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| Expression, Purification and Biological Activity of Arginine Mutants of Bacillus subtilis RecQ Helicase |
| WU Hai-li1, ZHANG San-jun2, DU Bing1, QIAN Min1, REN Hua1 |
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 arginine residues of Bacillus subtilis RecQ play important roles in ATP hydrolyzation and binding activities. The DNA corresponding to the coding sequence of the Bacillus subtilis RecQ helicase gene was amplified by PCR from the chromosome DNA of Bacillus subtilis 168, the sequence size is about 1.5kb. The arginine residues (arg319 and arg322) of Bacillus subtilis RecQ were mutated separately or simultaneously to alanine residues by overlapping PCR method, then wild type and mutants were subcloned into the expression vector pET24a(+).The recombinant proteins were induced to express in E.coli BL21(DE3)with IPTG. All of the proteins obtained in vitro were with above 90% purity and good solubility, and then the ATP hydrolysis of wild type and mutants were detected. The results showed that Bacillus subtilis RecQ and mutants had DNA-dependent ATPase activity in concentration-dependent manner. However, the ATP hydrolysis activities of mutants were significantly reduced compared to the wild RecQ. The consequence state that the two arginine residues took important part in interacting RecQ helicaese with ATP. These results are helpful to study the structures and functions of other members of the RecQ family helicases.
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Received: 23 September 2013
Published: 25 December 2013
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