Overview of the Characterization of Recombinant Proteins

Nancy D. Denslow1, Paul T. Wingfield2, Keith Rose3

1 University of Florida, Gainesville, 2 National Institutes of Health, Bethesda, 3 University of Medical Centre (CMU), Geneva
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.1
DOI:  10.1002/0471140864.ps0701s00
Online Posting Date:  May, 2001
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This overview provides guidelines for the characterization of recombinantly expressed proteins (e.g., verifying primary structure and appropriate post‐translational modifications), along with methodologies for characterizing the proteins according to size, X‐ray structure, absorbance, biological activity, and subunit structure. A flow chart presents a suggested path for fully characterizing recombinant protein and involves equipment for HPLC, mass spectrometry, circular dichroism, NMR and fluorescence spectroscopy. Also covered are sources and consequences of contamination in protein solutions.

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Table of Contents

  • Common Sources of Contamination
  • Chemical Identity
  • Physical and Conformational Characterization
  • Basic Plan of Action
  • Analysis of N‐Terminal Heterogeneity of Recombinant Proteins Produced in E. Coli
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Ackers, G.K. 1970. Analytical gel chromatography of proteins. Adv. Protein Chem. 24:342‐443.
   Allet, B., Payton, M., Mattaliano, R.J., Gronenborn, A.M., Clore, G.M., and Wingfield, P.T. 1988. Purification and characterization of the DNA‐binding protein ner of bacteriophage mu. Gene 65:259‐268.
   Ballery, N., Desmadril, M., Minard, P., and Yon, J.M. 1993. Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: Chemical reactivity of genetically introduced cysteinyl residues during the folding process. Biochemistry 32:708‐714.
   Bradshaw, R.A. and Stewart, A.E. 1994. Analysis of proteins modifications: Recent advances in detection, characterization and mapping. Measurement as a tool for studying proteins. Curr. Opin. Biotechnol. 5:85‐93.
   Bristow, A.F. 1989. Purification of proteins for therapeutic use. In Protein Purification Applications: A Practical Approach (E.L.V. Harris and S. Angal, eds.) pp. 29‐44. IRL Press, Oxford.
   Bussian, B.M. and Sander, C. 1989. How to determine protein secondary structure in solution by Raman spectroscopy: Practical guide and test case DNase I. Biochemistry 28:4271‐4277.
   Carr, S.A., Hemling, M.E., Bean, M.F., and Roberts, G.D. 1991. Integration of mass spectrometry in analytical biotechnology. Anal. Chem. 63:2802‐2824.
   Chaiken, I., Rose, S., and Karlsson, R. 1992. Analysis of macromolecular interactions using immobilized ligands. Anal. Biochem. 201:197‐210.
   Chen, R.F., Endelhoch, H., and Steiner, R.F. 1969. Fluorescence of proteins. In Physical Principles and Techniques of Protein Chemistry (S.J. Leach, ed.) pp. 171‐244. Academic Press, New York.
   Clore, G.M. and Gronenborn, A.M. 1992. Methods of structural analysis of proteins. Part 2—Nuclear magnetic resonance. In Protein Engineering: A Practical Approach (A.R. Rees, M.J.E. Sternberg, and R. Wetzel, eds.) pp. 33‐56. IRL Press, Oxford.
   Clore, G.M. and Gronenborn, A.M. 1994. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239:349‐363.
   Cooper, A. and Johnson, C.M. 1994. Microcalorimetry; differential scanning calorimetry; and isothermal titration calorimetry. Methods Mol. Biol. 22:109‐150.
   Cunningham, B.C. and Wells, J.A. 1993. Comparison of a structural and functional epitope. J. Mol. Biol. 234:554‐563.
   Donovan, J.W. 1969. Ultraviolet absorption. In Physical Principals and Techniques of Protein Chemistry, Part A (S.J. Leach, ed.) pp. 101‐170. Academic Press, New York.
   Donovan, J.W. 1973. Ultraviolet difference spectroscopy—new techniques and applications. Methods Enzymol. 27:497‐548.
  Ducruix, A. and Giege, R. (eds.). 1992. Crystallization of Nucleic Acids and Proteins: A Practical Approach. IRL Press, Oxford.
   Eftink, M.R. and Ghiron, C.A. 1981. Fluorescence quenching studies with proteins. Anal. Biochem. 114:199‐227.
   Englander, S.W. and Kallenbach, N.R. 1984. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 16:521‐655.
  FDA (Food and Drug Administration). FDA Handbook. Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology. Office of Biologics Research and Review, Center for Drugs and Biologics, Food and Drug Administration, Bethesda, Md.
   Fersht, A. 1985. Enzymes: Structure and Mechanism, 2nd Edition. Freeman, New York.
   Figuet, B., Djavadi‐Ohaniance, L., and Goldberg, M.E. 1989. Immunological analysis of protein conformation. In Protein Structure: A Practical Approach (T.E. Creighton, ed.) pp. 287‐310. IRL Press, Oxford.
   Garnick, R.L., Rose, M.J., and Baffi, R.A. 1991. Characterization of proteins from recombinant DNA manufacture. In Drug Biotechnology Regulation: Scientific Basis and Practices. (Y.‐Y. H. Chiu and J.L. Gueriguian, eds.) pp. 263‐313. Marcel Decker, New York.
   Geisow, M.J. 1991. Characterizing recombinant proteins. Bio/Technology 9:921‐924.
   Goldberg, M.E. 1991. Investigating protein conformation, dynamics and folding with monoclonal antibodies. Trends Biochem. Sci. 16:358‐362.
   Goldenberg, D.P. 1989. Analysis of protein conformation by gel electrophoresis. In Protein Structure: A Practical Approach (T.E. Creighton, ed.) pp. 225‐250. IRL Press, Oxford.
   Hampton Research Macromolecular Crystallization Reagent Kits: Crystal Screen I and II. Hampton Research, Calif.
   Harding, S.E. 1994. Sedimentation velocity; sedimentation equilibrium. Methods Mol. Biol. 22:61‐83.
   Harding, S.E. 1994. Classical light scattering and dynamic light scattering. Methods Mol. Biol. 22:61‐83.
   Haris, P.I. and Chapman, D. 1992. Does Fourier‐transform infrared spectroscopy provide useful information on protein structures? Trends Biochem. Sci. 17:328‐333.
   Haris, P.I. and Chapman, D. 1994. Analysis of polypeptide and protein structures using Fourier‐transform infrared spectroscopy. Methods Mol. Biol. 22:183‐202.
   Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., and Kaminogawa, S. 1993. Unfolding/refolding studies on bovine β‐lactoglobulin with monoclonal antibodies as probes. J. Biol. Chem. 268:22414‐22419.
   Haugland, R.P. 1994. Handbook of Fluorescent Probes and Research Chemicals, 5th Ed. Molecular Probes, Eugene, Oreg.
   Johnson, W.C. 1990. Protein secondary structure and circular dichroism: A practical guide. Proteins Struct. Funct. Genet. 7:205‐214.
   LeGendre, N., Mansfield, M. Weiss, A., and Matsudaira, P. 1993. Purification of proteins and peptides by SDS‐PAGE. In A Practical Guide to Protein and Peptide Purification for Microsequencing (P. Matsudaira, ed.) pp. 74‐101. Academic Press, San Diego.
   McRee, D.E. 1993. Practical Protein Crystallography. Academic Press, San Diego.
   McRorie, D.K. and Voelker, P.J. 1993. Self‐associating systems in the analytical ultracentrifuge. Vol. II, Analytical Ultracentrifugation Series. Beckman Instruments, Fullerton, Calif.
   Mercier, J.‐C., Grosclaude, F., and Ribadeau‐Dumas, B. 1971. Primary structure of bovine S1 casein. Complete sequence. Eur. J. Biochem. 23:41‐51.
   Mulkerrin, M.G. and Cunningham, B.C. 1993. Spectral analysis of site‐directed mutants of human growth hormone. In Protein Folding: In Vivo and In Vitro (J.L. Cleland, ed.) pp. 240‐253. ACS Symposium Series 526. American Chemical Society, Washington, DC.
   Pappin, D.J.C., Hojrup, P., and Bleasby, A.J. 1993. Rapid identification of proteins by peptide‐mass fingerprinting. Curr. Biol. 3:327‐333.
   Pennica, D., Kohr, W.J., Fendly, B.M., Shire, S.J., Raab, H.E., Borchardt, P.E., Lewis, M., and Goeddel, D.V. 1992. Characterization of a recombinant extracellular domain of the type I tumor necrosis factor receptor. Biochemistry 31:1134‐1141.
   Phillies, G.D. 1990. Quasielastic light scattering. Anal. Chem. 62:1049‐1057.
   Price, N.A. and Johnson, C.M. 1989. Proteinases as probes of conformation of soluble proteins. In Proteolytic Enzymes: A Practical Approach (R.J. Beynon and J.S. Bond, eds.) pp. 163‐179. IRL Press, Oxford.
   Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E., and Razgulyaev, O.I. 1990. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262:20‐24.
   Ralston, G. 1993. Introduction to analytical ultracentrifugation. Vol. 1, Analytical Ultracentrifugation Series. Beckman Instruments, Fullerton Calif.
   Rose, K., Anderegg, R., Wells, T.N., and Proudfoot, A.E. 1992a. Human interleukin‐5 expressed in E. coli has N‐terminal modifications. Biochem. J. 286:825‐828.
   Rose, K., Simona, M. G., Savoy, L.‐A., Green, B. M., Gronenborn, A.M., Clore, G.M., and Wingfield, P.T. 1992b. Pyruvic acid is attached to the amino terminus of the recombinant‐DNA derived DNA‐binding protein ner of bacteriophage mu. J. Biol Chem. 267:19101‐19106.
   Scoble, H.A. and Martin, S.A. 1990. Characterization of recombinant proteins. Methods Enzymol. 193:519‐536.
   Shire, S.J. 1992. Determination of the molecular weight of glycoproteins by analytical ultracentrifugation. Beckman Technical Information, Publication DS‐837. Spinco Business Unit, Palo Alto, Calif.
   Strickland, E.H. 1974. Aromatic contributions to circular dichroism spectra of proteins. Crit. Rev. Biochem. 2:113‐175.
   Sturtevant, J.M. 1987. Biochemical applications of differential scanning calorimetry. Ann. Rev. Phys. Chem. 38:463‐488.
   Surewicz, W.K., Mantsch, H.H., and Chapman, D. 1993. Determination of protein secondary structure by Fourier transform infrared spectroscopy: A critical assessment. Biochemistry 32:389‐393.
   Thomas, D., Schultz, P., Steven, A.C., and Wall, J.S. 1994. Mass analysis of biological macromolecular complexes by STEM. Biol. Cell 80:181‐192.
   Van Holde, K.E. 1975. Sedimentation analysis of proteins. In The Proteins, 3rd Edition, Vol. 1 (H. Neurath, ed.) pp. 225‐291. Academic Press, N.Y.
   Varley, P.G. 1994. Fluorescence spectroscopy. Methods Mol. Biol. 22:203‐218.
   Walsh, G. and Headon, D.R. 1994. Therapeutic proteins: Special considerations. In Protein Biotechnology. pp. 119‐162. John Wiley & Sons, Chichester, U.K.
   Wang, R. and Chait, B.T. 1994. High‐accuracy mass measurement as a tool for studying proteins. Curr. Opin. Biotechnol. 5:77‐84.
   Wang, R., Chait, B.T., and Kent, S.B.H. 1994. Protein ladder sequencing towards automation in techniques. In Protein Chemistry V (J.W. Crabb, ed.) pp. 19‐26. Academic Press, New York.
   Wetlaufer, D.B. 1962. Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem. 17:303‐390.
   Wettenhall, R.E. and Cohen, P. 1982. Isolation and characterization of cyclic AMP–dependent phosphorylation sites from rat liver ribosomal protein S6. FEBS Lett. 140:263‐269.
   Williams, R.W. 1986. Protein secondary structure analysis using Raman amide I and III spectra. Methods Enzymol. 130:311‐331.
   Wood, S.P. 1989. Purification for crystallography. In Protein Purification Applications. A Practical Approach (E.L.V. Harris and S. Angal, eds.) pp. 45‐58. IRL Press, Oxford.
   Yang, J.T., Wu, C.‐S., and Martinez, H. 1986. Calculation of protein conformation from circular dichroism. Methods Enzymol. 130:208‐297.
Key References
   Cantor, C.R. and Schimmel, P.R. 1980. Biophysical Chemistry. Part II: Techniques for the Study of Biological Structure and Function. Freeman, San Francisco.
   Fairly advanced text that includes descriptions of the spectroscopic and ultracentrifugation methods.
   Freifelder, D. 1982. Physical Biochemistry: Applications to Biochemistry and Molecular Biology. Freeman, N.Y.
   Excellent introductory text with especially strong sections on spectroscopy and centrifugation. Read this book first.
   Kyte, J. 1995. Structure in Protein Chemistry. Garland Publishing, N.Y.
   Structural biology emphasis, with a clear and concise explanation of X‐ray diffraction analysis. Excellent illustrations.
   Campbell, I.D. and Dwek, R.A. 1984. Biological Spectroscopy. Benjamin/Cummings Company, Menlo Park, Calif.
   Very good explanations of the theoretical principles, along with many practical applications.
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