Article info 2015 3 (3)    003  (03)   pp.  138 ~ 142
Title 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
Authors Young Jun An1, Min-Kyu Kim1, Jung Min Song2, Mee Hye Kang2, Youn-Ho Lee2 and Sun-Shin Cha1,3,4*
Institutions 1Marine Biotechnology Research Center and 2Marine Ecosystem and Biological Center, Korea Institute of Ocean Science and Technology, Ansan 426-744, Korea, 3Ocean Science and Technology School, Korea Maritime University, Pusan 606-791, Korea, 4Department of Marine Biotechnology, University of Science and Technology, Daejeon 305-333, Korea. *Correspondence: chajung@kiost.ac.kr
Abstract The CaCel gene product from Antarctic springtail Cryptopygus antarcticus (CaCel) belongs to the glycoside hydrolase family 45 (GH45) type endo-β-1,4-glucanase. Since the production of soluble recombinant CaCel was not successful at the temperature 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 growth rate, the induction time was expanded to 9 days and the 18-liter culture volume was necessary to get enough soluble protein for crystallization. Crystals of CaCel were grown in droplets under Al’s Oil that allows vapor diffusion. In spite of small 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.