A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.
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.
Department of Biomedical and Pharmaceutical Sciences, Idaho State University, 1311 E. Central Drive, Meridian, ID 83642-7991, USA.
Jesse A
JA
Jones
Department of Biomedical and Pharmaceutical Sciences, Idaho State University, 1311 E. Central Drive, Meridian, ID 83642-7991, USA.
Emily
E
Price
Department of Biomedical and Pharmaceutical Sciences, Idaho State University, 1311 E. Central Drive, Meridian, ID 83642-7991, USA.
Donovan
D
Miller
Department of Biomedical and Pharmaceutical Sciences, Idaho State University, 1311 E. Central Drive, Meridian, ID 83642-7991, USA. Electronic address: khevener@pharmacy.isu.edu.
Kirk E
KE
Hevener
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D013295:Streptococcus mutans / Q000201:enzymology*
D001426:Bacterial Proteins; D004264:DNA Topoisomerases, Type I
Autoinduction; Characterization; Purification; Streptococcus mutans; Topoisomerase I; Type IA topoisomerase