Expression and Purification of the Helicase-like Subdomains, H1 and H23, of Reverse Gyrase from A. fulgidus for Heteronuclear NMR study
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Reverse gyrase is a hyperthermophile specific protein which introduces positive supercoils into DNA molecules. Reverse gyrase consists of an N-terminal helicase-like domain and a C-terminal topoisomerase domain. The helicase-like domain shares the three-dimensional structure with two tandem RecA-folds (H1 and H2), in which the subdomain H2 is interrupted by the latch domain (H3). To understand the physical property of the hyperthermophile-specific protein, two subdomains af_H1 and af_H23 have been cloned into E. coli expression vector, pET28a. The 15N-labeled af_H1 and af_H23 proteins were expressed and purified for heteronuclear NMR study. The af_H1 protein exhibits the well-dispersion of amide signals in its 1H/15N-HSQC spectra and thus further NMR study continues to be progressed.
Abstract
Reverse gyrase is a hyperthermophile specific protein which introduces positive supercoils into DNA molecules. Reverse gyrase consists of an N-terminal helicase-like domain and a C-terminal topoisomerase domain. The helicase-like domain shares the three-dimensional structure with two tandem RecA-folds (H1 and H2), in which the subdomain H2 is interrupted by the latch domain (H3). To understand the physical property of the hyperthermophile-specific protein, two subdomains af_H1 and af_H23 have been cloned into E. coli expression vector, pET28a. The 15N-labeled af_H1 and af_H23 proteins were expressed and purified for heteronuclear NMR study. The af_H1 protein exhibits the well-dispersion of amide signals in its 1H/15N-HSQC spectra and thus further NMR study continues to be progressed.
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