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Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

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Abstract

The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable Km values (55.7 and 39 μM, respectively). Furthermore, inhibition studies using benzamidine resulted in a Ki of 195 μM for native uPA, while recombinant uPA had a Ki of 112 μM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.

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Abbreviations

CHO-S:

Chinese hamster ovary-S cells

PAI-1:

type-1 plasminogen activator inhibitor

PNGase F:

peptide-N-glycosidase F

SDS-PAGE:

sodium dodecylsulfate-polyacrylamide gel electrophoresis

uPA:

urokinase-type plasminogen activator

References

  • M.O. Agaphonov N.V. Romanova P.M. Trushkina V.N. Smirnov M.D. Ter-Avanesyan (2002) ArticleTitleAggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum BMC Mol. Biol. 3 15 Occurrence Handle10.1186/1471-2199-3-15 Occurrence Handle12366865

    Article  PubMed  Google Scholar 

  • D.S. Alexander J.D. Sipley J.P. Quigley (1998) ArticleTitleAutoactivation of avian urokinase-type plasminogen activator (uPA). A novel mode of initiation of the uPA/plasmin cascade J. Biol. Chem. 273 7457–7461 Occurrence Handle10.1074/jbc.273.13.7457 Occurrence Handle1:CAS:528:DyaK1cXit1emu7s%3D Occurrence Handle9516444

    Article  CAS  PubMed  Google Scholar 

  • P.A. Andreasen L. Kjoller L. Christensen M.J. Duffy (1997) ArticleTitleThe urokinase-type plasminogen activator system in cancer metastasis: a review Int. J. Cancer 72 1–22 Occurrence Handle10.1002/(SICI)1097-0215(19970703)72:1<1::AID-IJC1>3.0.CO;2-Z Occurrence Handle1:CAS:528:DyaK2sXkslahsbk%3D Occurrence Handle9212216

    Article  CAS  PubMed  Google Scholar 

  • A.A. Bergwerff J. Oostrum ParticleVan J.P. Kamerling J.F. Vliegenthart (1995) ArticleTitleThe major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated(alpha 2–6)-sialylated or (alpha 1–3)-fucosylated N-acetylgalactosamine(beta 1–4)-N-acetylglucosamine elements Eur. J. Biochem. 228 1009–1019 Occurrence Handle10.1111/j.1432-1033.1995.tb20351.x Occurrence Handle1:CAS:528:DyaK2MXksl2qtbY%3D Occurrence Handle7737145

    Article  CAS  PubMed  Google Scholar 

  • N. Brünner C. Pyke C.H. Hansen J. Romer J. Grondahl-Hansen K. Dano (1994) ArticleTitleUrokinase plasminogen activator (uPA) and its type 1 inhibitor (PAI-1): regulators of proteolysis during cancer invasion and prognostic parameters in breast cancer Cancer Treat. Res. 71 299–309 Occurrence Handle7946954

    PubMed  Google Scholar 

  • T.H. Bugge L.R. Lund K.K. Kombrinck B.S. Nielsen K. Holmbäck A.F. Drew M.J. Flick D.P. Witte K. Danø J.L. Degen (1998) ArticleTitleReduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice Oncogene 16 3097–3104 Occurrence Handle10.1038/sj.onc.1201869 Occurrence Handle1:CAS:528:DyaK1cXkt1Omur4%3D Occurrence Handle9671388

    Article  CAS  PubMed  Google Scholar 

  • V.C. Ciccarone Y. Chu K.P. Schifferli J.P. Pichet P. Hawley-Nelson K. Evans L. Roy S. Bennett (1999) ArticleTitleLipofectamine 2000 reagent for rapid, efficient transfection of eukaryotic cells Focus 21 54–56

    Google Scholar 

  • J. Davis M.R. Wagner W. Zhang F. Xu W.E. Nostrand ParticleVan (2003) ArticleTitleAmyloid beta-protein stimulates the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in human cerebrovascular smooth muscle cells J. Biol. Chem. 278 19054–19061 Occurrence Handle1:CAS:528:DC%2BD3sXjvVSks70%3D Occurrence Handle12754271

    CAS  PubMed  Google Scholar 

  • M.J. Duffy (2001) ArticleTitleClinical uses of tumor markers: a critical review Crit. Rev. Clin. Lab. Sci. 38 225–262 Occurrence Handle1:CAS:528:DC%2BD3MXlsVymsbc%3D Occurrence Handle11451209

    CAS  PubMed  Google Scholar 

  • M.J. Duffy (2002) ArticleTitleUrokinase plasminogen activator and its inhibitorPAI-1, as prognostic markers in breast cancer: from pilot to level 1 evidence studies Clin. Chem. 48 1194–1197 Occurrence Handle1:CAS:528:DC%2BD38XlslKksb8%3D Occurrence Handle12142372

    CAS  PubMed  Google Scholar 

  • M.J. Duffy T.M. Maguire E.W. McDermott N. O′Higgins (1999) ArticleTitleUrokinase plasminogen activator: a prognostic marker in multiple types of cancer J. Surg. Oncol. 71 130–135 Occurrence Handle10.1002/(SICI)1096-9098(199906)71:2<130::AID-JSO14>3.0.CO;2-9 Occurrence Handle1:CAS:528:DyaK1MXktlShu7k%3D Occurrence Handle10389872

    Article  CAS  PubMed  Google Scholar 

  • T.L. Frandsen C. Holst-Hansen B.S. Nielsen I.J. Christensen J.R. Nyengaard P. Carmeliet N. Brünner (2001) ArticleTitleDirect evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model Cancer Res. 61 532–537 Occurrence Handle1:CAS:528:DC%2BD3MXht1Gms7c%3D Occurrence Handle11212246

    CAS  PubMed  Google Scholar 

  • L.S. Gutierrez A. Schulman T. Brito-Robinson F. Noria V.A. Ploplis F.J. Castellino (2000) ArticleTitleTumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1 Cancer Res. 60 5839–5847 Occurrence Handle1:CAS:528:DC%2BD3cXnvVSnt7g%3D Occurrence Handle11059781

    CAS  PubMed  Google Scholar 

  • C.L. Hall R. Tsan G. Mugnai A. Mazar R. Radinsky C.A. Pettaway (2004) ArticleTitleEnhanced invasion of hormone refractory prostate cancer cells through hepatocyte growth factor (HGF) induction of urokinase-type plasminogen activator (u-PA) Prostate 59 167–176 Occurrence Handle10.1002/pros.20009 Occurrence Handle1:CAS:528:DC%2BD2cXktl2lsLw%3D Occurrence Handle15042617

    Article  CAS  PubMed  Google Scholar 

  • C.M. Hekman D.J. Loskutoff (1988) ArticleTitleKinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator Arch. Biochem. Biophys. 262 199–210 Occurrence Handle10.1016/0003-9861(88)90182-8 Occurrence Handle1:CAS:528:DyaL1cXhvVChs7c%3D Occurrence Handle3128176

    Article  CAS  PubMed  Google Scholar 

  • M.A. Helenius O.R. Saramèaki M.J. Linja T.L. Tammela T. Visakorpi (2001) ArticleTitleAmplification of urokinase gene in prostate cancer Cancer Res. 61 5340–5344 Occurrence Handle1:CAS:528:DC%2BD3MXlsVChsr8%3D Occurrence Handle11454671

    CAS  PubMed  Google Scholar 

  • B.A. Katz R. Mackman C. Luong K. Radika A. Martelli P.A. Sprengeler J. Wang H. Chan L. Wong (2000) ArticleTitleStructural basis for selectivity of a small moleculeS1-binding, submicromolar inhibitor of urokinase-type plasminogen activator Chem. Biol. 7 299–312 Occurrence Handle10.1016/S1074-5521(00)00104-6 Occurrence Handle1:CAS:528:DC%2BD3cXisFynuro%3D Occurrence Handle10779411

    Article  CAS  PubMed  Google Scholar 

  • H. Lee I. Struman C. Clapp J. Martial R.I. Weiner (1998) ArticleTitleInhibition of urokinase activity by the antiangiogenic factor 16K prolactin: activation of plasminogen activator inhibitor 1 expression Endocrinology 139 3696–3703 Occurrence Handle1:CAS:528:DyaK1cXlsFOisbo%3D Occurrence Handle9724020

    CAS  PubMed  Google Scholar 

  • C. Lenich R. Pannell J. Henkin V. Gurewich (1992) ArticleTitleThe influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase Thromb. Haemost. 68 539–544 Occurrence Handle1:CAS:528:DyaK3sXjs1Gmug%3D%3D Occurrence Handle1455401

    CAS  PubMed  Google Scholar 

  • X.K. Li H.R. Lijnen L. Nelles M.H. Hu D. Collen (1992) ArticleTitleBiochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator Biochim. Biophys. Acta 1159 37–43 Occurrence Handle1:CAS:528:DyaK38XmsVOlu7o%3D Occurrence Handle1390910

    CAS  PubMed  Google Scholar 

  • H.R. Lijnen B. Hoef ParticleVan D. Collen (1986) ArticleTitleComparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator Biochim. Biophys. Acta 884 402–408 Occurrence Handle1:CAS:528:DyaL2sXmtlGgsA%3D%3D Occurrence Handle3778931

    CAS  PubMed  Google Scholar 

  • V. Nienaber J. Wang D. Davidson J. Henkin (2000) ArticleTitleRe-engineering of human urokinase provides a system for structure-based drug design at high resolution and reveals a novel structural subsite J. Biol. Chem. 275 7239–7248 Occurrence Handle10.1074/jbc.275.10.7239 Occurrence Handle1:CAS:528:DC%2BD3cXhvVGku7c%3D Occurrence Handle10702294

    Article  CAS  PubMed  Google Scholar 

  • S. Ohta H. Fuse Y. Fujiuchi O. Nagakawa Y. Furuya (2003) ArticleTitleClinical significance of expression of urokinase-type plasminogen activator in patients with prostate cancer Anticancer Res. 23 2945–2950 Occurrence Handle1:CAS:528:DC%2BD3sXntVCns7s%3D Occurrence Handle12926141

    CAS  PubMed  Google Scholar 

  • D. Paar D. Maruhn (1980) ArticleTitleSpectrometric determination of urokinase in urine after gel filtration, using the chromogenic substrate S-2444 J. Clin. Chem. Clin. Biochem. 18 557–562 Occurrence Handle1:CAS:528:DyaL3cXlvF2rsrg%3D Occurrence Handle7003055

    CAS  PubMed  Google Scholar 

  • G. Pejler J.O. Winberg T.T. Vuong F. Henningsson L. Uhlin-Hansen K. Kimata S.O. Kolset (2003) ArticleTitleSecretion of macrophage urokinase plasminogen activator is dependent on proteoglycans Eur. J. Biochem. 270 3971–3980 Occurrence Handle10.1046/j.1432-1033.2003.03785.x Occurrence Handle1:CAS:528:DC%2BD3sXotFKru78%3D Occurrence Handle14511379

    Article  CAS  PubMed  Google Scholar 

  • M.S. Pepper (2001) ArticleTitleExtracellular proteolysis and angiogenesis Thromb. Haemost. 86 346–355 Occurrence Handle1:CAS:528:DC%2BD3MXlsVektrs%3D Occurrence Handle11487024

    CAS  PubMed  Google Scholar 

  • M. Renatus W. Bode R. Huber J. Stürzebecher M.T. Stubbs (1998) ArticleTitleStructural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase J. Med. Chem. 41 5445–5456 Occurrence Handle10.1021/jm981068g Occurrence Handle1:CAS:528:DyaK1cXns1ylt7w%3D Occurrence Handle9876114

    Article  CAS  PubMed  Google Scholar 

  • P.C. Roche J.D. Campeau S.T. Shaw SuffixJr. (1983) ArticleTitleComparative electrophoretic analysis of human and porcine plasminogen activators in SDS-polyacrylamide gels containing plasminogen and casein Biochim. Biophys. Acta 745 82–89 Occurrence Handle1:CAS:528:DyaL3sXksV2nt7c%3D Occurrence Handle6342680

    CAS  PubMed  Google Scholar 

  • T.W. Rockway V.L. Giranda (2003) ArticleTitleInhibitors of the proteolytic activity of urokinase type plasminogen activator Curr. Pharm. Des. 9 1483–1498 Occurrence Handle10.2174/1381612033454649 Occurrence Handle1:CAS:528:DC%2BD3sXksFGntro%3D Occurrence Handle12871064

    Article  CAS  PubMed  Google Scholar 

  • K.T. Sabapathy M.S. Pepper F. Kiefer U. Möhle-Steinlein F. Tacchini-Cottier I. Fetka G. Breier W. Risau P. Carmeliet R. Montesano (1997) ArticleTitlePolyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system J. Cell Biol. 137 953–963 Occurrence Handle10.1083/jcb.137.4.953 Occurrence Handle1:CAS:528:DyaK2sXjtleltL0%3D Occurrence Handle9151696

    Article  CAS  PubMed  Google Scholar 

  • E. Sarubbi M.L. Nolli F. Robbiati A. Soffientini F. Parenti G. Cassani (1989) ArticleTitleThe differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1 Thromb. Haemost. 62 927–933 Occurrence Handle1:CAS:528:DyaK3cXnsVGjsA%3D%3D Occurrence Handle2512681

    CAS  PubMed  Google Scholar 

  • M. Schmitt O. Wilhelm F. Jänicke V. Magdolen U. Reuning H. Ohi N. Moniwa H. Kobayashi U. Weidle H. Graeff (1995) ArticleTitleUrokinase-type plasminogen activator (uPA) and its receptor (CD87): a new target in tumor invasion and metastasis J. Obstet. Gynaecol. 21 151–165 Occurrence Handle1:STN:280:BymC3sjksFA%3D Occurrence Handle8556577

    CAS  PubMed  Google Scholar 

  • R.L. Shapiro J.G. Duquette D.F. Roses I. Nunes M.N. Harris H. Kamino E.L. Wilson D.B. Rifkin (1996) ArticleTitleInduction of primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient and wild-type mice: cellular blue nevi invade but do not progress to malignant melanoma in uPA-deficient animals Cancer Res. 56 3597–3604 Occurrence Handle1:CAS:528:DyaK28Xks1eltr8%3D Occurrence Handle8758932

    CAS  PubMed  Google Scholar 

  • S. Sheng (2001) ArticleTitleThe urokinase-type plasminogen activator system in prostate cancer metastasis Cancer Metastasis Rev. 20 287–296 Occurrence Handle10.1023/A:1015539612576 Occurrence Handle1:CAS:528:DC%2BD38XkvVWqs78%3D Occurrence Handle12085967

    Article  CAS  PubMed  Google Scholar 

  • G. Spraggon C. Phillips U.K. Nowak C.P. Ponting D. Saunders C.M. Dobson D.I. Stuart E.Y. Jones (1995) ArticleTitleThe crystal structure of the catalytic domain of human urokinase-type plasminogen activator Structure 3 681–691 Occurrence Handle10.1016/S0969-2126(01)00203-9 Occurrence Handle1:CAS:528:DyaK2MXntF2rsrw%3D Occurrence Handle8591045

    Article  CAS  PubMed  Google Scholar 

  • G.J. Steffens W.A. Günzler F. Ötting E. Frankus L. Flohé (1982) ArticleTitleThe complete amino acid sequence of low molecular mass urokinase from human urine Hoppe Seylers Z Physiol. Chem. 363 1043–1058 Occurrence Handle1:CAS:528:DyaL38XmtFWitLw%3D Occurrence Handle6754572

    CAS  PubMed  Google Scholar 

  • R.W. Stephens N. Brünner F. Jänicke M. Schmitt (1998) ArticleTitleThe urokinase plasminogen activator system as a target for prognostic studies in breast cancer Breast Cancer Res. Treat. 52 99–111 Occurrence Handle10.1023/A:1006115218786 Occurrence Handle1:STN:280:DyaK1M7mtl2mug%3D%3D Occurrence Handle10066075

    Article  CAS  PubMed  Google Scholar 

  • R.W. Stephens J. Pöllänen H. Tapiovaara K.C. Leung P.S. Sim E.M. Salonen E. Renne N. Behrendt K. Danø A. Vaheri (1989) ArticleTitleActivation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants J. Cell Biol. 108 1987–1995 Occurrence Handle10.1083/jcb.108.5.1987 Occurrence Handle1:CAS:528:DyaL1MXhvFShsLY%3D Occurrence Handle2523891

    Article  CAS  PubMed  Google Scholar 

  • J. Stürzebecher F. Markwardt (1978) ArticleTitleSynthetic inhibitors of serine proteinases. 17. The effect of benzamidine derivatives on the activity of urokinase and the reduction of fibrinolysis Pharmazie 33 599–602 Occurrence Handle733877

    PubMed  Google Scholar 

  • D.C. Stump H.R. Lijnen D. Collen (1986) ArticleTitlePurification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures J. Biol. Chem. 261 1274–1278 Occurrence Handle1:CAS:528:DyaL28XosVahsQ%3D%3D Occurrence Handle3080423

    CAS  PubMed  Google Scholar 

  • P. Wang J. Zhang Z. Sun Y. Chen V. Gurewich J.N. Liu (2000) ArticleTitleCatalytic and fibrinolytic properties of recombinant urokinase plasminogen activator from E. colimammalian, and yeast cells Thromb. Res. 100 461–467 Occurrence Handle1:CAS:528:DC%2BD3MXitlehug%3D%3D Occurrence Handle11150590

    CAS  PubMed  Google Scholar 

  • Y. Wang (2001) ArticleTitleThe role and regulation of urokinase-type plasminogen activator receptor gene expression in cancer invasion and metastasis Med. Res. Rev. 21 146–170 Occurrence Handle11223863

    PubMed  Google Scholar 

  • T.C. Wun L. Ossowski E. Reich (1982) ArticleTitleA proenzyme form of human urokinase J. Biol. Chem. 257 7262–7268 Occurrence Handle1:CAS:528:DyaL38Xks1ygsLg%3D Occurrence Handle6806270

    CAS  PubMed  Google Scholar 

  • H. Yang J. Henkin K.H. Kim J. Greer (1990) ArticleTitleSelective inhibition of urokinase by substituted phenylguanidines: quantitative structure-activity relationship analyses J. Med. Chem. 33 2956–2961 Occurrence Handle10.1021/jm00173a008 Occurrence Handle1:CAS:528:DyaK3cXlvVOju7c%3D Occurrence Handle2231595

    Article  CAS  PubMed  Google Scholar 

  • M. Zang H. Trautmann C. Gandor F. Messi F. Asselbergs C. Leist A. Fiechter J. Reiser (1995) ArticleTitleProduction of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium Biotechnology (N Y) 13 389–392 Occurrence Handle10.1038/nbt0495-389 Occurrence Handle1:CAS:528:DyaK2MXkslKntrw%3D

    Article  CAS  Google Scholar 

  • E. Zeslawska A. Schweinitz A. Karcher P. Sondermann S. Sperl J. Stürzebecher U. Jacob (2000) ArticleTitleCrystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design J. Mol. Biol. 301 465–475 Occurrence Handle10.1006/jmbi.2000.3966 Occurrence Handle1:CAS:528:DC%2BD3cXltlCjsLo%3D Occurrence Handle10926521

    Article  CAS  PubMed  Google Scholar 

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  1. Department of Biochemistry and Molecular Biology, School of Medicine, University of New Mexico, MSC08 4670, Albuquerque, NM, 87131-0001, USA

    Erica Atkins, Stephanie Zamora, Britny J. Candia, Amy Baca & Robert A. Orlando

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  1. Erica Atkins
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  2. Stephanie Zamora
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  3. Britny J. Candia
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  4. Amy Baca
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  5. Robert A. Orlando
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Correspondence to Robert A. Orlando.

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Atkins, E., Zamora, S., Candia, B.J. et al. Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator. Cytotechnology 49, 25–37 (2005). https://doi.org/10.1007/s10616-005-4637-7

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