SpringerProtocols: Abstract: Production of Coronavirus Nonstructural Proteins in Soluble Form for Crystallization

13. Production of Coronavirus Nonstructural Proteins in Soluble Form for Crystallization

By: Yvonne Piotrowski¹, Rajesh Ponnusamy¹, Stephanie Glaser¹, Anniken Daabach¹, Ralf Moll¹, Rolf Hilgenfeld¹

Abstract

Full Text

Download PDF (770K)

For biophysical investigations on viral proteins, in particular for structure determination by X-ray crystallography, relatively large quantities of purified protein are necessary. However, expression of cDNAs coding for viral proteins in prokaryotic or eukaryotic systems is often not straightforward, and frequently the amount and/or the solubility of the protein obtained are not sufficient. Here, we describe a number of protocols for production of nonstructural proteins of coronaviruses that have proven to be efficient in increasing expression yields or solubilities.

Affiliation(s): (1) Center for Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Lübeck, Lübeck, Germany

Book Title: SARS- and Other Coronaviruses: Laboratory Protocols

Series: Methods in Molecular Biology | Volume: 454 | Year: 2008 | Page Range: 1-21 | DOI: 10.1007/978-1-59745-181-9_13

Subject: Infectious Diseases

Key Words: nonstructural proteins - human coronavirus - expression - solubility - crystallization

References

Download References

1.	Groneberg, D. A., Hilgenfeld, R., and Zabel, P. (2005) Molecular mechanisms of severe acute respiratory syndrome (SARS). Respir. Res. 6, 8–23.

2.	van der Hoek, L., Pyrc, K., and Berkhout, B. (2006) Human coronavirus NL63, a new respiratory virus. FEMS Microbiol. Rev. 30, 760–773.

3.	van der Hoek, L., Sure, K., Ihorst, G., Stang, A., Pyrc, K., Jebbink, M. F., Petersen, G., Forster, J., Berkhout, B., and Überla, K. (2005) Croup is associated with the novel coronavirus NL63. PLoS Med. 2, 764–770.

4.	Dominguez, S. R., Anderson, M. S., Glodé, M. P., Robinson, C. C., and Holmes, K. V. (2006) Blinded case-control study of the relationship between human coronavirus NL63 and Kawasaki syndrome. J. Infect. Dis. 194, 1697–1701.

5.	Stadler, K., Masignani, V., Eickmann, M., Becker, S., Abrignani, S., Klenk, H. D., and Rappuoli, R. (2003) SARS—Beginning to understand a new virus. Nature Rev. Microbiol. 1, 209–218.

6.	Lindwall, G., Chau, M.-F., Gardner, S. R., and Kohlstaedt, L. A. (2000) A sparse matrix approach to the solubilization of overexpressed proteins. Protein Eng. 13, 67–71.

7.	Prilusky, J., Felder, C. E., Zeev-Ben-Mordehai, T., Rydberg, E. H., Man, O., Beckmann, J. S., Silmann, I., and Sussman, J. L. (2005) FoldIndex^©: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21, 3435–3438.

8.	Zhai, Y., Sun, F., Li, X., Pang, H., Xu, X., Bartlam, M., and Rao, Z. (2005) Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer, Nature Struct. Mol. Biol. 12, 980–986.

9.	Smyth, D. R., Mrozkiewicz, M. K., McGrath, W. J., Listwan, P., and Kobe, B. (2003) Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 12, 1313–1322.

10.	Hogg, T., and Hilgenfeld, R. (2007) Protein crystallography in drug design. In: Tayler, J. B., and Triggle, D. J. (eds.) Comprehensive Medicinal Chemistry II. Elsevier, Amsterdam, pp. 875–900.

11.	McPherson, A. (1999) Strategies and special approaches in growing crystals. In Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, New York, pp. 271–329.

12.	Jancarik, J., and Kim, S.-H. (1991) Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Cryst. 24, 409–411.

13.	Mesters, J. R. (2007) Practical protein crystallization. In: Drenth, J. (ed.) Principles of Protein X-Ray Crystallography 3rd Ed.. Springer, New York, pp. 297–304.

Comments (Loading...)

Post comment/View all comments