Purification & characterization of PceC protein involved in Dehalorespiration by Desulfitobacterium & Dehalobacter spp

Organohalide respiration (previously named as dehalorespiration) is a process during which microbes derive energy by the reductive dehalogenation of chlorinated organic pollutants and use them as terminal electron acceptors, hence helping in environmental clean-up. This amazing process is of utmost significance in today’s world, where chlorinated compounds are released from every major source of industrial effluents and lead to the pollution of all forms of environment from land to water. Such chlorinated compounds are considered very toxic to animals as well to humans. However fundamental knowledge on the microbiology of this process is still lacking, therefore a characterization of the proteins involved in organohalide respiration is required. This Master project was focused on the characterization of the conserved protein PceC from Dehalobacter/Desulfitobacterium. Sequence analysis revealed that PceC is homologous to CprC encoded in the chlorophenol reductive dehalogenase operon which has been postulated to be a membrane-bound FMN-binding transcription regulator of the NosR/NirI family. The specific goal of this work was the heterologous production in and purification from E. coli of the predicted soluble FMN-binding domain of PceC in order to raise anti-PceC antibodies which would further allow an accurate detection and localization of native PceC in the membranes of Dehalobacter and Desulfitobacterium. Optimization of various steps was performed from expression of PceC, its pre-purification using detergents, solubilization of inclusion bodies & their re-folding on column during affinity chromatographic purification. We were able to produce a relatively pure PceC protein, in a stable state. The protein was then outsourced for antibody production.