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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Abstract

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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Materials

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Literature Cited

Literature Cited
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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.
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   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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