中国水稻科学2011，Vol.25Issue(3)：326-330,5.DOI:10.3969/j.issn.1001-7216.2011.03.015
新霉素磷酸转移酶基因nptⅡ的原核表达、蛋白纯化及其活性鉴定
Prokaryotic Expression, Purification and Activity Assay of Neomycin Phosphotransferase Ⅱ(npt Ⅱ)Gene
摘要
Abstract
Neomycin phosphotransferaseⅡ (npt Ⅱ ) gene, cloned from a pCAMBIA1305 vector by PCR method, was inserted into a pET30a (+) vector to construct the recombinant vector pET30a-npt Ⅱ. Then the recombinant vector was transformed into an Escherichia coli strain BL21 (DE3) and the expression of the NPT Ⅱ protein was successfully induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG). The NPT Ⅱ 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 NPT Ⅱ protein can inactivate the kanamycin at concentrations up to 30 μg/mL.关键词
新霉素磷酸转移酶基因/原核表达/包涵体/可溶性蛋白/纯化Key words
neomycin phosphotransferase gene/ prokaryotic expression/ inclusion body/ soluble protein/ purification分类
农业科技引用本文复制引用
王玲,刘连盟,傅强,黄世文..新霉素磷酸转移酶基因nptⅡ的原核表达、蛋白纯化及其活性鉴定[J].中国水稻科学,2011,25(3):326-330,5.基金项目
转基因生物新品种培育重大专项资助项目(2009ZX08012 010B,2008ZX08012-04,2008ZX08001-002) （2009ZX08012 010B,2008ZX08012-04,2008ZX08001-002）
中央级公益性科研院所专项基金资助项目(2009RG004-4). （2009RG004-4）
