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Prokaryotic Soluble Expression of a Novel Ribonuclease Gene Rdrlec from Rana dybowskii |
TAO Feng-yun, YIN Xue-wei, LI Dan, JIANG Dan, ZHAO Wei |
Biochemical Engineering College of Beijing Union University, Beijing 100023, China |
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Abstract Much attention has been paid to ribonucleases from amphibian Rana species for their significant anti-tumor activity. Rdrlec is a novel RNase gene form Rana dybowskii and its biological function has not been identified. Getting a great deal of high purity wild type recombinant protein is the basis for its function study. Rdrlec gene was adjusted according to Escherichia coli codon bias without changing its amino acids. The synthetic gene was inserted to the pET-32a (+) expression vector through the EcoR I and Hind III site, and the resulting recombination expression plasmid was named pET32-Rdrlec and transferred into Escherichia coli BL21 (DE3) strains. After induced with 0.4 mmol/L IPTG at 30℃ for 6 h, the fusion protein was found expressed mainly in soluble form. The cell lysate was loading to Ni-NTA affinity chromatography and Sephadex G75 chromatography, and the fusion protein showed a single band on SDS-PAGE. The wild type recombinant Rdrlec protein was released and purified after enterokinase digesting, and it showed enzymatic activity to degrade RNA into nucleotides, which shows that this molecule has formed the correct spatial structure. The successful expression of wild type recombinant Rdrlec protein has providing the raw material for the subsequent structure, function and application study.
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Received: 08 October 2012
Published: 25 January 2013
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