Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae.
Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI-MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 A. A complete diffraction data set was collected to 2.14 A resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.
Songsiriritthigul
Chomphunuch
C
School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Yuvaniyama
Jirundon
J
Robinson
Robert C
RC
Vongsuwan
Archara
A
Prinz
Heino
H
Suginta
Wipa
W
D020816:Amino Acid Motifs; D002122:Calcium Chloride / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D014733:Vibrio / Q000201:enzymology*; D014961:X-Ray Diffraction; D014965:X-Rays
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D006160:Guanosine Triphosphate; D002688:Chitinases; D020558:GTP Phosphohydrolases; D002122:Calcium Chloride