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Expression of the Human Tissue-Plasminogen Activator in Hairy Roots of Oriental Melon (Cucumis melo)

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Abstract

The human tissue plasminogen activator (t-PA) is a thrombolytic protein that plays a key role in fibrinolysis by converting the plasminogen into plasmin which degrades fibrin clots in blood vessels. The t-PA gene encoding the human tissue plasminogen activator was cloned and expressed under transcriptional control of single and multiple rooting loci promoter (rolD) promoters in hairy roots derived from Oriental melon. t-PA gene integration in the genome of hairy roots was confirmed by PCR. Increased transcript and protein expression levels was observed in the transgenic hairy roots expressing t-PA under the control of single and dual rolD promoters, respectively. Enzyme-linked immunosorbent assay revealed the highest levels of the recombinant t-PA accumulation in transgenic hairy roots carrying the t-PA transgene under the control of single and dual rolD (0.17–0.15 μg/mg) promoters as compared to triple and quadruple rolD (0.07–0.03 μg/mg) promoters. Fibrinolytic activity indicated the proper function of the expressed recombinant t-PA in fibrin lysis. These results demonstrated that hairy roots could serve as alternatives to transgenic plants for expression of mammalian genes.

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References

  • Astrup T, Mullertz S (1952) The fibrin plate method for estimating of fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  PubMed  CAS  Google Scholar 

  • Cartwright T (1992) Production of tPA from animal cell culture. In: Spier RE, Griffiths JB (eds) Anim cell biotechnol London. Academic, UK, pp 218–245

    Google Scholar 

  • Collen D, Bounameaux H, DeCock F et al (1986) Analysis of coagulation and fibrinolysis during intravenous infusion of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction. Circulation 73:511–517

    Article  PubMed  CAS  Google Scholar 

  • Cui L, Chen Y, Shen G, Zhao L, Tang K (2010) Expression of bioactive thymosin alpha 1 (Tα1) in marker-free transgenic lettuce (Lactuca sativa). Plant Mol Biol Rep. doi:10.1007/s11105-010-0246-5

    Google Scholar 

  • Ebert KM, Selgrath JP, DiTullio P et al (1991) Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression. Biotechnology 9:835–838

    Article  PubMed  CAS  Google Scholar 

  • Elmayan T, Tepfer M (1995) Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Trans Res 4:388–396

    Article  CAS  Google Scholar 

  • Gaume A, Komarnytsky S, Borisjuk N et al (2003) Rhizosecretion of recombinant proteins from plant hairy roots. Plant Cell Rep 21:1188–1193

    Article  PubMed  CAS  Google Scholar 

  • Griffiths JB, Electricwala A (1987) Production of tissue plasminogen activators from animal cells. Adv Biochem Eng Biotechnol 34:147–166

    PubMed  CAS  Google Scholar 

  • Hahn BS, Sim JS, Kim HM et al (2009) Expression and characterization of human tissue-plasminogen activator in transgenic tobacco plants. Plant Mol Biol Rep 27:209–216

    Article  CAS  Google Scholar 

  • Jarvis DL, Summers MD (1989) Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. Mol Cell Biol 9:214–223

    PubMed  CAS  Google Scholar 

  • Keivan MA, Vahid K, Davami F et al (2010) Cloning and expression of functional full-length human tissue plasminogen activator in Pichia pastoris. Appl Biochem Biotechnol 162:2037–2048

    Article  Google Scholar 

  • Kim JY, Fogarty EA, Lu FJ, Zhu H, Wheelock GD, Henderson LA et al (2005) Twin-arginine translocation of active human tissue plasminogen activator in Escherichia coli. Appl Environ Microbiol 71:8451–8459

    Article  PubMed  CAS  Google Scholar 

  • Ko SM, Yoo BH, Lim JM, Oh KH, Liu JI, Kim SW, Liu JR, Choi KS, Yoon ES (2009) Production of fibrinolytic enzyme in plastid-transformed tobacco plants. Plant Mol Biol Rep 27:448–453

    Article  CAS  Google Scholar 

  • Leach F, Aoyagi K (1991) Promoter analysis of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes: enhancer and tissue-specific DNA determinants are dissociated. Plant Sci 79:69–76

    Article  CAS  Google Scholar 

  • Liang Y, Zhao S, Zhang X (2009) Antisense suppression of cycloartenol synthase results in elevated ginsenoside levels in Panax ginseng hairy roots. Plant Mol Biol Rep 27:298–304

    Article  CAS  Google Scholar 

  • Manosroi J, Tayapiwatana C, Gotz F, Werner RG, Manosroi A (2001) Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli. Appl Environ Microbiol 67:2657–2664

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nandeshwar SB, Moghe S, Chakrabarty PK, Deshattiwar MK, Kranthi K, Anandkumar P, Mayee CD, Khadi BM (2009) Agrobacterium-mediated transformation of cry1Ac gene into shoot-tip meristem of diploid cotton Gossypium arboreum cv. RG8 and regeneration of transgenic plants. Plant Mol Biol Rep 27:549–557

    Article  CAS  Google Scholar 

  • Nazari R, Davoudi N (2010) Cloning and expression of truncated form of tissue plasminogen activator in Leishmania tarentolae. Biotechnol Lett. doi:10.1007/s10529-010-0470-y

    PubMed  Google Scholar 

  • Pak HK, Sim JS, Rhee Y, Ko HR, Ha SH, Yoon MS, Kang CH, Lee S, Kim YH, Hahn BS (2009) Hairy roots induction in Oriental melon (Cucumis melo) by A. rhizogenes and production of the root-knot nematode. Plant Cell Tiss Organ Cult 98:219–228

    Article  CAS  Google Scholar 

  • Parry SK, Sharma SB, Terzaghi EA (1994) Construction of a bidirectional promoter probe vector and its use in analyzing Nod gene-expression in Rhizobium loti. Gene 150:105–109

    Article  PubMed  CAS  Google Scholar 

  • Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA et al (1983) Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 301:214–221

    Article  PubMed  CAS  Google Scholar 

  • Qiu J, Swartz JR, Georgiou G (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 64:4891–4896

    PubMed  CAS  Google Scholar 

  • Shanks JV, Morgan J (1999) Plant “hairy root” culture. Curr Opin Biotechnol 10:151–155

    Article  PubMed  CAS  Google Scholar 

  • Sharp JM, Doran PM (2001) Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnol Prog 17:979–992

    Article  PubMed  CAS  Google Scholar 

  • Silberstein L, Kovalski I, Huang R, Anagostou K, Kyle Jahn M, Perl-Treves R (1999) Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and RAPD markers. Sci Hort 79:101–111

    Article  CAS  Google Scholar 

  • Sim JS, Pak HK, Kim DS, Lee SB, Kim YH, Hahn BS (2009) Expression and characterization of synthetic heat-labile enterotoxin B subunit and hemagglutinin–neuraminidase-neutralizing epitope fusion protein in Escherichia coli and tobacco chloroplasts. Plant Mol Biol Rep 27:388–399

    Article  CAS  Google Scholar 

  • Sivakumar G (2006) Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol J 1:1419–1427

    Article  PubMed  CAS  Google Scholar 

  • Srivastava S, Srivastava A (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotech 27:29–43

    Article  CAS  Google Scholar 

  • Streatfield SJ (2005) Regulatory issues for plant-made pharmaceuticals and vaccines. Exp Rev Vacc 4:591–601

    Article  Google Scholar 

  • Thorup JE, McDonald KA, Jackman AP et al (1994) Ribosome-inactivating protein production from Trichosanthes kirilowii plant cell cultures. Biotechnol Prog 10:345–352

    Article  CAS  Google Scholar 

  • Unni SC, Soniya EV (2010) Transgenic Cucumis sativus expressing the hepatitis B surface antigen. Plant Mol Biol Rep 28:627–634

    Article  CAS  Google Scholar 

  • Upshall A, Kumar AA, Bailey MC, Parker MD, Favreau MA (1987) Secretion of active human tissue plasminogen activator from the filamentous fungus Aspergillus nidulans. Biotechnology 5:1301–1304

    Article  CAS  Google Scholar 

  • Van Zonneveld AJ, Chang GT, van den Berg J et al (1986) Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe. Biochem J 235:385–390

    PubMed  Google Scholar 

  • Wang B, Jiang YN, Yao LM, Wu TL (2009) Improved production of the human granulocyte-macrophage colony stimulating factor in transgenic Arabidopsis thaliana seeds using a dual sorting signal peptide. Plant Mol Biol Rep 27:462–468

    Article  CAS  Google Scholar 

  • Woods RR, Geyer BC, Mor TS (2008) Hairy root organ cultures for the production of human acetylcholinesterase. BMC Biotechnol 8:95

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank Ms. Kyung-Il Lee (NAAS) for technical assistance. This project (PJ006652) was supported by grant (to BSH) from the National Academy of Agricultural Science, RDA, Korea. This study was supported by 2011 Postdoctoral Fellowship Program (to HA) of National Academy of Agricultural Science, RDA, Korea.

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Authors and Affiliations

  1. National Academy of Agricultural Science, Suwon, 441-707, South Korea

    Sol Kang, Hemavathi Ajjappala, Hyun-Hui Seo, Joon-Soo Sim, Sang-Hong Yoon, Bon-Sung Koo, Yong-Hwan Kim & Bum-Soo Hahn

  2. Department of Genetic Engineering, Sungkyunkwan University, Suwon, 440-746, South Korea

    Sukchan Lee

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  1. Sol Kang
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  2. Hemavathi Ajjappala
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  3. Hyun-Hui Seo
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  4. Joon-Soo Sim
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  5. Sang-Hong Yoon
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  6. Bon-Sung Koo
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  7. Yong-Hwan Kim
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  8. Sukchan Lee
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  9. Bum-Soo Hahn
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Correspondence to Bum-Soo Hahn.

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Sol Kang and Hemavathi Ajjappala equally contributed to this work.

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Kang, S., Ajjappala, H., Seo, HH. et al. Expression of the Human Tissue-Plasminogen Activator in Hairy Roots of Oriental Melon (Cucumis melo). Plant Mol Biol Rep 29, 919–926 (2011). https://doi.org/10.1007/s11105-011-0303-8

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