Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

Autor: Afful D; Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 30602, USA., Cai L; Department of Computer Sciences, University of Georgia, Athens, GA, 30602, USA., Momany C; Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 30602, USA. Electronic address: cmomany@uga.edu.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2019 Nov; Vol. 163, pp. 105448. Date of Electronic Publication: 2019 Jul 04.
DOI: 10.1016/j.pep.2019.105448
Abstrakt: The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital and community acquired infections worldwide - making the organism an important public health pathogen. We developed a method for producing high quantities of highly pure and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes that employed two-vector systems for expressing the core enzyme (α, β, β', and ω subunits) and for expressing the holoenzyme complex (core + σ 70 ). Unlike other RNAP expression approaches, we used a low temperature autoinduction system in E. coli with T7 promoters that produced high cell yields and stable protein expression. The purification strategy comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange) with yields of up to 11 mg per 500 mL culture. Purified holoenzyme and reconstituted holoenzyme from core and σ 70 were highly active at transcribing both small and large-sized DNA templates, with a determined elongation rate of ~18 nt/s for the holoenzyme. The successful purification of the P. aeruginosa RNAP provides a gateway for studies focusing on in vitro transcriptional regulation in this pathogen.
(Copyright © 2019 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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