Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus.
Capsular polysaccharides (CPs) are important virulence factors of Staphylococcus aureus. The biosynthesis of type 5 and type 8 CPs (CP5 and CP8), which are produced by most clinical isolates of S. aureus, is catalyzed by 16 CP-assembling proteins. One of these proteins is the enzyme CapF, which catalyzes the synthesis of UDP-N-acetyl-L-fucosamine, a component of both CP5 and CP8. Here, the cloning, expression, purification, crystallization and diffraction analysis of CapF are reported. Optimization of the crystallization conditions by differential scanning calorimetry afforded a crystal of selenomethionine-substituted CapF that diffracted to a resolution of 2.80 A. The crystal belongs to space group P3(2)21, with unit-cell parameters a = b = 119.6, c = 129.5 A.
Miyafusa
Takamitsu
T
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 277-8562, Japan.
Tanaka
Yoshikazu
Y
Kuroda
Makoto
M
Ohta
Toshiko
T
Tsumoto
Kouhei
K
D017738:Alkanesulfonic Acids / Q000737:chemistry; D016880:Anisotropy; D016667:Bacterial Capsules / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D003460:Crystallization; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009025:Morpholines / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology*; D014961:X-Ray Diffraction
D017738:Alkanesulfonic Acids; D002021:Buffers; D009025:Morpholines; C004550:2-(N-morpholino)ethanesulfonic acid