Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli

Microbial pro-transglutaminase (MTG) from Streptomyces mobaraensis was cloned and expressed for the first time as a soluble protein at high levels in Escherichia coli. According to SDS-PAGE, more than 90% of the transglutaminase was produced in soluble form. This high solubility was obtained either by using a lactose auto-induction medium at a cultivation temperature of 28 degrees C or a temperature shift strategy comprising of growth at 37 degrees C and a post-induction temperature of 24 degrees C in a conventional LB medium and IPTG as the inductor. Using the auto-induction procedure the specific activity of MTG was as high as 627 U g(-1) CDM after activation by cleaving off the pro-sequence. Using shake flask cultivation, yields of 1460 U 1(-1) bioreactor volume were obtained. This amount of soluble MTG is sufficient to enable high-throughput screening of mutant libraries without the restraint of refolding. Preceding optimization experiments of the transglutaminase expression included variation of the inductor type, temperature after expression, the localization as well as the supply of rare t-RNAs. Almost exclusively insoluble inclusion bodies were formed by IPTG induction at 37 degrees C. Transport to the periplasm using a pelB leader sequence failed and the supply of rare t-RNAs by E. coli Rosetta(DE3) did not enhance the soluble fraction of the protein. Thirty percent soluble transglutaminase were obtained by lowering the growth temperature from 37 to 24 degrees C after induction with IPTG when a pe1B leader sequence was used. (C) 2006 Elsevier Inc. All rights reserved.