Expression, purification and crystallization of an extended-spectrum beta-lactamase from Klebsiella oxytoca.
OXY-1a is an extended-spectrum beta-lactamase from the conditional pathogenic bacterium Klebsiella oxytoca. OXY-1a is responsible for the antibiotic resistance of this pathogen. A soluble form of OXY-1a with a His tag at its C-terminus was overexpressed in Escherichia coli. The recombinant protein was purified and crystallized at room temperature using PEG 4000 as the main precipitant. Two crystal forms were obtained from the same growth conditions. One was orthorhombic, with crystals that diffracted to better than 1.9 A, while the other was tetragonal, with crystals that only diffracted to about 3.0 A. Complete data sets were collected from both crystal forms. The orthorhombic crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.54, b = 73.43, c = 84.56 A, while the tetragonal crystal has unit-cell parameters a = b = 73.72, c = 96.81 A. The asymmetric unit of the orthorhombic crystal is estimated to contain one OXY-1a molecule, giving a crystal volume per protein weight (V(M)) of 2.25 A(3) Da(-1) and a solvent content of 45%.
Wu
Shang Wei
SW
Laboratory of Microbiology, The Rockefeller University, New York, New York 10021-6399, USA.
Liang
Yu-He
YH
Su
Xiao-Dong
XD
D018360:Crystallography, X-Ray; D041121:Klebsiella oxytoca / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D001618:beta-Lactamases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D012997:Solvents; D011092:Polyethylene Glycols; D001618:beta-Lactamases