期刊名称：Pakistan Journal of Analytical & Environmental Chemistry
印刷版ISSN：1996-918X
电子版ISSN：2221-5255
出版年度：2019
卷号：20
期号：2
页码：169-176
DOI：10.21743/pjaec/2019.12.21
语种：English
出版社：National Centre of Excellence in Analytical Chemsitry
摘要：DNA repair proteins in halophilic organisms are interesting to study in the context of understanding the dynamics of protein-DNA interaction and their adaptation to perform biochemical activities at high osmolarity. Successful expression and purification of halophilic proteins is often challenging particularly when they are over-expressed in non-halophilic heterologous host. In the present study,radA from Haloferax volcanii was cloned and overexpressed in E. coli. Although,radA was over-expressed as a soluble protein in E. coli but purification of RadA seemed challenging. Various strategies were therefore implemented to attain maximum possible purification of RadA. The purification of RadA using Immobilized Metal Affinity Chromatography (IMAC) followed by Size Exclusion Chromatography (SEC) was initially adopted. The SDS-PAGE and agarose gel analysis of the representative fractions from SEC indicated this to be an unsuccessful strategy due to high affinity of protein with the DNA from host. The refolding strategy employing denaturation of the RadA in urea along with benzonase treatment was attempted to chop down the contaminating host DNA. This was observed an effective method as the subsequent analysis of the representative fractions from SEC indicated RadA at about 90% purity that can possibly be suitable for further biochemical and structural analysis..
