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| Expression, Purification and Biological Activity of Escherichia coli RecQ Helicase |
| HUANG Zhen-rong1, ZHANG San-jun1, QIAN Min2, REN Hua 1 |
1. School of Life Science, East China Normal University, Shanghai 200241, China;
2. State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, China |
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Abstract RecQ helicases are one of the most important macromolecules in the process of molecular metabolism. They play essential roles in maintaining the stability of the genetic materials in cells. The DNA corresponding to the coding sequence of the E.coli RecQ helicase gene was amplified by PCR from the chromosome DNA of Escherichia coli DH5α. The amplified product was subcloned into the expression vector pET24a(+). The recombinant protein was induced to express in Escherichia coli BL21(DE3) with IPTG at low temperature. E. coli RecQ helicase was with above 90% purity and good solubility was obtained in vitro. With biochemical and biophysical techniques, E. coli RecQ helicase was shown to have DNA-dependent ATPase activity in concentration-dependent manner. It was easier to bind forked ds DNA than ssDNA and blunt end dsDNA in the same protein concentration. E.coli RecQ helicase also has ATP-dependent DNA unwinding activity and time-dependent DNA annealing activity. These results are helpful to study the structures and functions of other members of the RecQ family helicases.
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Received: 18 December 2012
Published: 25 March 2013
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