Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin

  • Melina Laguia-Becher
  • Zurima Zaldúa
  • Weijie Xu
  • Patricia Laura Marconi
  • William Velander
  • María Alejandra AlvarezEmail author
Plant Tissue Culture


The serine protease α-thrombin (FIIa) plays a fundamental role in blood clotting. In the present report, a FIIa precursor (pFIIa) was expressed in Nicotiana benthamiana Domin. The expression construct featured the Kozak consensus sequence and the 2S2 Arabidopsis thaliana (L.) Heynh. signal peptide to direct the protein into the secretory pathway (sec-pFIIa). A version carrying the KDEL endoplasmic reticulum (ER) retention signal (pFIIa-ER) was also constructed. Transient expression of pFIIa in N. benthamiana leaves was achieved by Agrobacterium tumefaciens infiltration. The influence of post-transcriptional gene silencing (PTGS) was analyzed by co-infiltrating with an A. tumefaciens strain carrying the construct for the Turnip Crinkle Virus-coat protein (TCV-CP) known for interfering with PTGS. Reverse transcription polymerase chain reaction and Western blot analyses confirmed the presence of the corresponding messenger RNA and the recombinant pFIIa protein in plant extracts. A positive effect of the addition of the PTGS inhibitor was demonstrated. The accumulation of sec-pFIIa and pFIIa-ER was estimated to be 6 μg g−1 fresh weight (FW) (0.07% (w/w) total protein concentration; TPC) and 17 μg g−1 FW (0.21% (w/w) TPC), respectively. Furthermore, stably transformed callus and suspension cultures were obtained. The recombinant protein was detected only in the biomass of the pFIIa-ER cell suspension line at a concentration of 0.25 μg mL−1 (0.017% (w/w) of total soluble protein). This appears to be the first report describing the expression of a precursor of FIIa in plants.


Alpha-thrombin Plant-made recombinant protein Agrobacterium tumefaciens infiltration Post-transcriptional gene silencing Plant cell suspension cultures 



The authors thank Dr. Satyanarayana Tatineni from the University of Nebraska for kindly providing the PZP-TCV-CP plasmid and Ms. Isabel Rillo for her advice and careful revision of the English language. MAA, MLB, and PM are members of Consejo Nacional de Ciencia y Tecnología (CONICET) from Argentina.

Funding information

This article received funding from Agencia Nacional de Ciencia y Tecnología (PICT 2010-00552) and Nebraska University (21-1106-4006-2).


  1. Adams TE, Huntington JA (2016) Structural transitions during prothrombin activation: on the importance of fragment 2. Biochimie 122:235–242CrossRefPubMedPubMedCentralGoogle Scholar
  2. Albarracín RM, Becher ML, Farran I, Sander VA, Corigliano MG, Yácono ML, Pariani S, López ES, Veramendi J, Clemente M (2015) The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts. Biotechnol J 10:748–759CrossRefPubMedGoogle Scholar
  3. Alvarez MA, Nigra HM, Giulietti AM (1993) Solasodine production by Solanum eleagnifolium Cav. in vitro cultures: influence of plant growth regulators, age and inoculum size. Large-scale production. Nat Prod Lett 3:9–19CrossRefGoogle Scholar
  4. Batra J, Rathore AS (2016) Glycosylation of monoclonal antibody products: current status and future prospects. Biotechnol Prog 32:1091–1102CrossRefPubMedGoogle Scholar
  5. Bertani G (1951) Studies on lysogenesis I : the mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedPubMedCentralGoogle Scholar
  6. Boivin EB, Lepage E, Matton DP, De Crescenzo G, Jolicoeur M (2010) Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co-transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol Prog 26:1534–1543CrossRefPubMedGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Casademunt E, Martinelle K, Jernberg M, Winge S, Tiemeyer M, Biesert L, Knaub S, Schröder WO (2012) The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics. Eur J Haemat 89:165–176CrossRefGoogle Scholar
  9. Choi EH, Kim YJ, Kim JM, Hong HJ, Han MH, Kim J (1989) Cloning and expression of human prethrombin 2 cDNA in Escherichia coli. Korean Biochem J 22:154–160Google Scholar
  10. Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38:101–109CrossRefPubMedGoogle Scholar
  11. Del L, Yácono M, Farran I, Becher ML, Sander V, Sánchez VR, Martín V, Veramendi J, Clemente M (2012) A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice. Plant Biotechnol J 10:1136–1144CrossRefGoogle Scholar
  12. DiBella EE, Maurer MC, Scherag HA (1995) Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J Biol Chem 270:163–169CrossRefPubMedGoogle Scholar
  13. Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426–432CrossRefPubMedGoogle Scholar
  14. Ferraro G, Becher ML, Angel SO, Zelada A, Mentaberry AN, Clemente M (2008) Efficient expression of a Toxoplasma gondii dense granule Gra4 antigen in tobacco leaves. Exp Parasitol 120:118–122CrossRefPubMedGoogle Scholar
  15. Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back. Bio/Technology 12:883–889Google Scholar
  16. Fischer R, Schillberg S, Buyel JF, Twyman RM (2013) Commercial aspects of pharmaceutical protein production in plants. Curr Pharm Des 19:5471–5477CrossRefPubMedGoogle Scholar
  17. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 30:434–439CrossRefPubMedGoogle Scholar
  18. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158CrossRefPubMedGoogle Scholar
  19. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37CrossRefPubMedPubMedCentralGoogle Scholar
  20. Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D, Faye L (2010) Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 8:564–587CrossRefPubMedGoogle Scholar
  21. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994CrossRefPubMedGoogle Scholar
  22. Heemskerk J, Bevers E, Lindhout T (2002) Platelet activation and blood coagulation. Thromb Haemost 88:186–193CrossRefPubMedGoogle Scholar
  23. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) Green: a versatile and flexible binary Ti vector for agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832CrossRefPubMedGoogle Scholar
  24. Holly DC, Foster DC (1996) Methods for producing thrombin. US Patent Number 005527692AGoogle Scholar
  25. Huang TK, Plesha MA, Falk BW, Dandekar AM, McDonald KA (2009) Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures. Biotechnol Bioeng 102:508–520CrossRefPubMedGoogle Scholar
  26. James E, Lee JM (2006) Loss and recovery of protein productivity in genetically modified plant cell lines. Plant Cell Rep 25:723–727CrossRefPubMedGoogle Scholar
  27. Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195CrossRefPubMedGoogle Scholar
  28. Krebbers E, Herdies L, De Clerq A, Seurinck J, Leemans J, Van Damme J, Segura M, Gheysen G, Van Montagu M, Vandekerckhove J (1988) Determination of the processing sites of an Arabidopsis 2S albumin and characterization of the complete gene family. Plant Physiol 87:859–866CrossRefPubMedPubMedCentralGoogle Scholar
  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  30. Laguia-Becher M, Martín V, Kraemer M, Corigliano M, Yacono ML, Goldman A, Clemente M (2010) Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice. BMC Biotechnol 15:10–52Google Scholar
  31. Le Bonniec BF, Guinto ER, Esmon CT (1992) Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. structural link between the autolysis loop and the Tyr-pro-pro-Trp insertion of thrombin. J Biol Chem 267:19341–19348PubMedGoogle Scholar
  32. Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Lainé A, Gomord V, Faye L (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38:31–48CrossRefPubMedGoogle Scholar
  33. Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 23Google Scholar
  34. Liu D, Shi L, Han C, Yu J, Li D, Zhang Y (2012) Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS One 7:e46451CrossRefPubMedPubMedCentralGoogle Scholar
  35. López J, Lencina F, Petrucceli S, Marconi PM, Alvarez MA (2010) Influence of the KDEL signal, DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term Nicotiana tabacum cell suspension culture. Plant Cell Tissue Organ Cult 103:307–314CrossRefGoogle Scholar
  36. Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: The green perspective BioMed Res Int Article ID 136419Google Scholar
  37. Moura RR, Melo LM, de Figueirêdo Freitas VJ (2011) Production of recombinant proteins in milk of transgenic and non-transgenic goats. Braz Arc Biol Technol 54(5) 927–938Google Scholar
  38. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  39. Narasimhulu SB, Deng X-B, Sarria R, Gelvin SB (1996) Early transcription of agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8:873–886CrossRefPubMedPubMedCentralGoogle Scholar
  40. Nelson G, Marconi P, Periolo O, La Torre J, Alvarez MA (2012) Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines. Vaccine 30:4499–4504CrossRefPubMedGoogle Scholar
  41. Nocarova E, Fischer L (2009) Cloning of transgenic tobacco BY-2 cells; an efficient method to analyze and reduce high natural heterogeneity of transgene expression. BMC Plant Biol 9(44):44CrossRefPubMedPubMedCentralGoogle Scholar
  42. Oates AM, Kupczyk M, Kannelos J (2001) Method for the production of thrombin, US Patent Number 6168938B1Google Scholar
  43. Osadská M1, Boňková H, Krahulec J, Stuchlík S, Turňa J (2014) Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli. Appl Microbiol Biotechnol 98:9259–9270CrossRefPubMedGoogle Scholar
  44. Qu F, Ren T, Morris TJ (2003) The coat protein of Turnip crinkle virus suppresses post-transcriptional gene silencing at an early initiation step. J Virol 77:511–522CrossRefPubMedPubMedCentralGoogle Scholar
  45. Rech E, Vianna G, Murad A, Cunha N, Lacorte C, Araujo A, Brigido M, Waters M, Fontes A, O’Keefe B, Simpson A, Caballero O (2014) Recombinant proteins in plants. BMC Proc 2014 8(Suppl 4):O1Google Scholar
  46. Russo G, Gast A, Schlaeger EJ, Angiolillo A, Pietropaolo C (1997) Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin. Protein Expr Purif 10:214–225CrossRefPubMedGoogle Scholar
  47. Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36:2367–2379CrossRefPubMedGoogle Scholar
  48. Sack M, Hofbauer A, Fischer R, Stoger E (2015) The increasing value of plant-made proteins. Curr Opin Biotechnol 32:163–170CrossRefPubMedGoogle Scholar
  49. Santagostino E, Jacobs IC, Voigt C, Feussner A, Limsakun T (2014) Pharmacokinetic results of two phase III clinical studies of coagulation factor IX (recombinant) albumin fusion protein (rIX-FP) in previously treated patients with hemophilia B (PROLONG-9FP). Blood 124:1491Google Scholar
  50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  51. Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832CrossRefPubMedGoogle Scholar
  52. So IS, Lee S, Kim SW, Hahm KS, Kim J (1992) Purification and activation of recombinant human prethrombin 2 produced in E. coli. Korean Biochem J 25:60–65Google Scholar
  53. Soejima K, Mimura N, Yonemura H, Nakatake H, Imamura T, Nozaki C (2001) An efficient refolding method for the preparation of recombinant human Prethrombin-2 and characterization of the recombinant-derived a-thrombin. Biochem 130:269–277CrossRefGoogle Scholar
  54. Stam M, Mol JNM, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12CrossRefGoogle Scholar
  55. Sudarshana MR, Plesha MA, Uratsu SL, Falk BW, Dandekar AM, Huang TK, McDonald KA (2006) A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues. Plant Biotechnol J 4:551–559PubMedGoogle Scholar
  56. Tatineni S, Qu F, Li R, Morris TJ, French R (2012) Triticum mosaic poacevirus enlists P1 rather than HC-pro to suppress RNA silencing-mediated host defense. Virology 433:104–115CrossRefPubMedGoogle Scholar
  57. Twyman RM, Schillberg S, Fischer R (2012) The production of vaccines and therapeutic antibodies in plants. In: Wang A, Ma S, (eds.), Molecular farming in plants: recent advances and future prospects. Springer, Dordrecht, Netherlands, pp 145–159Google Scholar
  58. Ullrich KK, Hiss M, Rensing SA (2015) Means to optimize protein expression in transgenic plants. Curr Opin Biotechnol 32:61–67CrossRefPubMedGoogle Scholar
  59. Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091PubMedGoogle Scholar
  60. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 5:949–956CrossRefGoogle Scholar
  61. Yonemura H, Imamura T, Soejima K, Nakahara Y, Morikawa W, Ushio Y, Kamachi Y, Nakatake H, Sugawara K, Nakagaki T, Nozaki C (2004) Preparation of recombinant α-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. J Biochem 135:577–582CrossRefPubMedGoogle Scholar
  62. Zanetti ME, Chang IF, Gong F, Galbraith DW, Bailey-Serres J (2005) Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiol 138:624–635CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Departamento de Investigaciones Bioquímicas y Farmacológicas, Laboratorio de Biotecnología VegetalCABAArgentina
  2. 2.Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET)CABAArgentina
  3. 3.Department of Biochemical EngineeringNebraska UniversityLincolnUSA
  4. 4.Farmacobotánica y Farmacognosia, Farmacia y Bioquímica, Facultad de Ciencias de la SaludUniversidad Maimónides-CEBBADCABAArgentina

Personalised recommendations