Chapter 3.1 Preparing recombinant proteins for X‐ray crystallography

Crystallography of biological macromolecules

Second Online Edition (2012)

Part 3. Techniques of molecular biology

  1. S. H. Hughes1,
  2. A. M. Stock2

Published Online: 14 APR 2012

DOI: 10.1107/97809553602060000810

International Tables for Crystallography

International Tables for Crystallography

How to Cite

Hughes, S. H. and Stock, A. M. 2012. Preparing recombinant proteins for X‐ray crystallography. International Tables for Crystallography. F:3:3.1:75–91.

Author Information

  1. 1

    National Cancer Institute, Frederick Cancer R&D Center, Frederick, MD 21702–1201, USA

  2. 2

    Center for Advanced Biotechnology and Medicine, Howard Hughes Medical Institute and University of Medicine and Dentistry of New Jersey – Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854–5627, USA

Publication History

  1. Published Online: 14 APR 2012



It is possible to use recombinant DNA techniques to produce most proteins in quantities sufficient for crystallography. This chapter discusses issues (and problems) relevant to choosing methods appropriate for preparing recombinant proteins for X‐ray crystallography. The aim is to help readers understand both the extant problems and the available solutions, so that, armed with a general understanding of the issues, they can more easily confront a variety of specific projects. Specific topics covered include protein engineering an expression construct, expression systems, protein purification and the characterization of the purified product.


  • E. coli expression systems;
  • affinity chromatography;
  • baculovirus expression systems;
  • chromatography;
  • expression systems;
  • insect cell–virus expression systems;
  • mammalian‐cell expression systems;
  • protein expression;
  • protein folding;
  • protein purification;
  • protein refolding;
  • storage of proteins;
  • tags;
  • yeasts as expression systems