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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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[1] Leone E, D’Alessio G. Structure and properties of seminal ribonuclease. Biochem Soc Trans, 1977,5 (2):466-470.
[2] Beintema J J, Gaastra W, Lenstra J A, et al. The molecular evolution of pancreatic ribonuclease. J Mol Evol, 1977,10 (1):49-71.
[3] Pizzo E, Buonanno P, Di Maro A, et al. Ribonucleases and angiogenins from fish. J Biol Chem, 2006,281 (37):27454-27460.
[4] Ardelt W, Mikulski S M, Shogen K. Amino acid sequence of an anti-tumor protein from Rana pipiens oocytes and early embryos. Homology to pancreatic ribonucleases. J Biol Chem, 1991,266 (1):245-251.
[5] Beintema J J, Broos J, Meulenberg J, et al. The amino acid sequence of snapping turtle (Chel·ra serpentina) ribonuclease. Eur J Biochem, 1985,153 (2):305-312.
[6] Nitto T, Dyer K D, Czapiga M, et al. Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus. J Biol Chem, 2006,281 (35):25622-25634.
[7] Zhu C F, Liu Q, Zhang L, et al. RNase9, an androgen-dependent member of the RNase A family, is specifically expressed in the rat epididymis. Biol Reprod, 2007,76 (1):63-73.
[8] 陶凤云,赵伟,马润宇. 核糖核酸酶A超家族成员杀菌作用研究进展. 中国抗生素杂志,2009,34(6):321-325, 379. Tao F Y, Zhao W, Ma R Y. Progress in bacterictdal activity of ribonuclease A superfamily menbers. Chinese Journal of Antibiotics, 2009, 34(6): 321-325, 379.
[9] Monti D M, Yu W, Pizzo E, et al. Characterization of the angiogenic activity of zebrafish ribonucleases. FEBS J, 2009,276 (15):4077-4090.
[10] Lee I. Ranpirnase (Onconase), a cytotoxic amphibian ribonuclease, manipulates tumour physiological parameters as a selective killer and a potential enhancer for chemotherapy and radiation in cancer therapy. Expert Opin Biol Ther, 2008,8 (6):813-827.
[11] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry,1976, 72:248-254.
[12] Rosenberg H F. Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. J Biol Chem, 1995,270 (14):7876-7881.
[13] Ardelt W, Shogen K, Darzynkiewicz Z. Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes. Curr Pharm Biotechnol, 2008,9 (3):215-225.
[14] Liao Y D, Huang H C, Leu Y J, et al. Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog). Nucleic Acids Res, 2000,28 (21):4097-4104.
[15] 陶凤云,赵伟,林强,等. 中国林蛙卵核糖核酸酶的分离纯化及其抗肿瘤作用. 中国生物工程杂志, 2010, 30(5):103-109. Tao F Y, Zhao W, Lin Q, et al. Algtotoxic ribonucleose isolated from occytes of Chinese Nothease Frog Rana dybowckii. China Biotechnology, 2010,30(5):103-109.
[16] Tao F, Fan M, Zhao W, et al. A novel cationic ribonuclease with antimicrobial activity from Rana dybowskii. Biochem Genet, 2011, 49(5-6): 369-384.
[17] Lou Y C, Huang Y C, Pan Y R, et al. Roles of N-terminal pyroglutamate in maintaining structural integrity and pKa values of catalytic histidine residues in bullfrog ribonuclease 3. J Mol Biol, 2006,355 (3):409-421.
[18] Gorbatyuk V Y, Tsai C K, Chang C F, et al. Effect of N-terminal and Met23 mutations on the structure and dynamics of onconase. J Biol Chem, 2004,279 (7):5772-5780.
[19] Leland PA, Schultz L W, Kim B M, et al. Ribonuclease A variants with potent cytotoxic activity. Proc Natl Acad Sci U S A, 1998,95 (18):10407-10412.
[20] Gagliardo B, Faye A, Jaouen M, et al. Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone. FEBS J, 2008, 275:3793-3803.
[21] Stewart E J, Aslund F, Beckwith J. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. The EMBO journal, 1998, 17:5543-5550. |
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