丹参二萜合酶基因CPS4的原核表达体系优化及活性蛋白纯化
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  • 英文篇名:Heterologous Expression and Purification of Recombinant CPS4 from Salvia miltiorrhiza
  • 作者:靳保龙 ; 崔光红 ; 党伯岳 ; 刘世会 ; 漆小泉
  • 英文作者:JIN Bao-long;CUI Guang-hong;DANG Bo-yue;LIU Shi-hui;QI Xiao-quan;College of Pharmacy,Guizhou University;Key Laboratory of Plant Molecular Physiology,Institute of Botany,Chinese Academy of Sciences;National Resource Center of Chinese Materia Medica,China Academy of Chinese Medical Sciences;
  • 关键词:丹参 ; 原核表达 ; 正交试验 ; 纯化
  • 英文关键词:Salvia miltiorrhiza;;prokaryotic expression;;orthogonal experiment;;purifying
  • 中文刊名:ZSFX
  • 英文刊名:Chinese Journal of Experimental Traditional Medical Formulae
  • 机构:贵州大学药学院;中国科学院植物研究所;中国中医科学院中药资源中心;
  • 出版日期:2014-05-20
  • 出版单位:中国实验方剂学杂志
  • 年:2014
  • 期:v.20
  • 基金:国家自然科学基金项目(81001604);; 中国博士后科学基金(2012T50159);; 贵州省中药现代化科技产业研究开发专项项目(黔科合ZY字[2013]3009号)
  • 语种:中文;
  • 页:ZSFX201410030
  • 页数:5
  • CN:10
  • ISSN:11-3495/R
  • 分类号:102-106
摘要
目的:优化丹参柯巴基焦磷酸合酶基因家族新基因CPS4的原核表达体系并对重组蛋白进行纯化。方法:对2种原核表达菌株进行比较。对诱导条件,包括异丙基-β-D-硫代半乳糖苷(IPTG)诱导浓度、诱导时宿主菌的密度(A600)和诱导时间进行正交试验。利用HisTrap HP蛋白纯化柱对重组蛋白进行纯化。结果:Escherichia coli Tuner(DE3)表达菌株能诱导产生更多的可溶重组蛋白。最佳诱导表达条件为诱导时宿主菌的A600值0.7,IPTG浓度0.2 mmol·L-1,诱导表达时间10 h。HisTrap HP蛋白纯化柱纯化时梯度洗脱能得到纯度高的重组蛋白。结论:利用此优化体系可得到足量用于后续研究的重组蛋白,为其他二萜类合酶基因的原核表达提供参考。
        Objective: To find the optimal heterologous expression conditions and purification system for the new gene CPS4 from Salvia miltiorrhiza. Method: Compare two different prokaryotic expression strains and optimizing the induction conditions including A600values of Escherichia coli,isopropyl-beta-D-galactose and glycosides(IPTG) concentration and induction time with orthogonal experiment. The recombinant protein was purified by HisTrap HP. Result: E. coli Tuner(DE3) induced more soluble recombinant protein than BL21(DE3). The optimal expression conditions are A600is 0. 7,IPTG concentration is 0. 2 mmol·L-1and induction time for 10 h. The recombinant protein was purified by gradient elution with HisTrap HP. Conclusion: This optimal system can obtain enough protein for further study and provide a reference for the prokaryotic expression of the other diterpene synthase.
引文
[1]Trapp S C,Croteau R B.Genomic organization of plant terpene synthases and molecular evolutionary implications[J].Genetics,2001,158(2):811.
    [2]Xu M M,Matthew L H,Sladjana P,et al.Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products[J].Plant J,2004,39(3):309.
    [3]Ma Y M,Yuan L C,Wu B,et al.Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza[J].J Exp Bot,2012,63(7):2809.
    [4]Gao W,Hillwig M L,Huang L Q,et al.A functional genomics approach to tanshinone biosynthesis provides stereochemical insights[J].Org Lett,2009,11(22):5170.
    [5]Patnaik P R.Investigation of induction effect on the steady state performance of a continuous fermentation for recombinantβ-ga1actosidase[J].Process Biochem,2001,11(36):1069.
    [6]高伟,崔光红,孔建强,等.丹参柯巴基焦磷酸合酶基因的优化表达、纯化及抗体制备[J].药学学报,2008,43(7):766.
    [7]Liu T,Zhu P,Cheng K D,et al.Molecular cloning,expression and characterization of hyoscyamine 6β-hydroxylase from hairy roots of Anisodus tanguticus[J].Planta Med,2005,71(3):249.
    [8]徐端正.生物统计在实验和临床药理学中的应用[M].北京:科学出版社,2004:144.
    [9]Sambrook J,Fritsch E F,Maniatis T.Molecular cloning,a laboratory manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989:16.
    [10]Fanger B O.Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents[J].Anal Bio Chem,1987,162(1):11.
    [11]Matthew L H,Xu M M,Tomonobu T,et al.Domain loss has independently occurred multiple times in plant terpene synthase evolution[J].Plant J,2011,68(6):1051.
    [12]Michael J W,Maria P,Shawn R C,et al.Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli[J].Appl Environ Microbiol,1997,63(11):4313.
    [13]Baneyx F.Recombinant protein expression in Escherichia coli[J].Curr Opin Biotechnol,1999,10(5):411.
    [14]余光清,蒋诗琴,李雍龙.可溶性重组日本血吸虫26kDa GST的优化表达[J].中国人兽共患病学报,2006,22(8):770.
    [15]Yasukawa T,Kanei I C,Maekawa T,et al.Increase of solubility of foreign proteins in Escherichia coil by coprodution of the bacterial thioredoxin[J].J Biol Chem,1995,270(43):25328.
    [16]Li J L,Chen Q Q,Jin Q P,et al.CPS2 is potentially involved in the biosynthesis of pharmacologically active Isodon diterpenoids rather than gibberellin[J].Phytochemistry,2012,76(4):32.
    [17]Hans P S,Kim K M.Advanced genetic strategies for recombinant protein expression in Escherichia coli[J].J Biotechnol,2005,115(2):113.

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