Strategies for the Reconstitution and Purification ofHaloarchael Protein RadA

DNA repair proteins in halophilic organisms are interesting to study in the context ofunderstanding the dynamics of protein-DNA interaction and their adaptation to performbiochemical activities at high osmolarity. Successful expression and purification of halophilicproteins is often challenging particularly when they are over-expressed in non-halophilicheterologous host. In the present study,radAfromHaloferax volcaniiwas cloned andoverexpressed inE. coli. Although,radAwasover-expressed as a soluble protein inE. colibutpurification of RadA seemed challenging. Various strategies were therefore implemented to attainmaximum possible purification ofRadA. The purification ofRadAusing Immobilized MetalAffinity Chromatography (IMAC) followed by Size Exclusion Chromatography (SEC) wasinitially adopted. The SDS-PAGE and agarose gel analysis of the representative fractions fromSEC indicated this to be an unsuccessful strategy due to high affinity of protein with the DNAfrom host. The refolding strategy employing denaturation of theRadAin urea along withbenzonase treatment was attempted to chop down the contaminating host DNA. This wasobserved an effective method as the subsequent analysis of the representative fractions from SECindicatedRadA at about 90% puritythatcanpossiblybesuitable for further biochemical andstructural analysis.