Characterization of the predicted membrane anchor of reductive dehalogenases

BACKGROUND Organohalide respiration (OHR) is a metabolic process that couples the reduction of halogenated organic compounds to energy conservation in a few phylogenetically diverse anaerobic bacteria. In this study, tetrachloroethene (= perchloroethene, PCE) was selected as model organohalide compound, as it represents a major groundwater pollutant, as a result of inappropriate use and storage. The minimal and highly conserved gene pool among OHR bacteria consists of a 2-gene operon encoding for four enzymes which are responsible for the reductive dehalogenation activity including PceA, which is the key reductive dehalogenase; and PceB, a predicted membrane anchor for PceA at the cytoplasmic membrane. In the present project, we aim at understanding the role of PceB by studying the protein interaction between PceB and PceA from Dehalobacter restrictus. MATERIALS AND METHODS GST-fusion protein with the second loop of PceB (L2, presumed responsible of the interaction with PceA) is designed in order to be produced in E. coli in a soluble form, and to be purified for in vitro experiments. At the same time, we produce and purify a soluble form of PceA by using cloning techniques to allow to it to be matured in E. coli. Then, pull-down experiments are performed to trap PceA or PceB by fixing one or the other protein. Additional in vitro experiments such as far western blot with anti-PceA antibody will be able to be carried on to confirm the interaction between PceB and PceA. RESULTS Preliminaries results shows that we are capable of produce a soluble form of PceA when it is co-expressed with its chaperone PceT in E. coli. We are currently waiting for our results of the expression of the fusion protein GST-L2 in E. coli.