Strategies for the Reconstitution and Purification of Haloarchael Protein RadA

The reconstitution and purification of Haloarchael RadA

  • Bushra Bano Patoli Institute of Microbiology, University of Sindh, Jamshoro.
  • Atif Ahmed Patoli Institute of Microbiology, University of Sindh, Jamshoro.

Abstract

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.

Published
Dec 23, 2019
How to Cite
PATOLI, Bushra Bano; PATOLI, Atif Ahmed. Strategies for the Reconstitution and Purification of Haloarchael Protein RadA. Pakistan Journal of Analytical & Environmental Chemistry, [S.l.], v. 20, n. 2, p. 169-176, dec. 2019. ISSN 2221-5255. Available at: <http://www.pjaec.pk/index.php/pjaec/article/view/494>. Date accessed: 20 feb. 2020. doi: http://dx.doi.org/10.21743/pjaec/2019.12.21.