Affinity Purification of Natural Ligands

John H.T. Luong1, William H. Scouten2

1 National Research Council Canada, Biotechnology Research Institute, Montreal, Quebec, Canada, 2 University of Texas, San Antonio, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.3
DOI:  10.1002/0471140864.ps0903s52
Online Posting Date:  May, 2008
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Abstract

Immobilization of proteins, nucleic acids, and other bioligands is not always straightforward since they are often large molecules with numerous chemically reactive groups that can all participate in the immobilization process through physical adsorption, ionic binding, or covalent linkage. Protocols for some of the most frequently used matrix‐activation systems are described in this unit. For agarose, protocols are given for cyanogen bromide, p‐nitrophenyl chloroformate, tresyl chloride, and cyanuric chloride. Tosyl chloride is used to activate cellulose, and cyanuric chloride is also used to activate aminopropyl silica gel. Activation of magnetic beads with cyanogen bromide is described, and a protocol is provided for reacting the aldehyde groups of glyoxal agarose beads with the primary amine groups of ligands, with subsequent reduction of the formed Schiff base to yield a stable matrix‐ligand bond. Curr. Protoc. Protein Sci. 52:9.3.1‐9.3.22. © 2008 by John Wiley & Sons, Inc.

Keywords: affinity separation; biomolecules; ligands; matrices; agarose; activation; coupling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cyanogen Bromide Activation of Beaded Agarose Gel for Coupling to Macromolecules
  • Basic Protocol 2: p‐Nitrophenyl Chloroformate Activation of Beaded Agarose Gel for Coupling to Macromolecules
  • Basic Protocol 3: Tresyl Chloride Activation of Beaded Agarose Gel for Coupling to Macromolecules
  • Basic Protocol 4: Tosyl Chloride Activation of Cellulose for Coupling to Macromolecules
  • Basic Protocol 5: Cyanuric Chloride Activation of Beaded Agarose Gel for Coupling to Macromolecules
  • Basic Protocol 6: Cyanuric Chloride Activation of Aminopropyl Silica Gel for Coupling to Macromolecules
  • Basic Protocol 7: Cyanogen Bromide Activation of Magnetizable Cellulose/Iron Oxide Particles for Coupling to Macromolecules
  • Basic Protocol 8: Coupling Ligands to Glyoxal Agarose Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cyanogen Bromide Activation of Beaded Agarose Gel for Coupling to Macromolecules

  Materials
  • Agarose gel (e.g., Sepharose CL‐4B)
  • 2 M and 5 M potassium phosphate, pH 12.1
  • 2 M sodium carbonate
  • 2 g/ml CNBr in anhydrous acetonitrile (see recipe)
  • 1 M NaOH
  • 0.2 M sodium bicarbonate, pH 9.0
  • Sample to be coupled: typically 1 to 10 mg/ml protein, 10 to 200 mg/ml small ligand, or 0.1 to 10 mg/ml nucleic acid
  • 0.1 M potassium phosphate, pH 7.0 ( appendix 2E)
  • Solvent‐ and CNBr‐resistant gloves
  • Suction‐filter apparatus

Basic Protocol 2: p‐Nitrophenyl Chloroformate Activation of Beaded Agarose Gel for Coupling to Macromolecules

  Materials
  • Agarose gel (e.g., Sepharose CL‐4B)
  • 30:70 and 70:30 (v/v) acetone/water
  • Acetone, reagent grade, room temperature and ice cold
  • Anhydrous acetone (dried over 4 Å molecular sieves; see recipe)
  • Anhydrous acetonitrile, room temperature, and 4°C (see recipe)
  • p‐Nitrophenyl chloroformate, 800 mg in 12 ml anhydrous acetonitrile
  • Dimethylaminopyridine
  • 5% acetic acid in dioxane, ice cold
  • Methanol, ice cold
  • Anhydrous isopropanol, ice cold
  • 0.5 and 0.1 M potassium phosphate, pH 7.5 ( appendix 2E)
  • 0.1 M ethanolamine, pH 7.5
  • 0.1 M NaOH
  • Sintered glass funnel
  • Suction funnel
  • End‐over‐end mixer (e.g., Sepco or equivalent)

Basic Protocol 3: Tresyl Chloride Activation of Beaded Agarose Gel for Coupling to Macromolecules

  Materials
  • Agarose gel (e.g., Sepharose CL‐4B)
  • 30:70 and 70:30 (v/v) acetone/water
  • Acetone, reagent grade, room temperature and ice cold
  • Anhydrous acetone (dried over 4 Å molecular sieves)
  • Anhydrous acetonitrile, room temperature, and 4°C (see recipe)
  • Pyridine (stored over NaOH pellets; see recipe)
  • Tresyl chloride
  • 5 mM and 1 mM HCl
  • Protein ligand sample: typically 1 to 10 mg/ml of protein or 10 to 50 mg/ml low‐molecular‐weight nucleophilic ligand
  • 0.2 M potassium phosphate, pH 8.0 ( appendix 2E)
  • 1 M glycine, pH 8.0

Basic Protocol 4: Tosyl Chloride Activation of Cellulose for Coupling to Macromolecules

  Materials
  • Cellulose (Sigma and high purity cellulose powders should be used; Sigmacell cellulose, type 20, 20 µm: microgranular is for column chromatography and fibrous (medium) is for general column chromatography)
  • Pyridine
  • Tosyl chloride
  • Acetone
  • 0.1 M bicarbonate buffer, pH 8.5
  • 0.5% (w/v) trypsin
  • 0.85% NaCl in water (saline)
  • 50 mM carbonate buffer, pH 11

Basic Protocol 5: Cyanuric Chloride Activation of Beaded Agarose Gel for Coupling to Macromolecules

  Materials
  • Cyanuric chloride
  • Dioxane
  • Toluene
  • Aminopropyl silica gel, commercially available (Sigma) or prepared in the laboratory (see recipe)
  • Acetone
  • Ligand of interest
  • Temperature‐regulated bath
NOTE: A rotatory motion is preferred to mix the ligand with the matrix since magnetic stirring might damage the matrix and/or the ligand.

Basic Protocol 6: Cyanuric Chloride Activation of Aminopropyl Silica Gel for Coupling to Macromolecules

  Materials
  • Magnetizable Cellulose/Iron Oxide Particles (50:50, particle size ranging from 1 to 10 µm with >75% between 2 to 4 µm; Cortex Biochem)
  • 50 mM phosphate buffer, pH 11.5 ( appendix 2E)
  • Cyanogen bromide (CNBr), solid
  • 2 M NaOH
  • 0.1 M bicarbonate buffer, pH 8.6
  • Ligand of interest (e.g., antiserum or antibody)
  • Ethanolamine
  • 0.1 M sodium acetate buffer, pH 4.0
  • 0.1% sodium azide
  • End‐over‐end mixer (e.g., Sepco or equivalent)
  • 100‐ml beaker
  • Magnetic block

Basic Protocol 7: Cyanogen Bromide Activation of Magnetizable Cellulose/Iron Oxide Particles for Coupling to Macromolecules

  Materials
  • Alkaline buffer (see recipe)
  • Cyanoborohydride stock solution (see recipe)
  • Glyoxal agarose beads (Gentaur or Hispanagar)
  • Ligand, dissolved in water, phosphate‐buffered saline, or coupling buffer (no amine‐containing buffers)
  • Ethanolamine
  • Preservative‐containing buffer
  • Sintered glass filter
  • Filter funnel
CAUTION: Cyanoborohydride contains a cyanide group which can be liberated as a highly toxic gas if acidified. It is therefore imperative that cyanoborohydride solutions, like cyanogen bromide solutions, NEVER be acidified (i.e., pH lowered below pH 7.0). In addition, all coupling reactions should be performed in the hood as an added safety precaution.
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Figures

Videos

Literature Cited

   Afeyan, N.B., Gordon, N.F., Maszaroff, I., Varady, L. Fulton, S.P., Yang, Y.B., and Regnier, F.E. 1990. Flow‐through particles for the high‐performance liquid chromatographic separation of biomolecules: Perfusion chromatography. J. Chromatogr. 519: 1‐29.
   Afeyan, N.B., Fulton, S.P., and Regnier, F.E. 1991. Perfusion chromatography packing materials for protein and peptides. J. Chromatogr. 544: 257‐279.
   Axen, R., Porath, J., and Ernback, S. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214: 1302‐1304.
   Bergold, A. and Scouten, W.H. 1983. Boronate chromatography. In Solid Phase Biochemistry: Analytical and Synthetic Aspects (W.H. Scouten, ed.) pp. 149‐188. John Wiley & Sons, New York.
   Bethell, G.S., Ayers, J.S., Hancock, W.S., and Hearn, M.T.W. 1979. A novel method of activation of cross‐linked agaroses with 1,1′‐carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J. Biol. Chem. 254: 2572‐2574.
   BioProbe International. 1986. U.S. Patent 4,582,875.
   Clonis, Y.D., Atkinson, A., Bruton, C.J., and Lowe, C.R. 1987. Reactive Dyes in Protein and Enzyme Technology. Stockton Press, N.Y.
   Coleman, P.L., Walker, M.M., Milbroth, D.S., Stauffer, D.M., Rasmussen, J.K., Krepski, L.R., and Heilmann, S.M. 1990. Immobilization of protein‐A at high‐density on azolactone—functional polymeric beads and their use in affinity chromatography. J. Chromatogr. 512: 345‐363.
   Cuatrecasas, P. 1970. Protein‐purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245: 3059‐3065.
   Domen, P.L., Nevens, J.R., Mallia, A.F., Hermanson, G.T., and Klenk, D.C. 1990. Site‐directed immobilization of proteins. J. Chromatogr. 510: 293‐302.
   Drobnik, J., Labsky, J., Kudwasarova, H., Saudek, V., and Svec, F. 1982. The activation of hydroxy groups of carriers with 4‐nitrophenyl and N‐hydroxysuccinimide chloroformates. Biotechnol. Bioeng. 24: 487‐493.
   Fieser, L.F. and Fieser, M. 1967. Reagents for Organic Synthesis. John Wiley & Sons, New York.
   Gribnau, T.C.J. 1977. Coupling of effector‐molecules to solid supports. Ph.D. Thesis, University of Nijmegen, Nijmegen, The Netherlands.
   Grubhofer, N. and Schleith, L. 1953. Modifizierte Ionenaustauscher als spezifische Adsorbentien. Naturwissenschaften 40: 508.
   Guisan, J.M. 1988. Aldehyde‐agarose gels as activated supports for immobilization‐stabilization of enzymes. Enz. Microbial. Technol. 10: 375‐382.
   Hearn, M.T.W. 1987. Carbonyldiimidazole‐mediated immobilization of enzymes and affinity ligands. Methods Enzymol. 135: 102‐117.
   Hermanson, G.T., Mallia, A.K., and Smith, P.K. 1992. Immobilized Affinity Ligand Techniques. Academic Press, New York.
   Hornby, W.E. and Goldstein, L. 1976. Immobilization of enzymes on nylon. Methods Enzymol. 44: 118‐134.
   Inman, J.K. and Dintzis, H.M. 1969. The derivatization of cross‐linked polyacrylamide beads. Controlled introduction of functional groups for the preparation of special‐purpose, biochemical adsorbents. Biochemistry 8: 4074‐4082.
   Kohn, J. and Wilchek, M. 1982. Mechanism of activation of Sepharose and Sephadex by cyanogen‐bromide. Enzyme Microb. Technol. 4: 161‐163.
   Kohn, J. and Wilchek, M. 1983a. Para‐nitrophenylcyanate—an efficient, convenient and nonhazardous substitute for cyanogen‐bromide as an activating agent for Sepharose. Appl. Biochem. 8: 277‐285.
   Kohn, J. and Wilchek, M. 1983b. Activation of polysaccharide resins by CNBr. In Solid Phase Biochemistry: Analytical and Synthetic Aspects (W.H. Scouten, ed.) pp. 599‐630. John Wiley & Sons, New York.
   Kohn, J. and Wilchek, M. 1984. The use of cyanogen bromide and other novel cyanylating agents for the activation of polysaccharide resins. Appl. Biochem. Biotechnol. 9: 285‐305.
   Lawson, T.G., Regnier, F.E., and Weith, H.L. 1983. Separation of synthetic oligonucleotides on columns of macro particulate silica coated with crosslinked polyethylene imine. Anal. Biochem. 133: 85‐93.
   Lily, M.D. 1976. Enzymes immobilized to cellulose. Methods Enzymol. 44: 46‐53.
   March, S.C., Parikh, I., and Cuatrecasas, P. 1974. Cyanogen‐bromide activation of agarose for affinity chromatography. Anal. Biochem. 60: 149‐152.
   Miron, T. and Wilchek, M. 1987. Immobilization of protein and ligands using chloroformates. Methods Enzymol. 135: 84‐90.
   Murayama, K., Shimada, K., and Yamamoto, T. 1978. Modification of immunoglobulin‐G using specific reactivity of sugar moiety. J. Immunochem. 15: 523‐528.
   National Research Council, Committee on Hazardous Substances in the Laboratory. 1981. Prudent Practices for Handling Hazardous Chemicals in Laboratories. National Academy Press, Washington, D.C.
   Ngo, T.T. and Lenhoff, H.M. 1980. Immobilization of enzymes through activated peptide bonds of protein supports. J. Appl. Biochem. 2: 373‐379.
   Ngo, T.T., Laidler, K.J., and Yam, C.F. 1979. Kinetics of acetylcholinesterase immobilized on polyethylene tubing. Can. J. Biochem. 57: 1200‐1203.
   Nilsson, K. and Mosbach, K. 1980. p‐Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins. Eur. J. Biochem. 112: 397‐402.
   Nilsson, K. and Mosbach, K. 1981. Immobilization of enzymes and affinity ligands to various hydroxyl group carrying supports using high reactive sulfonyl chlorides. Biochemistry 102: 449‐457.
   Nilsson, K. and Mosbach, K. 1987. Tresyl chloride‐activated supports for enzyme immobilization. Methods Enzymol. 135: 65‐78.
   Parikh, I., March, S., and Cuatrecasas, P. 1974. Topics in the methodology of substitution reactions with agarose. Methods Enzymol. 34: 70‐102.
   Peška, J., Štamberg, J., Hradil, J., and Ilavsky, M. 1976. Cellulose in bead form: Properties related to chromatographic uses. J. Chromatogr. 125: 455‐469.
   Porath, J. 1974. Preparation of cyanogen bromide‐activated agarose gels. Methods Enzymol. 34: 13‐30.
   Porath, J., Aspberg, K., Drevin, H., and Axen, R. 1973. Preparation of cyanogen bromide activated agarose gels. J. Chromatogr. 86: 53‐56.
   Regnier, F.E. 1991. Perfusion chromatography. Nature 350: 634‐635.
   Rozprimova, L., Franek, F., and Kubanek, V. 1978. Utilization of powder polyester in making insoluble antigens and pure antibodies. Czech Epidemiol. Mikrobiol. Immunol. 27: 335‐341.
   Sallee, C.J. and Russell, D.F. 1993. Embedding of neural tissue in agarose or glyoxal agarose for vibratome sectioning. Biochem. Histochem. 68: 360‐368.
   Scouten, W.H. 1981. Affinity Chromatography: Bioselective Absorption on Inert Matrices. Wiley‐Interscience, New York.
   Scouten, W.H. 1983. Solid Phase Biochemistry: Analytical and Synthetic Aspects. John Wiley & Sons, New York.
   Scouten, W.H. 1987. Immobilization Techniques for Enzymes. Methods Enzymol. 135: 30‐65.
   Scouten, W.H., Van den Tweel, W., Kromenburg, H., and Dekker, M. 1987. Colored sulfonyl chloride as an activating agent for hydroxylic matrices. Methods Enzymol. 135: 79‐84.
   Shainoff, J.R. 1980. Zonal immobilization of proteins. Biochem. Biophys. Res. Commun. 95: 690‐695.
   Shainoff, J.R. 1981. Glyoxal agarose. United States Patent 4,275,196
   Shlomo, M. and Sturchak, S. 1996. Bioactive conjugates of cellulose with amino compounds. United States Patent 5516673.
   Stults, N.L., Lin, P., Hardy, M., Lee, Y.G., Uchida, Y., Tsukada, Y., and Sugimori, T. 1983. Immobilization of proteins on partially hydrolyzed agarose beads. Anal. Biochem. 135: 392‐400.
   Sun, S.F., Yang, G.L., Liu, H.Y., Sun, H.W., and Liu, C.F. 2002. A new method to immobilize enzyme and its application to the papain. Chem. J. on Internet 4: 48‐54 (http://www.chemistrymag.org/cji/2002/04a048pe.htm).
   Sundberg, L. and Porath, J. 1974. Preparation of adsorbents for biospecific affinity chromatography: Attachment of group‐containing ligands to insoluble polymers by means of bifunctional oxiranes. J. Chromatogr. 90: 87‐98.
   Turkova, J. 1993. Bioaffinity Chromatography, 2nd ed. Elsevier, Amsterdam.
   Ugelstad, J., Soderberg, L., Berge, A., and Bergstrom, J. 1983. Monodisperse polymer particles: A step forward for chromatography. Nature 303: 95‐96.
   Vogel, A. 1978. Textbook of Practical Organic Chemistry, 4th ed. Longman, New York.
   Voivodov, K., Chan, W.‐H., and Scouten, W.H. 1993. Chemical approaches to oriented immobilization. Makromol. Chem. 70/71: 275‐283.
   Wilchek, M. and Miron, T. 1982. Immobilizations of enzymes and affinity ligands onto agarose via stable and uncharged carbamate linkages. Biochem. Int. 4: 629‐635.
   Yamamoto, K., Yamazaki, A., Takeuchi, M., and Tanaka, A. 2006. A versatile method of identifying specific binding proteins on affinity resins. Anal. Biochem. 352: 15‐23.
   Yang, Y. and Chase, H.A. 1998. Immobilization of enzymes on poly(vinyl alcohol)‐coated perfluorocarbon supports. A comparison of techniques for the immobilisation of trypsin and a‐amylase on solid and liquid poly(vinyl alcohol)‐coated perfluorocarbons. Biotechnol. Appli. Biochem. 27: 295‐216.
Key References
   Hermanson, et al., 1992. See above.
  A very good practical laboratory manual with good description of methods.
   Scouten, W.H. 1981. See above.
  An older, simple approach with a good mix of theory and practical laboratory methods.
   Turkova, J. 1993. See above.
  The most comprehensive volume available at the time of its writing. It remains a key to all of the preceding literature.
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