Expression, purification and crystallisation of phosphorylase kinase catalytic domain.
The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.
Owen
D J
DJ
Laboratory of Molecular Biophysics, University of Oxford, U.K.
Papageorgiou
A C
AC
Garman
E F
EF
Noble
M E
ME
Johnson
L N
LN
D000818:Animals; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
D010764:Phosphorylase Kinase