Development of a novel in vitro assay for the evaluation of integron DNA integrase activity

Tohidi, F. and Rajabnia, R. and Taravati, A. and Behdani, M. and Shokrollahi, N. and Sadeghnia, H. R. and Jamialahmadi, K. (2016) Development of a novel in vitro assay for the evaluation of integron DNA integrase activity. Biotechnology and Biotechnological Equipment, 30 (3). pp. 585-591.

[img] Text
Development of a novel in vitro assay for the evaluation of integron DNA integrase activity.pdf

Download (772kB)

Abstract

Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI) that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment. © 2016 The Author(s). Published by Taylor & Francis.

Item Type: Article
Additional Information: Export Date: 16 February 2020 Correspondence Address: Jamialahmadi, K.; Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical SciencesIran; email: JamialahmadiKh@mums.ac.ir
Uncontrolled Keywords: Antibiotic resistance integrase site-specific recombination Affinity chromatography Assays Cloning DNA Electrophoresis Electrophoretic mobility Escherichia coli Genes Nickel Polymerase chain reaction Proteins Purification Electrophoretic mobility shift assay Nitrilotriacetic acid Recombinant protein expression Recombination activity Sodium dodecyl sulphate Recombinant proteins
Subjects: QZ pathology-neoplasms-Genetics
Divisions: Mashhad University of Medical Sciences
Depositing User: mr lib4 lib4
Date Deposited: 04 Mar 2020 04:21
Last Modified: 04 Mar 2020 04:21
URI: http://eprints.mums.ac.ir/id/eprint/13238

Actions (login required)

View Item View Item