Trigger factor-dependent refolding of bacterial luciferases in Escherichia coli: Kinetics, efficiency, and effect of bichaperone system

Abstract

The main parameters of the trigger-factor-dependent refolding process of thermally inactivated bacterial luciferases were determined. It has been demonstrated that TF-dependent refolding is less efficient and more time consuming than DnaKJE-dependent refolding. An increase in the cellular concentration of TF was found to result in a dramatic decrease in the maximum level of refolding of thermally inactivated bacterial luciferases. Additionally, the efficiency of TF-dependent refolding was shown to decrease with an increase in the thermal stability of the substrate, that is, the level of TF-dependent refolding is significantly higher for thermolabile luciferases than for thermostable luciferases. For example, the maximum TF-dependent refolding level was determined as 30–40% for thermolabile luciferases from Aliivibrio fischeri and Photobacterium leiognathi, 10% in the case of luciferase from Vibrio harveyi, which is characterized by an average thermal stability, and finally 0.5% in the case of highly stable at high temperatures luciferase from Photorhabdus luminescens. An effect of the DnaKJE-ClpB bichaperone system on the efficiency of TF-dependent refolding was investigated. The ClpB component of the bichaperone system was shown to negatively affect the process efficiency, that is, TF-dependent refolding of bacterial luciferases was found to be far more efficient in E. coli clpB::kan cell strains than in E. coli clpB+ strains.