Open AccessExpression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae
Author(s) -
Songsiriritthigul Chomphunuch,
Yuvaniyama Jirundon,
Robinson Robert C.,
Vongsuwan Archara,
Prinz Heino,
Suginta Wipa
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309105027831
Subject(s) - chitinase , crystallization , crystallography , expression (computer science) , vibrio , materials science , microbiology and biotechnology , chemistry , biology , biochemistry , bacteria , enzyme , genetics , computer science , organic chemistry , programming language
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 CaCl 2 . The crystals belonged to the tetragonal space group P 422, with two molecules per asymmetric unit and unit‐cell parameters a = b = 127.64, c = 171.42 Å. A complete diffraction data set was collected to 2.14 Å resolution using a Rigaku/MSC R‐AXIS IV ++ detector system mounted on an RU‐H3R rotating‐anode X‐ray generator.