
Received: May 3, 2013; Accepted: June 3, 2013
Abstract
Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and
metastasis. Targeting the excessive activation of this system as well as the proliferation of the
tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt
the tumor development. In this regard, the amino-terminal fragment (ATF) of urokinase has
been confirmed as effective to inhibit the proliferation, migration, and invasiveness of cancer
cells via interrupting the interaction of uPA and uPAR. Previous studies indicated that ATF
expressed in Escherichia coli was mainly contained in inclusion bodies and also lacked
posttranslational modifications. In this study, the biologically active and soluble ATF was
cloned and expressed in Pichia pastoris. The recombinant protein was purified to be
homogenous and confirmed to be biologically active. The yield of the active ATF was about
30 mg/l of the P. pastoris culture medium. The recombinant ATF (rATF) could efficiently
inhibit angiogenesis, endothelial cell migration, and tumor cell invasion in vitro. Furthermore,
it could inhibit in vivo xenograft tumor growth and prolong the survival of tumor-bearing
mice significantly by competing with uPA for binding to cell surfaces. Therefore, P. pastoris is
a highly efficient and cost-effective expression system for large-scale production of
biologically active rATFs for potential therapeutic application.
Keywords: the amino-terminal fragment of urokinase, Pichia pastoris, recombinant protein production, anti-angiogenesis, tumor therapy
References
- Andreasen PA, Kjoller L, Christensen L, Duffy MJ. 1997. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72: 1-22.
- Behrendt N, Ronne E, Ploug M, Petri T, Lober D, Nielsen LS, et al. 1990. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J. Biol. Chem. 265: 6453-6460.
- Binder BR, Mihaly J, Prager GW. 2007. uPAR-uPA-PAI-1 interactions and signaling: a vascular biologist's view. Thromb. Haemost. 97: 336-342.
- Brondyk WH. 2009. Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol. 463: 131147.
- Cereghino JL, Cregg JM. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45-66.
- Choong PF, Nadesapillai AP. 2003. Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis. Clin. Orthop. Relat. Res. S46-S58.
- Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. 2009. Expression in the yeast Pichia pastoris. Methods Enzymol. 463: 169-189.
- Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. 1985. Plasminogen activators, tissue degradation, and cancer. Adv. Cancer Res. 44: 139-266.
- Dass K, Ahmad A, Azmi AS, Sarkar SH, Sarkar FH. 2008. Evolving role of uPA/uPAR system in human cancers. Cancer Treat. Rev. 34: 122-136.
- Demain AL, Vaishnav P. 2009. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27: 297-306.
- Foekens JA, Peters HA, Look MP, Portengen H, Schmitt M, Kramer MD, et al. 2000. The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res. 60: 636-643.
- Goldstein D, Gofrit O, Nyska A, Benita S. 2007. Anti-HER2 cationic immunoemulsion as a potential targeted drug delivery system for the treatment of prostate cancer. Cancer Res. 67:269-275.
- Guo Y, Higazi AA, Arakelian A, Sachais BS, Cines D, Goldfarb RH, et al. 2000. A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 14: 1400-1410.
- Kim TD, Song KS, Li G, Choi H, Park HD, Lim K, et al. 2006. Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer. BMC Cancer 6: 211.
- Kobayashi H, Gotoh J, Shinohara H, Moniwa N, Terao T. 1994. Inhibition of the metastasis of Lewis lung carcinoma by antibody against urokinase-type plasminogen activator in the experimental and spontaneous metastasis model. Thromb. Haemost. 71: 474-480.
- Koolwijk P, van Erck MG, de Vree WJ, Vermeer MA, Weich HA, Hanemaaijer R, et al. 1996. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J. Cell Biol. 132:1177-1188.
- Laufs S, Schumacher J, Allgayer H. 2006. Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease. Cell Cycle 5: 1760-1771.
- Li H, Griscelli F, Lindenmeyer F, Opolon P, Sun LQ, Connault E, et al. 1999. Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum. Gene Ther. 10: 30453053.
- Li H, Soria C, Griscelli F, Opolon P, Soria J, Yeh P, et al. 2005. Amino-terminal fragment of urokinase inhibits tumor cell invasion in vitro and in vivo: respective contribution of the urokinase plasminogen activator receptor-dependent or -independent pathway. Hum. Gene Ther. 16: 1157-1167.
- Mars WM, Jo M, Gonias SL. 2005. Activation of hepatocyte growth factor by urokinase-type plasminogen activator is ionic strength-dependent. Biochem. J. 390: 311-315.
- Mohanam S, Chandrasekar N, Yanamandra N, Khawar S, Mirza F, Dinh DH, et al. 2002. Modulation of invasive properties of human glioblastoma cells stably expressing amino-terminal fragment of urokinase-type plasminogen activator. Oncogene 21: 7824-7830.
- Moreau P, Voillat L, Benboukher L, Mathiot C, Dumontet C, Robillard N, et al. 2007. Rituximab in CD20 positive multiple myeloma. Leukemia 21: 835-836.
- Odekon LE, Sato Y, Rifkin DB. 1992. Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity. J. Cell Physiol 150: 258-263.
- Pollanen J, Stephens RW, Vaheri A. 1991. Directed plasminogen activation at the surface of normal and malignant cells. Adv. Cancer Res. 57: 273-328.
- Rabbani SA, Gladu J. 2002. Urokinase receptor antibody can reduce tumor volume and detect the presence of occult tumor metastases in vivo. Cancer Res. 62: 2390-2397.
- Shih T, Lindley C. 2006. Bevacizumab: an angiogenesis inhibitor for the treatment of solid malignancies. Clin. Ther. 28: 1779-1802.
- Sidenius N, Blasi F. 2003. The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metastasis Rev. 22: 205222.
- Sun Q, Xu Q, Dong X, Cao L, Huang X, Hu Q, et al. 2008. A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy. Int. J. Cancer 123: 942-950.
- Sun QM, Cao L, Fang L, Chen C, Dai J, Chen LL, et al. 2005. Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization. Protein Expr. Purif. 39: 288-295.
- Wang X, Hou M, Tan L, Sun X, Zhang Y, Li P, et al. 2005. A hybrid protein of the amino-terminal fragment of urokinase and mutant plasminogen activator inhibitor-2 efficiently inhibits tumor cell invasion and metastasis. J. Cancer Res. Clin. Oncol. 131: 129-136.
- Zhao G, Yuan C, Bian C, Hou X, Shi X, Ye X, et al. 2006. Protein expression and preliminary crystallographic analysis of amino-terminal fragment of urokinase-type plasminogen activator. Protein Expr. Purif. 49: 71-77.
