Chinese Journal OF Rice Science

Chinese Journal OF Rice Science >

2011 , Vol. 25 >Issue 3: 326 - 330

DOI: https://doi.org/10.3969/j.issn.1001-7216.2011.03.015

研究简报

Prokaryotic Expression, Purification and Activity Assay of Neomycin Phosphotransferase Ⅱ (nptⅡ) Gene

Expand
  • China National Rice Research Institute, Hangzhou 310006, China； *Corresponding author, E-mail: swhuang666@sohu.com

Received date: 1900-01-01

  Revised date: 1900-01-01

  Online published: 2011-05-10

Fold

Abstract

Neomycin phosphotransferaseⅡ (nptⅡ) gene, cloned from a pCAMBIA1305 vector by PCR method, was inserted into a pET30a (+) vector to construct the recombinant vector pET30anptⅡ. Then the recombinant vector was transformed into an Escherichia coli strain BL21 (DE3) and the expression of the NPTⅡ protein was successfully induced by adding isopropylβDthiogalactopyranoside (IPTG). The NPTⅡ fusion protein was produced in the form of the solubility or inclusion body with molecular weight around 35 kD, and a yield of approximately 45%. The inclusion bodies were solubilized and denatured by addition of 5 mmol/L dithiothreitol (DDT) and 1% sodium lauroyl sarcosine (SKL), then renatured by dialysis. The solubilized proteins were purified by Ni2+NTA affinity chromatography at approximately 95% purity. An in vitro assay revealed that, the NPTⅡ protein can inactivate the kanamycin at concentrations up to 30 μg/mL.

Key words： neomycin phosphotransferase gene; prokaryotic expression; inclusion body; soluble protein; purification

Cite this article

WANG Ling,LIU Lian-meng,FU Qiang,HUANG Shi-wen* . Prokaryotic Expression, Purification and Activity Assay of Neomycin Phosphotransferase Ⅱ (nptⅡ) Gene[J]. Chinese Journal OF Rice Science, 2011 , 25(3) : 326 -330 . DOI: 10.3969/j.issn.1001-7216.2011.03.015

References

[1]Anfossi L, Tozzi C, Giraudi G, et al. Evaluation of procedures for the extraction and purification of neomycin phosphotransferase Ⅱ from genetically modified agrobacterium. Ann Chim, 2004, 94(1/2): 93-94.
[2]王关林, 方宏筠. 植物基因工程. 2版. 北京: 科学出版社, 2002： 527-528.
[3]Miki B, McHugh S. Selectable marker genes in transgenic plants: Application, alternatives and biosafety. J Biotechnol, 2004, 107(3): 193-232.
[4]Fuchs R L, Ream J E, Hammond B G, et al. Safety assessment of the neomycin phosphotransferase II (NPTII) protein. Nat Biotechnol, 1993, 11: 1543-1547.
[5]Fuchs R L, Heeren R A, Gustafson M E, et al. Purification and characterization of microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein. Nat Biotechnol, 1993, 11: 1537-1542.
[6]Bennett P M, Livesey C T, Nathwani D, et al. An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: Report of the Working Party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother, 2004, 53(3): 418-431.
[7]Daniell H, Muthukumar B, Lee S B. Marker free transgenic plants: Engineering the chloroplast genome without the use of antibiotic seletion. Curr Genet, 2001, 39: 109-116.
[8]李文凤, 季静, 王罡, 等. 提高转基因植物标记基因安全性策略的研究进展. 中国农业科学, 2010, 43(9): 1761-1770.
[9]邓欣, 赵延昌, 高必达. 转基因抗虫棉叶围卡那霉素抗性细菌种群动态及nptII基因漂移研究. 中国农业科学, 2007, 40(11): 2488-2494.
[10]Hoffmann T, Golz C, Schieder O. Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Curr Genet, 1994, 27: 70-76.
[11]Nielsen K M, van Elsas J D, Smalla K. Transformation of Acinetobacter sp. strain BD413 (pFG4D nptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl Environ Microbiol, 2000, 66(3): 1237-1242.
[12]McDonnell R E, Clark R D, Smith W A, et al. A simplified method for the detection of neomycin phosphotransferase Ⅱ activity in transformed plant tissues. Plant Mol Biol Rep, 1987, 5: 380-386.
[13]Baszczynski C L. A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues. Biochem Cell Biol, 1990, 68(6): 983-987.
[14]McKenzie M J, Mett V, Jameson P E. Modified ELISA for the detection of neomycin phosphotransferase II in transformed plants pecies. Plant Cell Rep, 2000, 9: 286-289.
[15]Eom S H, Reisch B I. Inhibitors of neomycin phosphotransferase II enzyme-linked immunosorbent assay in grapevine (Vitis vinifera L.) leaves. Vitis, 2008, 47(1): 7-9.
[16]Eom S H, Reisch B I. Quantificational analysis of NPT-Ⅱ protein from genetically modified Vitis vinifera L. Afr J Biotechnol, 2010, 9(23): 3468-3474.
[17]Aatsinki J T, Rajaniemi H J. An alternative use of basic pGEX vectors for producing both N- and C- terminal fusion proteins for production and affinity purification of antibodies. Prot Expr Purif, 2005, 40(2): 287-291.
[18]汪家政, 范伟. 蛋白质技术手册. 北京： 科学出版社, 2002: 42-46.
[19]Tabashnik B E. Communal benefits of transgenic corn. Science, 2010, 330(6001): 189-190.
[20]Wu K M, Lu Y H, Feng H Q, et al. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin containing cotton. Science, 2008, 321(5896): 1676-1678.
[21]Hoffmann F, van den Heuvel J, Zidek N, et al. Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Technol, 2004, 34: 235-241.
[22]Terpe K. Overview of bacterial expression systems of heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol, 2006, 72: 211-222.
[23]Suttnar J, Dyr J E, Hamsikova E, et al. Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger. J Chromatogr B Biomed Appl, 1994, 656(1): 123-126.
[24]Mergulhao F J M, Manteiro G A. Recombinant protein secretion in Escherichia coli. Biotechnol Adv, 2005, 23, 177-202.
[25]Esposito D, Chatterjee D K. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol, 2006, 17(4): 353-358.
[26]de Macro A. Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins. Nat Protoc, 2006, 1: 1538-1543.
Options
Outlines

/

〈 〉

Tel: 0571-63370278　E-mail: cjrs@263.net
Supported by Beijing Magtech Co., Ltd.