Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus.
The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow single well defined crystals in the microbatch system which belonged to the space group C2 and diffracted isotropically to approximately 2 A resolution.
Dale
G E
GE
F. Hoffmann-La Roche Ltd. Pharma Preclinical Research, CH-4070, Basel, Switzerland. glenn.dale@roche.com
Kostrewa
D
D
Gsell
B
B
Stieger
M
M
D'Arcy
A
A
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D016296:Mutagenesis; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology*
D001426:Bacterial Proteins; D010446:Peptide Fragments; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II