Extraction of Proteins from Plant Tissues

William Laing1, John Christeller1

1 The Horticultural and Food Research Institute of New Zealand, Palmerston North
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 4.7
DOI:  10.1002/0471140864.ps0407s38
Online Posting Date:  November, 2004
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Abstract

A basic protocol is described for extracting protein from plants. Suggestions are included for overcoming some of the common obstacles encountered (e.g., acidity, phenolics, cell walls).

Keywords: Plant protein extracts; proteinases; phenols; oxidizing agents; acids

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Frozen or fresh plant material
  • Liquid nitrogen
  • Extraction buffer (see recipe)
  • Mortar and pestle or tissue grinder (Freezer/mill from SPEX CertiPrep or equivalent)
  • 20‐ to 70‐µm pore size nylon mesh (screen printing mesh) or Calbiochem Miracloth for filtration
  • Small paintbrush
  • Refrigerated centrifuge and centrifuge tubes
NOTE: All operations at should be carried out at 0° to 4°C (i.e., in a cold room, or on ice) unless there are reasons to do otherwise. The extraction procedure should be performed rapidly to minimize exposure of proteins of interest to potentially damaging compounds and enzymes released upon cell breakage.
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Figures

Videos

Literature Cited

   Barraclough, D., Obenland, D., Laing, W., and Carroll, T. 2004. A method for quick and easy two‐dimensional electrophoresis of plant samples. Postharvest Biol. Technol. 32:175‐181.
   Bonin, C.P., Potter, I., Vanzin, G.F., and Reiter, W.D. 1997. The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP‐D‐mannose‐4,6‐dehydratase, catalyzing the first step in the de novo synthesis of GDP‐L‐fucose. Proc. Natl. Acad. Sci. U.S.A. 94:2085‐2090.
   Brovko, F.A. and Zagranichnaya, T.K. 1998. Separation of proteins from phenols in cereal leaf extract by hydrophobic interaction: Ammonium sulfate fractionation. Plant Physiol. Bioch. 36:773‐777.
   Che, P., Weaver, L.M., Wurtele, E.S., and Nikolau, B.J. 2003. The role of biotin in regulating 3‐methylcrotonyl‐coenzyme A carboxylase expression in Arabidopsis. Plant Physiol. 131:1479‐1486.
   Christeller, J.T., Laing, W.A., Ramsay, R.J., Cutfield, J., Cutfield, S., and Sullivan, P. 1998. Purification and characterization of an aspartic acid proteinase inhibitor from squash phloem exudate. Euro. J. Biochem. 254:160‐167.
   Delmas, F., Petit, J., Joubès, J., Séveno, M., Paccalet, T., Hernould, M., Lerouge, P. Mouras, A., and Chevalier, C. 2003. The gene expression and enzyme activity of plant 3‐deoxy‐D‐manno‐2‐octulosonic acid‐8‐phosphate synthase are preferentially associated with cell division in a cell cycle‐dependent manner. Plant Physiol. 133:348‐360.
   Geigenberger, P., Reimholz, R., Geiger, M., Merlo, L., Canale, V., and Stitt, M. 1997. Regulation of sucrose and starch metabolism in potato tubers in response to short‐term water deficit. Planta 201:502‐518.
   Heyen, B., Alsheikh, M.K., Smith, E.A., Torvik, C.F., Seals, D.F., and Randall, S.K. 2002. The calcium‐binding activity of a vacuole‐associated, dehydrin‐like protein is regulated by phosphorylation. Plant Physiol. 130:675‐687.
   Laing, W.A. and Christeller, J.T. 1997. A plant chloroplast glutamyl proteinase. Plant Physiol. 114:715‐722.
   Loomis, W.D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 31:528‐544.
   Lunn, J.E., Ashton, A.R., Hatch, M.D., and Heldt, H.W. 2000. Purification, molecular cloning, and sequence analysis of sucrose‐6F‐phosphate phosphohydrolase from plants. Proc. Natl. Acad. Sci. U.S.A. 97:12914‐12919.
   Makkar, H.P.S., Blümmel, M., and Becker, K. 1995. Formation of complexes between polyvinyl pyrrolidones or polyethylene glycols with tannins, and their implications in gas production and true digestibility in in vitro techniques. Br. J. Nutr. 73:897‐913.
   McDougall, G.J. 2000. A comparison of proteins from the developing xylem of compression and non‐compression wood of branches of sitka spruce (Picea sitchensis) reveals a differentially expressed laccase. J. Exp. Bot. 349:1395‐401.
   Melville, J.C. and Ryan, C.A. 1972. Chymotrypsin inhibitor I from potatoes: Large scale preparation and characterization of its subunit components. J. Biol. Chem. 247:3445‐53.
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   Murray, C. and Christeller, J.T. 1995. Purification of a trypsin inhibitor (PFTI) from pumpkin fruit phloem exudate and isolation of putative trypsin and chymotrypsin inhibitor cDNA clones. Biol. Chem. Hoppe Seyler 376:281‐287.
   Pastorello, E.A. and Trambaioli, C. 2001. Isolation of food allergens. J. Chromatogr. B. Biomed. Sci. Appl. 756:71‐84.
   Rassam, M. and Laing, W. 2003. Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochemistry 65:19‐30.
   Rontein, D., Wu, W.I., Voelker, D.R., and Hanson, A.D. 2003. Mitochondrial phosphatidylserine decarboxylase from higher plants: Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis. Plant Physiol. 132:1678‐1687.
   Ryan, S.N., McManus, M.T., and Laing, W.A. 2003. Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica). J. Biochem. 134:31‐42.
   Schägger, H., and von Jagow, G, 1987. Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368‐379.
   Schmalhausen, E.V., Pleten, A.P., and Muronetz, V.I. 2003. Ascorbate‐induced oxidation of glyceraldehyde‐3‐phosphate dehydrogenase. Biochem. Biophys. Res. Comm. 308:492‐496.
   Singh, B., Bhat, T.K., and Singh, B. 2003. Potential therapeutic applications of some antinutritional plant secondary metabolites. J. Agric. Food Chem. 51:5579‐5597.
   Spencer, C.M., Cai, Y., Martin, R., Gaffney, S.H., Goulding, P.N., Magnolato, D., Lilley, T.H., and Haslam, E. 1988. Polyphenol complexation—some thoughts and observations. Phytochemistry 27:2397‐2409.
   Turner, W.L. and Plaxton, W.C. 2001. Purification and characterization of banana fruit acid phosphatase. Planta 214:243‐249.
   Wolucka, B.A., Persiau, G., Van Doorsselaere, J., Davey, M.W., Demol, H., Vandekerckhove, J., Van Montagu, M., Zabeau, M., and Boerjan, W. 2001. Partial purification and identification of GDP‐mannose 3′′,5′′‐epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc. Natl. Acad. Sci. U.S.A. 9826:14843‐14848.
   Yang, P., Fu, H., Walker, J., Papa, C.M., Smalle, J., Ju, Y.M., and Vierstra, R.D. 2004. Purification of the Arabidopsis 26S proteasome: Biochemical and molecular analyses revealed the presence of multiple isoforms. J. Biol. Chem. 279:6401‐6413.
   Yuasa, K. and Maeshima, M. 2000. Purification, properties, and molecular cloning of a novel Ca2+‐binding protein in radish vacuoles. Plant Physiol. 124:1069‐1078.
Key References
   Loomis, 1974. See above.
  Describes in detail procedures to avoid the problems of phenolics.
   Barraclough et al., 2004. See above.
  Describes a robust method to process plant tissue for two‐dimensional electrophoresis.
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