Skip to main content
SpringerLink
Log in

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

In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price includes VAT (Finland)

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

References

  • Adams TE, Huntington JA (2016) Structural transitions during prothrombin activation: on the importance of fragment 2. Biochimie 122:235–242

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 10:748–759

    Article  PubMed  CAS  Google Scholar 

  • 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 3:9–19

    Article  Google Scholar 

  • Batra J, Rathore AS (2016) Glycosylation of monoclonal antibody products: current status and future prospects. Biotechnol Prog 32:1091–1102

    Article  PubMed  CAS  Google Scholar 

  • Bertani G (1951) Studies on lysogenesis I : the mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300

    PubMed  PubMed Central  CAS  Google Scholar 

  • 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 26:1534–1543

    Article  PubMed  CAS  Google Scholar 

  • 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 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • 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 89:165–176

    Article  CAS  Google Scholar 

  • 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 22:154–160

    CAS  Google Scholar 

  • 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 38:101–109

    Article  PubMed  CAS  Google Scholar 

  • 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 10:1136–1144

    Article  CAS  Google Scholar 

  • DiBella EE, Maurer MC, Scherag HA (1995) Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J Biol Chem 270:163–169

    Article  PubMed  CAS  Google Scholar 

  • Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426–432

    Article  PubMed  CAS  Google Scholar 

  • 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 120:118–122

    Article  PubMed  CAS  Google Scholar 

  • Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back. Bio/Technology 12:883–889

    Google Scholar 

  • Fischer R, Schillberg S, Buyel JF, Twyman RM (2013) Commercial aspects of pharmaceutical protein production in plants. Curr Pharm Des 19:5471–5477

    Article  PubMed  CAS  Google Scholar 

  • Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 30:434–439

    Article  PubMed  CAS  Google Scholar 

  • Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158

    Article  PubMed  CAS  Google Scholar 

  • Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 8:564–587

    Article  PubMed  CAS  Google Scholar 

  • Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  PubMed  CAS  Google Scholar 

  • Heemskerk J, Bevers E, Lindhout T (2002) Platelet activation and blood coagulation. Thromb Haemost 88:186–193

    Article  PubMed  CAS  Google Scholar 

  • 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 42:819–832

    Article  PubMed  CAS  Google Scholar 

  • Holly DC, Foster DC (1996) Methods for producing thrombin. US Patent Number 005527692A

  • 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 102:508–520

    Article  PubMed  CAS  Google Scholar 

  • James E, Lee JM (2006) Loss and recovery of protein productivity in genetically modified plant cell lines. Plant Cell Rep 25:723–727

    Article  PubMed  CAS  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  • 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 87:859–866

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • 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 15:10–52

    Google Scholar 

  • 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 267:19341–19348

    PubMed  Google Scholar 

  • 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 38:31–48

    Article  PubMed  CAS  Google Scholar 

  • 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 23

  • 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:e46451

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 103:307–314

    Article  CAS  Google Scholar 

  • 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 136419

  • 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) 927–938

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

    Article  CAS  Google Scholar 

  • Narasimhulu SB, Deng X-B, Sarria R, Gelvin SB (1996) Early transcription of agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8:873–886

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 30:4499–4504

    Article  PubMed  CAS  Google Scholar 

  • 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):44

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Oates AM, Kupczyk M, Kannelos J (2001) Method for the production of thrombin, US Patent Number 6168938B1

  • 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 98:9259–9270

    Article  PubMed  CAS  Google Scholar 

  • 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 77:511–522

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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):O1

    Google Scholar 

  • 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 10:214–225

    Article  PubMed  CAS  Google Scholar 

  • Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36:2367–2379

    Article  PubMed  CAS  Google Scholar 

  • Sack M, Hofbauer A, Fischer R, Stoger E (2015) The increasing value of plant-made proteins. Curr Opin Biotechnol 32:163–170

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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 124:1491

    Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108

    Article  PubMed  CAS  Google Scholar 

  • Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832

    Article  PubMed  CAS  Google Scholar 

  • 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 25:60–65

    CAS  Google Scholar 

  • 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 130:269–277

    Article  CAS  Google Scholar 

  • Stam M, Mol JNM, Kooter JM (1997) The silence of genes in transgenic plants. Ann Bot 79:3–12

    Article  CAS  Google Scholar 

  • 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 4:551–559

    PubMed  CAS  Google Scholar 

  • 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 433:104–115

    Article  PubMed  CAS  Google Scholar 

  • 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 145–159

  • Ullrich KK, Hiss M, Rensing SA (2015) Means to optimize protein expression in transgenic plants. Curr Opin Biotechnol 32:61–67

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091

    PubMed  CAS  Google Scholar 

  • 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 5:949–956

    Article  Google Scholar 

  • 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 135:577–582

    Article  PubMed  CAS  Google Scholar 

  • 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 138:624–635

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

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

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

Author information

Authors and Affiliations

  1. Departamento de Investigaciones Bioquímicas y Farmacológicas, Laboratorio de Biotecnología Vegetal, CEBBAD, Hidalgo, 775, CABA, Argentina

    Melina Laguia-Becher

  2. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Godoy Cruz, 2290, CABA, Argentina

    Melina Laguia-Becher, Patricia Laura Marconi & María Alejandra Alvarez

  3. Department of Biochemical Engineering, Nebraska University, Lincoln, NE, 68588, USA

    Zurima Zaldúa, Weijie Xu & William Velander

  4. Farmacobotánica y Farmacognosia, Farmacia y Bioquímica, Facultad de Ciencias de la Salud, Universidad Maimónides-CEBBAD, Hidalgo, 775, CABA, Argentina

    Patricia Laura Marconi & María Alejandra Alvarez

Authors
  1. Melina Laguia-Becher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zurima Zaldúa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Weijie Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patricia Laura Marconi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William Velander
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. María Alejandra Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to María Alejandra Alvarez.

Additional information

Editor: Baochun Li

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Laguia-Becher, M., Zaldúa, Z., Xu, W. et al. Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin. In Vitro Cell.Dev.Biol.-Plant 55, 88–98 (2019). https://doi.org/10.1007/s11627-018-09956-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-018-09956-0

Keywords

Search

Navigation