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Updated: Aug 11, 2026
Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
Published on: March 20, 2021
Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio
Chomphunuch Songsiriritthigul1, Jirundon Yuvaniyama, Robert C Robinson
1School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Abstract:
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.
