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

International Tables for Crystallography (2012). Vol. F, ch. 4.3, p. 129   | 1 | 2 |

Section 4.3.1. Introduction

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail: zsd4n@virginia.edu

4.3.1. Introduction

| top | pdf |

Following the dawn of recombinant technology brought about by the groundbreaking overexpression of synthetic genes coding for insulin and somatostatin in Escherichia coli (Goeddel et al., 1979[link]; Itakura et al., 1977[link]) and the subsequent discovery of the polymerase chain reaction (PCR; Mullis et al., 1986[link]; Saiki et al., 1985[link], 1988[link]), macromolecular crystallography was freed of its longstanding dependence on purified native protein samples for cryst­allization. Heterologous expression made it possible to generate samples of proteins and com­plexes that are found in only small or trace amounts in living cells and to engineer large and unstable proteins so that isolated domains or modified forms can be made available for crystallization. At the same time, the effort required for protein purification was dramatically reduced by the use of fusion proteins and affinity tags (Brewer et al., 1991[link]; Sassenfeld, 1990[link]; Malhotra, 2009[link]). As a consequence, the overwhelming majority of samples used today for crystallization are recombinantly derived proteins. However, even though material for crystallization is more easily available, the prep­aration of single, well diffracting crystals of the target macromolecule is still a time-consuming challenge.

Historically, two complementary approaches to protein crystallization were developed in parallel. Firstly, natural variations in the amino-acid sequences of homologues from different species were exploited to identify a target with suitable crystallization properties during the purification procedure (Kendrew et al., 1954[link]; Campbell et al., 1972[link]). The second approach was to extensively screen a specific target protein against a range of diverse precipitating agents, buffers and additives until the right conditions for crystallization were identified (Carter & Carter, 1979[link]; Jancarik & Kim, 1991[link]). These strategies remain the pillars of contemporary macromolecular crystallization. However, as the palette of molecular biology techniques expanded to include site-directed mutagenesis, ligation-independent cloning and other tools, it became possible to modify proteins with relative ease with the specific purpose of enhancing their propensity to crystallize or improving the diffraction quality of the resulting crystals. The early proof-of-principle of these capabilities was the crystallization of an engineered variant of human H-ferritin in which a single-site mutation, K86Q, was introduced to duplicate a crystal contact mediated by Cd2+ ions in the crystal structure of the homologous rat L-ferritin (Lawson et al., 1991[link]).

In this review, current progress in the methodologies of protein engineering used to enhance the crystallizability of targets that are recalcitrant to crystallization in their wild-type form is discussed. This burgeoning field is very broad and includes both general strategies that apply to a range of targets and many diverse approaches that only apply to specific proteins or protein families. Thus, owing to space limitations, the focus is on those techniques that have either been demonstrated to be of general utility or are at a point in their development to clearly have the potential to become widely used in the future. Understandably, only representative examples are provided.

References

Brewer, S. J., Haymore, B. L., Hopp, T. P. & Sassenfeld, H. M. (1991). Engineering proteins to enable their isolation in a biologically active form. Bioprocess Technol. 12, 239–266.
Campbell, J. W., Duée, E., Hodgson, G., Mercer, W. D., Stammers, D. K., Wendell, P. L., Muirhead, H. & Watson, H. C. (1972). X-ray diffraction studies on enzymes in the glycolytic pathway. Cold Spring Harb. Symp. Quant. Biol. 36, 165–170.
Carter, C. W. Jr & Carter, C. W. (1979). Protein crystallization using incomplete factorial experiments. J. Biol. Chem. 254, 12219–12223.
Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K. & Riggs, A. D. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl Acad. Sci. USA, 76, 106–110.
Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heyneker, H. L., Bolivar, F. & Boyer, H. W. (1977). Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science, 198, 1056–1063.
Jancarik, J. & Kim, S.-H. (1991). Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Cryst. 24, 409–411.
Kendrew, J. C., Parrish, R. G., Marrack, J. R. & Orlans, E. S. (1954). The species specificity of myoglobin. Nature (London), 174, 946–949.
Lawson, D. M., Artymiuk, P. J., Yewdall, S. J., Smith, J. M., Livingstone, J. C., Treffry, A., Luzzago, A., Levi, S., Arosio, P., Cesareni, G., Thomas, C. D., Shaw, W. V. & Harrison, P. M. (1991). Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts. Nature (London), 349, 541–544.
Malhotra, A. (2009). Tagging for protein expression. Methods Enzymol. 463, 239–258.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. & Erlich, H. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51, 263–273.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487–491.
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230, 1350–1354.
Sassenfeld, H. M. (1990). Engineering proteins for purification. Trends Biotechnol. 8, 88–93.








































to end of page
to top of page