Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme

Pro-transglutaminase from Streptomyces mobaraensis was expressed in Escherichia coli as a fusion protein carrying a C-terminal histidine tag (pro-MTG-His₆). The recombinant organism was cultivated in 15 L bioreactor scale and pro-MTG-His₆ was purified by immobilized metal affinity chromatography. Activation of the inactive pro-enzyme using trypsin resulted in an unexpected degradation of the transglutaminase and a concomitant loss of activity. Therefore, a set of commercially available proteases was investigated for their activation potential without destroying the target enzyme. Besides trypsin, chymotrypsin and proteinase K were found to activate but hydrolyze the (pro-MTG-His₆). Cathepsin B, dispase I, and thrombin were shown to specifically hydrolyze pro-MTG-His₆ without deactivation. TAMEP, the endogeneous protease from S. mobaraensis was purified for comparison and also found to activate the recombinant histidine-tagged transglutaminase without degradation. The TAMEP activated MTG-His₆ was purified and characterized. The specific activity (23 U/mg) of the recombinant histidine-tagged transglutaminase, the temperature optimum (50 °C), and the temperature stability (t_1/2 at 60 °C = 1.7 min) were comparable to the wild-type enzyme. A C-terminal peptide tag did neither affect the activity nor the stability but facilitated the purification. The purification of the histidine-tagged protein is possible before or after activation.