International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 3.1, p. 65   | 1 | 2 |

Section 3.1.1. Introduction

S. H. Hughesa and A. M. Stockb*

aNational Cancer Institute, Frederick Cancer R&D Center, Frederick, MD 21702-1201, USA, and bCenter 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
Correspondence e-mail:  stock@cabm.rutgers.edu

3.1.1. Introduction

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Preparing protein crystals appropriate for X-ray diffraction usually requires a considerable amount of highly purified protein. When crystallographic methods were first developed, the practitioners of the art were compelled to study proteins that could be easily obtained in large quantities in relatively pure form; the first proteins whose structures were solved by crystallographic methods were myoglobin and haemoglobin. Unfortunately, some of the most interesting proteins are normally present in relatively small amounts, which, while it did not prevent crystallographers from dreaming about their structures, prevented any serious attempts at crystallization. Recombinant DNA techniques changed the rules: it is now possible to instruct a variety of cells and organisms to make large amounts of almost any protein chosen by the investigator. Not only can specific proteins be expressed in large quantities, recombinant proteins can be modified in ways that make the task of the crystallographer simpler and can, in some cases, dramatically improve the quality of the resulting crystals. It is not our intention in writing this chapter to provide either a methods manual for those interested in expressing a particular protein or a complete compendium of the available literature. The literature is vast and complex, and, as we will discuss, the problems associated with expressing a particular protein are often idiosyncratic, making it difficult to provide a simple, comprehensive, methodological guide. What we intend is to discuss issues (and problems) relevant to choosing methods appropriate for preparing recombinant proteins for X-ray crystallography. In this way, we hope 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.

Fortunately, there are a large number of additional resources available to those who are interested in expressing and purifying recombinant proteins, but lack the expertise. These include numerous methods books (e.g. on molecular biology: Sambrook et al., 1989[link]; Ausubel et al., 1995[link]; on protein purification: Abelson & Simon, 1990[link]; Scopes, 1994[link]; Bollag et al., 1996[link]), useful reviews of the literature (cited throughout), formal courses (such as those offered by Cold Spring Harbor Laboratory), meetings (i.e. IBC's International Conference on Expression Technologies, Washington DC, 1997) and a specialized journal (Protein Expression and Purification). The pace of methodological development is rapid, and company catalogues, publications and web pages can provide extensive, useful, up-to-date information. In many cases, a convenient source of information is a nearby researcher whose own research depends on expressing and purifying recombinant proteins. Those who are serious about preparing recombinant proteins for crystallography, but have little or no experience, are strongly urged to avail themselves of these resources. In many cases the help of a knowledgeable colleague is the most valuable resource. In general, the literature provides a much better guide to what will work than what will fail; quite often, in designing a good strategy to produce a recombinant protein that is suitable for crystallography, it is more important to understand the potential pitfalls. Discussion with an experienced colleague is usually the best way to avoid the most obvious errors.

Section 3.1.2[link] gives an overview of the problem, Section 3.1.3[link] discusses engineering an expression construct, Section 3.1.4[link] discusses expression systems, Section 3.1.5[link] discusses protein purification and Section 3.1.6[link] discusses the characterization of the purified product.

References

Abelson, J. N. & Simon, M. I. (1990). Guide to protein purification. Methods Enzymol. 182, 1–894.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1995). Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology, 3rd ed. New York: Greene Publishing Associates and Wiley.
Bollag, D. M., Rozycki, M. D. & Edelstein, S. J. (1996). Protein methods. New York: Wiley-Liss.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: A laboratory manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press.
Scopes, R. K. (1994). Protein purification: principles and practice. New York: Springer-Verlag.








































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