Vapor batch crystallization and preliminary X-ray crystallographic analysis of a cold-active endo-β-1,4-glucanase that was produced through the cold temperature protein expression

Abstract

The CaCel gene product from Antarctic springtail Cryptopygus antarcticus (CaCel) belongs to the glycoside hydrolasefamily 45 (GH45) type endo-β-1,4-glucanase. Since the production of soluble recombinant CaCel was not successful at thetemperature range of 15-37oC, we further lowered the expression temperature. The Escherichia coli Rosetta-gami2 (DE3)strain harbouring an expression vector including the CaCel gene was cultured at 10oC. Due to the extremely low growthrate, the induction time was expanded to 9 days and the 18-liter culture volume was necessary to get enough solubleprotein for crystallization. Crystals of CaCel were grown in droplets under Al’s Oil that allows vapor diffusion. In spite ofsmall size, the crystal of CaCel, which belonged to the space group P3121, with unit-cell parameters a = 73.57, b = 83.93,c = 163.77 Å, diffracted to 2.6 Å resolution.