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. 4.3, p. 101   | 1 | 2 |

Section 4.3.3. Use of fusion proteins

D. R. Daviesa* and A. Burgess Hickmana

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA
Correspondence e-mail:  david.davies@nih.gov

4.3.3. Use of fusion proteins

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Fusion proteins have been frequently used in a variety of applications (reviewed by Nilsson et al., 1992[link]), such as preventing proteolysis, changing solubility and increasing stability. They have also been used – although less frequently – for crystallization. The disadvantage in the context of crystallography is that the length and flexibility of the linker chain often introduce mobility of one protein domain relative to the other, which can impede, rather than enhance, crystallization.

Donahue et al. (1994[link]) were able to determine the three-dimensional structure of the 14 residues representing the platelet integrin recognition segment of the fibrinogen γ chain by constructing a fusion protein with lysozyme, which was then crystallized from ammonium sulfate. Kuge et al. (1997[link]) successfully obtained crystals of a fusion protein consisting of glutathione S-transferase (GST) and the DNA-binding domain (residues 16–115) of the DNA replication-related element-binding factor, DREF, under crystallization conditions similar to those used for GST alone.

In many cases, a fusion protein is made to aid in the isolation and purification of the target protein, and the intervening linker is engineered to contain a proteolytically susceptible sequence. However, subsequent cleavage to separate the two proteins can introduce the possibility of accidental proteolysis elsewhere in the protein. This was observed with a fusion protein between thioredoxin and VanH, a D-lactate dehydrogenase, where attempts to remove the carrier resulted in non-specific proteolysis and VanH inactivation (Stoll et al., 1998[link]). Fortunately, cleavage was unnecessary, and conditions were identified under which the authors were able to crystallize the intact fusion protein.

A novel approach to crystallizing membrane proteins is provided by the fusion protein in which cytochrome [b_{562}] was inserted into a central cytoplasmic loop of the lactose permease from Escherichia coli (Privé et al., 1994[link]). Although crystals have not yet been reported, the cytochrome attachment provides increased solubility together with the ability to use the red colour to assay the progress of crystallization trials.

References

Donahue, J. P., Patel, H., Anderson, W. F. & Hawiger, J. (1994). Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen γ chain obtained by carrier protein-driven crystallization. Proc. Natl Acad. Sci. USA, 91, 12178–12182.
Kuge, M., Fujii, Y., Shimizu, T., Hirose, F., Matsukage, A. & Hakoshima, T. (1997). Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF. Protein Sci. 6, 1783–1786.
Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M. & Uhlén, M. (1992). Fusion proteins in biotechnology and structural biology. Curr. Opin. Struct. Biol. 2, 569–575.
Privé, G. G., Verner, G. E., Weitzman, C., Zen, K. H., Eisenberg, D. & Kaback, H. R. (1994). Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli. Acta Cryst. D50, 375–379.
Stoll, V. S., Manohar, A. V., Gillon, W., Macfarlane, E. L. A., Hynes, R. C. & Pai, E. F. (1998). A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization. Protein Sci. 7, 1147–1155.








































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