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| Expression and Purification Procedure of Nonspecific Endonuclease Sma and Its Performance Study |
| XU Yi-fan, LIU Ming-qiu |
| School of Life Sciences, Fudan University, Shanghai 200438, China |
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Abstract Objective:To express nonspecific endonuclease Serratia marcescens (Sma), gain the high purity expressed product, and determine its activity. Methods:Sma fragment was produced by PCR. The constructed recombinant plasmid pET28a-ompA-Sma was transformed into E. coli BL21(DE3) to express soluble and high-yielding Sma by optimizing different medium. Target protein was extracted by osmotic shock, purified by ion exchange. Compared Sma with commercial product by activity test of different temperature. Results:PCR and sequencing proved that recombinant plasmid was constructed correctly. The recombinants Sma at an expression level of 7 mg/L, gained 7 300kU Sma per liter media, super-reached a purity of 95% and a specific activity of 273U/μl (commercial product is 250U/μl) after purification. Conclusion:Sma was successfully expressed. The purified Sma showed a high purity and activity. Under various conditions, the activity is not lower than that of commercial products.
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Received: 06 June 2017
Published: 15 November 2017
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| [1] |
Barbara Yannelli R N, Paul E S, Burke A C, et al. Serratia marcescens. Clinical Microbiology Newsletter, 1987, 9(20):157-160.
|
|
|
| [2] |
Dake E, Hofmann T J, Mcintire S, et al. Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae. Journal of Biological Chemistry, 1988, 263(16):7691-7702.
|
|
|
| [3] |
Klint J K, Senff S, Saez N J, et al. Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PLoS ONE, 20138(5):e63865.
|
|
|
| [4] |
Rastgar J F, Karkhane A A, Yakhchali B, et al. A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli. Journal of Chromatograph B, 2007, 856(1):214-221.
|
|
|
| [5] |
Cole S T, Sonntag I, Henning U, et al. Cloning and expression in Escherichia coli K-12 of the genes for major outer membrane protein OmpA from Shigella dysenteriae, Enterobacter aerogenes, and Serratia marcescens. Journal of Bacteriology, 1982, 149(1):145-150.
|
|
|
| [6] |
Takayama Y, Akutsu H. Expression in periplasmic space of Shewanella oneidensis. Protein Expression and Purification, 2007, 56(1):80-84.
|
|
|
| [7] |
Smith S, Mahon V, Lambert M, et al. A molecular Swiss army knife:OmpA structure, function and expression. Fems Microbiology Letters, 2010, 273(1):1-11.
|
|
|
| [8] |
李振国,徐明波,牛罡,等.在大肠杆菌周质表达重组蛋白的研究进展.药物生物技术,2011, 18(1):73-76. Li Z G, Xu M B, Niu G. et al. Progress of recombinant protein expressed in E.coli periplasmic space. Pharmaceutical Biotechnology, 2011, 18(1):73-76.
|
|
|
| [9] |
Gao D W, Wen X H, Qian Y, et al. Comparative study on using carbon or nitrogen limited medium to culture white rot fungi for reactive brilliant red dye K-2BP decolotization under non-sterile conditions. Science in China Series B:Chemistry, 2007,50(5):718-724.
|
|
|
| [10] |
Gao D W, Wen X H, Qian Y, et al. Effect of nitrogen concentration in culture mediums on growth and enzyme production of Phanerochaete chrysosporium. Journal of Environmental Sciences, 2005, 17(2):190-193.
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