High-level production of active human TFPI-2 Kunitz domain in plant

  1. Williams, Laura 1
  2. Deana, Atilio 12
  3. Romero, Alicia 1
  4. Molina, Antonio 1
  5. Lunello, Pablo 1
  1. 1 Agrenvec S.L., Tres Cantos, Madrid 28760, Spain
  2. 2 Institut Pasteur de Montevideo
    info

    Institut Pasteur de Montevideo

    Montevideo, Uruguay

    ROR https://ror.org/04dpm2z73

Revista:
Protein Expression and Purification

ISSN: 1046-5928

Año de publicación: 2014

Volumen: 96

Páginas: 14-19

Tipo: Artículo

DOI: 10.1016/J.PEP.2014.01.011 GOOGLE SCHOLAR lock_openAcceso abierto editor

Otras publicaciones en: Protein Expression and Purification

Resumen

Plants are an attractive production system alternative to cell bioreactor not only because of its lower production costs, but also due to its lack of mammalian pathogens and contaminants, plant capacity to generate appropriate eukaryotic folding and in many cases correct post-translational modifications. In recent years, several recombinant proteins and antibodies have been introduced in the biopharmaceutical market, in particular in cancer therapeutics. Kunitz domain 1 (KD1), a domain of Human Tissue Factor Pathway Inhibitor-2 (TFPI-2), has an outstanding potential in cancer treatment because it is a potent inhibitor of extracellular serine proteinases involved in tumor progression and angiogenesis. We present here the expression and purification of active human KD1 in different Nicotiana species as hosts and its stability during the infection process using a construct derived from a Tobacco mosaic virus (TMV) vector. Our purification protocol allows to recover over 100 mg of active human KD1 per batch of 1 kg of plant tissue at about 97% purity. The yields are reproducible, being N. benthamiana the best system where higher levels of KD1 are obtained. Recombinant KD1 was also used to produce a high-sensitivity polyclonal antibody able to detect not only KD1 but also full-length TFPI-2. Finally, we show that this platform is a valuable alternative for the large scale production of KD1.

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Información de financiación

We would like to acknowledge Walter Kisiel (University of New Mexico, USA) for kindly providing us with human KD1 protein produced in bacteria. This work was partially supported by the venture capital company UNINVEST, through its UNIFONDO Fund; also by the Community of Madrid through the grant 47/2007 of the Consejería de Economía, co-financed by European FEDER funds.

Financiadores

Referencias bibliográficas

  • Bajaj, (2011), J. Biol. Chem., 286, pp. 4329, 10.1074/jbc.M110.191163
  • Daci, (1999), J. Bone Miner. Res., 14, pp. 946, 10.1359/jbmr.1999.14.6.946
  • Rao, (1999), Biochem. Biophys. Res. Commun., 255, pp. 94, 10.1006/bbrc.1999.0153
  • Chand, (2004), Blood, 103, pp. 1069, 10.1182/blood-2003-06-1930
  • Laskowski, (1980), Annu. Rev. Biochem., 49, pp. 593, 10.1146/annurev.bi.49.070180.003113
  • Potempa, (1994), J. Biol. Chem., 269, pp. 15957, 10.1016/S0021-9258(17)33954-6
  • Wiedow, (1990), J. Biol. Chem., 265, pp. 14791, 10.1016/S0021-9258(18)77182-2
  • Sprecher, (1994), Proc. Natl. Acad. Sci. U.S.A., 91, pp. 3353, 10.1073/pnas.91.8.3353
  • Miyagi, (1994), J. Biochem., 116, pp. 939, 10.1093/oxfordjournals.jbchem.a124648
  • Sierko, (2007), Semin. Thromb. Hemost., 33, pp. 653, 10.1055/s-2007-991532
  • Ivanciu, (2007), Arterioscler. Thromb. Vasc. Biol., 27, pp. 310, 10.1161/01.ATV.0000254147.89321.cf
  • Xu, (2006), Arterioscler. Thromb. Vasc. Biol., 26, pp. 2819, 10.1161/01.ATV.0000248731.55781.87
  • Konduri, (2001), Int. J. Oncol., 18, pp. 127
  • Guo, (2007), BMC Mol. Biol., 8, pp. 110, 10.1186/1471-2199-8-110
  • Crawley, (2002), Arterioscler. Thromb. Vasc. Biol., 22, pp. 218, 10.1161/hq0102.101842
  • Obata, (2005), Eye, 19, pp. 464, 10.1038/sj.eye.6701531
  • Horn, (2004), Plant Cell Rep., 22, pp. 711, 10.1007/s00299-004-0767-1
  • Nolasco, (1993), J. Virol. Methods, 45, pp. 201, 10.1016/0166-0934(93)90104-Y
  • Lizárraga, (1989), Plant Dis., 73, pp. 11, 10.1094/PD-73-0011
  • Lommel, (1982), Phytopathology, 72, pp. 1018, 10.1094/Phyto-72-1018
  • Platis, (2008), Curr. Med. Chem., 15–19, pp. 1940, 10.2174/092986708785132924
  • Avesani, (2007), Transgenic Res., 16, pp. 587, 10.1007/s11248-006-9051-1
  • Dolja, (1998), Virology, 252, pp. 269, 10.1006/viro.1998.9458
  • German-Retana, (2000), Mol. Plant Microbe Interact., 13, pp. 316, 10.1094/MPMI.2000.13.3.316
  • Touriño, (2008), Span. J. Agric. Res., 6, pp. 48, 10.5424/sjar/200806S1-373
  • Guo, (1998), Virus Res., 57, pp. 183, 10.1016/S0168-1702(98)00100-2
  • Goodin, (2008), Mol. Plant Microbe Interact., 21, pp. 1015, 10.1094/MPMI-21-8-1015
  • Chapman, (1992), Plant J., 2, pp. 549
  • Cruz, (1996), Proc. Natl. Acad. Sci. U.S.A., 93, pp. 6286, 10.1073/pnas.93.13.6286
  • Lucas, (2006), Virology, 344, pp. 169, 10.1016/j.virol.2005.09.026
  • Leisner, (1993), Trends Microbiol., 1, pp. 314, 10.1016/0966-842X(93)90009-G
  • Leisner, (1993), Bioessays, 15, pp. 741, 10.1002/bies.950151107
  • Samuel, (1934), Ann. Appl. Biol., 21, pp. 90, 10.1111/j.1744-7348.1934.tb06891.x
  • Lunello, (2007), Mol. Plant Microbe Interact., 20, pp. 1589, 10.1094/MPMI-20-12-1589