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Escherichia coli Expression, Purification and Functional Identification of Squalene Synthase from Artemisia annua L.
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF APPLIED & ENVIRONMENTAL BIOLOGY
- Issue:
- 3
- DOI:
- 10.3321/j.issn:1006-687X.2007.03.005
- Key Word:
- 鲨烯合酶;大肠杆菌表达;功能鉴定;青蒿
Abstract： 利用PCR方法,将经RACE方法克隆到的中药青蒿鲨烯合酶cDNA(AF302464)开放阅读框的3'末端截短99bp,插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间,构建N端和C端均携带有HIS6表达标签的鲨烯合酶重组表达载体pETSSA.将pETSSA转入大肠杆菌BL21(DE3),0.5 mmol/L IPTG(isopropyl-beta-D-thiogalactoside)28 ℃诱导重组鲨烯合酶的表达.表达产物经镍琼脂糖柱纯化.纯化蛋白加入酶促反应体系(含FPP和NADPH),GC-MS分析酶促反应体系的正己烷萃取物,结果显示重组鲨烯合酶可以催化FPP向鲨烯的转化.青蒿鲨烯合酶的功能鉴定为进一步利用反义或RNAi技术限制甾类生物合成,从而提高青蒿中的青蒿素含量提供了基础.
- [1]M.-A. Hartmann,L. Wentzinger,A. Hemmerlin,etc.Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin[C].2000.
- [2]耿飒,叶和春,李国凤,等.中药青蒿的生理生化特征及其研究进展[J].2002,(1).
- [3]Shin DH.,Devarenne TP.,Chappell J.,etc.Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor[J].1998,349(2).
- [4]G J, Kudakasseril,L, Lam,E J, Staba.Effect of Sterol Inhibitors on the Incorporation of 14C-Isopentenyl Pyrophosphate into Artemisinin by a Cell-Free System from Artemisia annua Tissue Cultures and Plants.[J].1987,53.
- [5]T R, Tansey,I, Shechter.Squalene synthase: structure and regulation.[J].2001,65.
- [6]HermanJ.Woerdenbag,JosF.J.Lüers,WimvanUden,etc.Production of the new antimalarial drug artemisinin in shoot cultures ofArtemisia annuaL.[J].1993,32(2).
- [7]Eggelte TA,van Agtmael-MA,van Boxtel-CJ.Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication.[J].1999,20(5).
- [8]U, V?geli,J, Chappell.Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor.[J].1988,88.
- [9]Israr M,Abdin MZ,Jain SK,etc.Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production.[J].2003,69(4).
- [10]G W Robinson,Y H Tsay,B K Kienzle,etc.Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.[J].1993,13(5).
- [11]Harada E,Jeong JH,Seo JW,etc.Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation[J].2005,66(8).