Expression of Tissue Plasminogen Activator in Transgenic Tobacco Plants by Signal Peptides Targeting for Delivery to Apoplast, Endoplasmic Reticulum and Cytosol Spaces

Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

Abstract:

Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: Recombinant tissue plasminogen activator, plant cell comportment, leader signals, transgenic tobacco.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.2363187

References:

[1] Collen D, Bounameaux H, DeCock F, Lijnen HR, Verstraete M. Analysis of coagulation and fibrinolysis during intravenous infusion of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction. Circulation 1986;73:511–7.
[2] Pennica D, Holmes WE, Kohr WJ, et al. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 1983; 301: 214–21.
[3] Collen D, Lijnen HR. Tissue-type plasminogen activator: a historical perspective and personal account. J Thromb Haemost. 2004;2:541–6. doi:10.1111/j.1538-7933.2004.00645.x.
[4] Manosroi J, Tayapiwatana C, Gotz F, Werner RG, Manosroi A. Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli. Appl Environ Microbiol. 2001;67:2657–64 doi:10.1128/AEM.67.6.2657-2664.2001.
[5] Kim JY, Fogarty EA, Lu FJ, Zhu H, Wheelock GD, Henderson LA, et al. Twin-arginine translocation of active human tissue plasminogen activator in Escherichia coli. Appl Environ Microbiol. 2005;71:8451–9. doi:10.1128/AEM.71.12.8451-8459.2005.
[6] Qiu J, Swartz JR, Georgiou G. Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol. 1998;64:4891–6.
[7] Cartwright T. Production of tPA from animal cell culture. In: Spier RE, Griffiths JB, editors. Animal cell biotechnology. London, UK: Academic Press; 1992. p. 218−245.
[8] Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, et al. Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res. 1999;8:441–9. doi:10.1023/A:1008969031219.
[9] Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Monecke M, et al. Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves. Proc Natl Acad Sci USA. 1999;96:11128–33. doi:10.1073/pnas.96.20.11128.
[10] Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, et al. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol. 2000;42:583– 90. doi:10.1023/A:1006301519427.
[11] Galeffi P, Lombardi A, DonatoMD, Latini A, SperandeiM, Cantale C, et al. Expression of single-chain antibodies in transgenic plants. Vaccine 2005;23:1823–7. doi:10.1016/j.vaccine.2004.11.025.
[12] 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.
[13] Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422.
[14] Zimmermann S, Schillberg S, Liao YC, Fischer R (1998) Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breeding 4:369–379.
[15] Hu D, Tan X, Sato T, Yamgata S, Yamagata T (2006). Apparent suppression of MMP-9 activity by GD1a as determined by gelatin zymography. Biochemical and Biophys. Res. Comm. 349: 426-431.
[16] Kozak M (1992). Regulation of translation in eukaryotic systems. Annu Rev. Cell. Biol. 25: 110-115.
[17] Jayaraj J, Devlin R, Punja Z (2008) Metabolic engineering of novel ketocarotenoid production in carrot plants. Transgenic Res (in press). doi:10.1007/s11248-007-9120-0.
[18] Kwon TH, Seo JE, Kim J, Lee JH, Jang YS, Yang MS (2003). Expression and Secretion of the Heterodimeric Protein Interleukin-12 in Plant Cell Suspension Culture. Biotechnol. Bioeng. 81: 870-877.
[19] Stevens LH, Stoopen GM, Elbers IJ,Molthoff JW, Bakker HA, Lommen A, et al. Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol. 2000;124:173–82. doi:10.1104/pp.124.1.173.
[20] Sharp JM, Doran PM. Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng. 2001;73:338–46. doi:10.1002/bit.1067.
[21] Hellwig S, Drossard J, Twyman RM, Fischer R. Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 2004;22:1415–22.
[22] Okamoto T, Nakayama H, Seta K, Isobe T, Minamikawa T (1994). Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vascular cysteine endopeptidase (SH-EP). FEBS letter 351: 31-34.