Open AccessCloning, expression, purification, crystallization and preliminary X‐ray analysis of Thermus aquaticus succinyl‐CoA synthetase
Author(s) -
Joyce Michael A.,
Brownie Edward R.,
Hayakawa Koto,
Fraser Marie E.
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309107017113
Subject(s) - thermus aquaticus , thermus , crystallization , proteolysis , protease , enzyme , thermophile , cloning (programming) , protein subunit , biochemistry , chemistry , biology , microbiology and biotechnology , gene , organic chemistry , computer science , programming language
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 Mn 2+ ‐GDP.