Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

doi:10.1107/S1744309107017113

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 May 1; 63(Pt 5): 399–402.

Published online 2007 April 14. doi: 10.1107/S1744309107017113

PMCID: PMC2335007

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

Michael A. Joyce,^a^‡ Edward R. Brownie,^b Koto Hayakawa,^b and Marie E. Fraser^b^*

^aDepartment of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

^bDepartment of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada

Correspondence e-mail: frasm/at/ucalgary.ca

^‡Current address: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Received January 31, 2007; Accepted April 5, 2007.

This article has been cited by other articles in PMC.

Abstract

Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the β-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 Å resolution, are of the protein in complex with Mn²⁺-GDP.

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