Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

Patoli, B.B., Winter, J.A. ORCID: 0000-0003-3582-7596, Patoli, A.A., Delahay, R.M. and Bunting, K.A., 2017. Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures. Microbiology. ISSN 1350-0872

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Abstract

The present study demonstrates the successful co-expression of two RecA-like proteins, RadA and RadB, from the halophilic euryarchaeon, Haloferax volcanii (Hvo). A co-expression approach was used to improve the purification of RadA by providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking and the physical interaction of Hvo RadA and Hvo RadB has not been demonstrated under either in vitro or in vivo conditions. Here, we describe for the first time co-expression of Hvo RadA with RadB and interaction of these proteins as a complex under in vitro conditions. Purification procedures were performed under conditions of high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadA-B complex estimated the ratio of RadA to RadB to be 4:1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside cofactor.

Item Type: Journal article
Publication Title: Microbiology
Creators: Patoli, B.B., Winter, J.A., Patoli, A.A., Delahay, R.M. and Bunting, K.A.
Publisher: The Microbiology Society
Date: 26 October 2017
ISSN: 1350-0872
Identifiers:
NumberType
10.1099/mic.0.000562DOI
Divisions: Schools > School of Science and Technology
Depositing User: Jonathan Gallacher
Date Added: 17 Nov 2017 09:39
Last Modified: 17 Nov 2017 09:39
URI: http://irep.ntu.ac.uk/id/eprint/32043

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