Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase.
Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture. Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A. A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution. X-ray data to 2.05 A for the structure of L. mexicana iPGAM complexed with 2-phosphoglycerate have also been collected.
Poonperm
Buabarn
B
Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland.
Guerra
Daniel G
DG
McNae
Iain W
IW
Fothergill-Gilmore
Linda A
LA
Walkinshaw
Malcolm D
MD
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D005988:Glyceric Acids / Q000737:chemistry; D006639:Histidine; D007894:Leishmania mexicana / Q000201:enzymology*; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment
D005988:Glyceric Acids; D011993:Recombinant Fusion Proteins; D006639:Histidine; C005156:3-phosphoglycerate; D010736:Phosphoglycerate Mutase