Cereal Research Communications
Volume/Issue:
Volume 39: Issue 3
Expression of barley leaf cysteine proteinase inhibitor as an active fusion protein in Escherichia coli cells
Authors:
A. Gholizadeh University of Tabriz Research Institute for Fundamental Sciences (RIFS) Tabriz Iran

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B. Baghbankohnehrouz University of Tabriz Department of Plant Breeding and Biotechnology Tabriz Iran

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Pages:
325–333
Online Publication Date:
10 Sep 2011
Publication Date:
01 Sep 2011
Article Category:
Research Article
DOI:
https://doi.org/10.1556/crc.39.2011.3.2
Keywords:
barley cystatin; proteinase inhibitor; fusion protein expression
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Nowadays, identification of the novel physio-biological and therapeutic functions of plant cysteine proteinase inhibitors “plant cystatins / phytocystatins” are the great of interests for molecular biologists. Whether for biochemical, structural or functional studies, their successful expression along with an easy purification method is required. To date, fusion tags are the best available tools that meet all those requirements. We report here the cloning and simple functional expression and purification of a barley putative cystatin in Escherichia coli cells. For the first time, a part of barley coding sequence containing a predicted active cystatin was amplified by polymerase chain reaction and expressed as maltose binding fusion protein in TB1 strain of E. coli cells using pMALc2X over-expression vector system without affecting the bacterial growth. The expressed product was purified by single step affinity chromatography from the soluble fraction of induced culture at a yield of about 37 mg/ liter of bacterial cell culture. The purified fused protein could efficiently inhibit papain activity in vitro without the cleavage of the fusion partner.

  • Abe, M., Abe, K., Kuroda, M., Arai, S. 1992. Corn kernel cysteine protease inhibitor as a novel cystatin superfamily member of plant cystatins. Eur. J. Biochem. 209:933–937.

    Arai S. , 'Corn kernel cysteine protease inhibitor as a novel cystatin superfamily member of plant cystatins ' (1992 ) 209 Eur. J. Biochem. : 933 -937 .

    • Search Google Scholar

  • Annadana, S., Schipper, B., Beekwilder, J., Outchkourov, N., Udayakumar, M., Jongsma, M.A. 2003. Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin. J. Biosci. Bioeng. 95:118–123.

    Jongsma M.A. , 'Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin ' (2003 ) 95 J. Biosci. Bioeng. : 118 -123 .

    • Search Google Scholar

  • Arai, S., Matsumoto, I., Emori, Y., Abe, K. 2002. Plant seed cystatins and their target enzymes of endogenous and exogenous origin. J. Agric. Food Chem. 50:6612–6617.

    Abe K. , 'Plant seed cystatins and their target enzymes of endogenous and exogenous origin ' (2002 ) 50 J. Agric. Food Chem. : 6612 -6617 .

    • Search Google Scholar

  • Barrett, A.J., Fritz, H., Grubb, A. 1986. Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin. Biochem. J. 236:312–318.

    Grubb A. , 'Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin ' (1986 ) 236 Biochem. J. : 312 -318 .

    • Search Google Scholar

  • Bateman, A., Birney, E., Cerruti, L., Burbin, R., Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Howe, K-L., Marsahall, M., Sonnhammer, E.L. 2002. The Pfam protein families database. Nucl. Acids Res. 30:276–280.

    Sonnhammer E.L. , 'The Pfam protein families database ' (2002 ) 30 Nucl. Acids Res. : 276 -280 .

    • Search Google Scholar

  • Belenghi, B., Acconcia, F., Trovato, M., Perazzolli, M., Bocedi, A., Polticelli, F., Ascenzi, P., Delledonne, M. 2003. AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur. J. Biochem. 270:2593–2604.

    Delledonne M. , 'AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death ' (2003 ) 270 Eur. J. Biochem. : 2593 -2604 .

    • Search Google Scholar

  • Birnboirm, H.C., Doly, J.A. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513–1523.

    Doly J.A. , 'A rapid alkaline extraction procedure for screening recombinant plasmid DNA ' (1979 ) 7 Nucl. Acids Res. : 1513 -1523 .

    • Search Google Scholar

  • Botella, M.A., Xu, Y., Prabha, T.N., Zhao, Y., Narasimhan, M.L., Wilson, K.A., Nielsen, S.S., Bressan, R.A., Hasegawa, P.M. 1996. Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate. Plant Physiol. 112:1201–1210.

    Hasegawa P.M. , 'Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate ' (1996 ) 112 Plant Physiol. : 1201 -1210 .

    • Search Google Scholar

  • Campos, R.G., Acosta, J.A.T., Arias, L.J.S., Lim, M.A.G. 1999. The use of cysteine proteinase inhibitor to engineer resistance against potyviroses in transgenic tobacco plants. Nat. Biotechnol. 17:1223–1226.

    Lim M.A.G. , 'The use of cysteine proteinase inhibitor to engineer resistance against potyviroses in transgenic tobacco plants ' (1999 ) 17 Nat. Biotechnol. : 1223 -1226 .

    • Search Google Scholar

  • Cipriani, G., Fuentes, S., Bello, V., Salazar, L.F., Ghislain, M., Zhang, D.P. 2000. Transgene expression of rice cysteine proteinase inhibitors for the development of resistance against sweetpotato feathery mottle virus. In: CIP (Centro Internacional de la Papa) Program Report 1999–2000. Lima, Peru, pp. 267–271.

  • Corre-Menguy, F., Cejudo, F.J., Mazubert, C., Vidal, J., Lelandais-Brière, C., Torres, G., Rode, A., Hartmann, C. 2002. Characterization of the expression of a wheat cystatin gene during caryopsis development. Plant Mol. Biol. 50:687–698.

    Hartmann C. , 'Characterization of the expression of a wheat cystatin gene during caryopsis development ' (2002 ) 50 Plant Mol. Biol. : 687 -698 .

    • Search Google Scholar

  • Diop, N.N., Kidric, M., Repellin, A., Gareil, M., d’Arcy-Lameta, A., Pham Thi, A.T., Zuily-Fodil, Y. 2004. A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett. 577:545–550.

    Zuily-Fodil Y. , 'A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves ' (2004 ) 577 FEBS Lett. : 545 -550 .

    • Search Google Scholar

  • Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W., Hofnung, M. 1984. Sequences of the malE gene and of its product. The Maltose-Binding Protein of Escherichia coli K12. J. Biol. Chem. 259:10606–10613.

    Hofnung M. , 'Sequences of the malE gene and of its product. The Maltose-Binding Protein of Escherichia coli K12 ' (1984 ) 259 J. Biol. Chem. : 10606 -10613 .

    • Search Google Scholar

  • Gaddour, K., Vicente-Carbajosa, J., Lara, P., Isabel-Lamoneda, I., Diaz, I., Carbonero, P. 2001. A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli. Plant Mol. Biol. 45:599–608.

    Carbonero P. , 'A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli ' (2001 ) 45 Plant Mol. Biol. : 599 -608 .

    • Search Google Scholar

  • Gianotti, A., Rios, M.W., Costa, A.S., Nogaroto, V., Carmona, A.K., Oliva, M.L.V., Andrade, S.S., Silva, F.H. 2006. Recombinant expression, purification and functional analysis of two novel cystatins from sugarcane (Sacchaum officinarum). Prot. Exp. Puri. 47:483–489.

    Silva F.H. , 'Recombinant expression, purification and functional analysis of two novel cystatins from sugarcane (Sacchaum officinarum) ' (2006 ) 47 Prot. Exp. Puri. : 483 -489 .

    • Search Google Scholar

  • Kellerman, O.K., Ferenci, T. 1982. Maltose-binding protein from E. coli. Methods Enzymol. 90:459–463.

    Ferenci T. , 'Maltose-binding protein from E. coli ' (1982 ) 90 Methods Enzymol. : 459 -463 .

    • Search Google Scholar

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

    Laemmli U.K. , 'Cleavage of structural proteins during the assembly of the head of bacteriophage T4 ' (1970 ) 227 Nature : 680 -685 .

    • Search Google Scholar

  • Martinez, M., Abraham, Z., Carbonero, P., Diaz, I. 2005. Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley. Mol. Gen. Genom. 273:423–432.

    Diaz I. , 'Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley ' (2005 ) 273 Mol. Gen. Genom. : 423 -432 .

    • Search Google Scholar

  • Martinez, M., Rubio-Somoza, I., Fuentes, R., Lara, P., Carbonero, P., Diaz, I. 2005. The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination. J. Exp. Bot. 56:547–556.

    Diaz I. , 'The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination ' (2005 ) 56 J. Exp. Bot. : 547 -556 .

    • Search Google Scholar

  • Massonneau, A., Condamine, P., Wisniewski, J.-P., Zivy, M., Rogowsky, P.M. 2005. Maize cystatins respond to developmental cues, cold stress and drought. Biochim. Biophys. Acta 1729:186–199.

    Rogowsky P.M. , 'Maize cystatins respond to developmental cues, cold stress and drought ' (2005 ) 1729 Biochim. Biophys. Acta : 186 -199 .

    • Search Google Scholar

  • Otto, H.H., Schirmeister, T. 1997. Cysteine proteases and their inhibitors. Chem. Rev. 97:133–171.

    Schirmeister T. , 'Cysteine proteases and their inhibitors ' (1997 ) 97 Chem. Rev. : 133 -171 .

    • Search Google Scholar

  • Rawling, N.D., Barrett, A.J. 1990. Evolution of proteins of the cystatin superfamily. J. Mol. Evol. 30:60–71.

    Barrett A.J. , 'Evolution of proteins of the cystatin superfamily ' (1990 ) 30 J. Mol. Evol. : 60 -71 .

    • Search Google Scholar

  • Rivard, D., Girard, C., Anguenot, R., Vézina, L.P., Trépanier S., Michaud, D. 2007. MsCYS1, a developmentally-regulated cystatin from alfalfa. Plant Physiol. Biochem. 45:508–514.

    Michaud D. , 'MsCYS1, a developmentally-regulated cystatin from alfalfa ' (2007 ) 45 Plant Physiol. Biochem. : 508 -514 .

    • Search Google Scholar

  • Sculachev, V. 2004. A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata L. Walp.) leaves. FEBS Lett. 577:545–550.

    Sculachev V. , 'A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata L. Walp.) leaves ' (2004 ) 577 FEBS Lett. : 545 -550 .

    • Search Google Scholar

  • Solomon, M., Belenghi, B., Delledonne, M., Menachem, E., Levine, A. 1999. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443.

    Levine A. , 'The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants ' (1999 ) 11 Plant Cell : 431 -443 .

    • Search Google Scholar
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Cover Cereal Research Communications
Cereal Research Communications A Quarterly of the Cereal Research Non-Profit Ltd. Company
Print ISSN:
0133-3720
Online ISSN:
1788-9170

 

 

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Cereal Research Communications
Language English
Size A4
Year of
Foundation		 1973
Volumes
per Year		 1
Issues
per Year		 4
Founder Akadémiai Kiadó
Founder's
Address		 H-1117 Budapest, Hungary 1516 Budapest, PO Box 245
Publisher Akadémiai Kiadó
Springer Nature Switzerland AG
Publisher's
Address		 H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
CH-6330 Cham, Switzerland Gewerbestrasse 11.
Responsible
Publisher		 Chief Executive Officer, Akadémiai Kiadó
ISSN 0133-3720 (Print)
ISSN 1788-9170 (Online)

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