Tables for
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 3.1.2. Overview

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:

3.1.2. Overview

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The idea that underlies the problem of expressing large amounts of a recombinant protein is straightforward: prepare a DNA segment that, when introduced into an appropriate host, will cause the abundant expression of the relevant protein. However, as the saying goes, `The devil is in the details.' Not only is it necessary to design the appropriate DNA segment, but also to introduce it into an appropriate host such that the host retains and faithfully replicates the DNA. The DNA segment must contain all of the elements necessary for high-level RNA expression; moreover, the RNA, when expressed, must be recognized by the translational machinery of the host. The recombinant protein, once expressed, needs to be properly folded either by the host or, if not properly folded in the host, by the experimentalist. If the protein is subject to post-translational modifications (cleavage, glycosylation, phosphorylation etc.) and the experimentalist wishes to retain these modifications, the appropriate signals must be present and the chosen host must also be capable of recognizing the signals. Once the recombinant protein is expressed, assuming it is reasonably stable in the chosen host, the protein must be purified; as we will discuss, recombinant proteins can be modified to simplify purification. Once purified, the quality of the protein preparation must be evaluated to ensure it is both relatively homogeneous and monodisperse.

While this chapter will be limited to discussions of the basic strategies for creating an expression vector, expressing the protein and purifying and characterizing the product, molecular biological methods can be used in other ways that are relevant to crystallography. In some cases, a protein in its natural form is not suitable for crystallization. Crystallographers have long used proteolytic digestion and/or glycolytic digestion to produce proteins suitable for crystallization from ones that are not. Such techniques have been used to good effect on recombinant proteins; however, the ability to modify the segment encoding the protein makes it possible to alter the protein in a variety of ways beyond simple enzymatic digestions. Specific examples of such applications are described in Chapter 4.3[link] .

Unfortunately, no single strategy for producing proteins for crystallization appears to be universally successful. Any particular protocol has the potential for displaying undesirable behaviour at any step during the process of expression, purification or crystallization. It is important to distinguish major and minor problems. If the problems are serious, it is often better to try an alternative strategy than to struggle with an inappropriate system. Because it is usually difficult to predict what will work and what will not, often the most expedient route to successful expression of a protein for crystallization is the simultaneous pursuit of several expression strategies with multiple protein expression constructs.

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