A simplified protocol for high-yield expression and purification of bacterial topoisomerase I

Jesse A. Jones, Emily Price, Donovan Miller, Kirk Hevener

Research output: Contribution to journalArticle

Abstract

Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes - topoisomerase I and topoisomerase III - share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity.

Original languageEnglish (US)
Pages (from-to)32-40
Number of pages9
JournalProtein Expression and Purification
Volume124
DOIs
StatePublished - Aug 1 2016

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Type I DNA Topoisomerase
Streptococcus mutans
Enzymes
Pharmaceutical Preparations
Infection

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

A simplified protocol for high-yield expression and purification of bacterial topoisomerase I. / Jones, Jesse A.; Price, Emily; Miller, Donovan; Hevener, Kirk.

In: Protein Expression and Purification, Vol. 124, 01.08.2016, p. 32-40.

Research output: Contribution to journalArticle

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