Expression of Trypanosoma congolense trypanothione reductase in Escherichia coli: overproduction, purification, and characterization.
Expression of Trypanosoma congolense trypanothione reductase in Escherichia coli: overproduction, purification, and characterization.
复制标题
刚果锥虫锥硫酮还原酶在大肠杆菌中的表达：过量生产、纯化和表征。
DOI：
10.1021/bi00438a013
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Walsh,CT
中科院分区：
文献类型：
--
作者：
Sullivan,FX;Shames,SL;Walsh,CT
材料与方法脱氧腺苷5 ′-α-[35 S]硫代三磷酸和Klenow聚合酶得自阿默舍姆。限制酶、T4 DNA连接酶和T4多核苷酸激酶从新英格兰生物实验室或国际生物技术公司获得。（IBI）。小牛肠碱性磷酸酶来自Boehringer-Mannheim或Stratagene。二异丙基（β-氰基乙基）亚磷酰胺、DEAE-Sephacel和2 β，5 β，-ADP-Sepharose来自Pharmacia。测序酶DNA测序试剂盒来自US Biochemicals。锥硫酮和脱氮锥硫酮（亨德森等人，1987b）是G博士的礼物。亨德森（洛克菲勒大学，纽约州纽约）。除非另有说明，所有其他生化试剂均购自Sigma Chemical Co.。载体和细菌菌株。M13 mpl8和M13 mpl9来自阿默舍姆。pKK 233 - 2和pUC 13来自Pharmacia。pBS+购自Stratagene。pGP 1 - 2（塔博尔& Richardson，1985），一种含有在温度敏感的cI 857 X阻遏物控制下的T7 RNA聚合酶基因的质粒，是S.塔博尔（哈佛医学院，波士顿，MA）。质粒pCGTR-2含有T.刚果锥虫硫酮还原酶基因在3.3-kb BamHl插入片段上（Shames等，1988年）。含有tac II启动子和强核糖体结合位点的质粒pSD8SP（Shindeling等人，这是一份来自L博士的礼物。Gold（科罗拉多州博尔德市科罗拉多大学）。e.大肠杆菌菌株JM 101和JM 105来自新英格兰Biolabs。e.大肠杆菌菌株SG5（Greer & Perham，1986），谷胱甘肽还原酶缺失突变体，是R. Perham（剑桥大学，剑桥，英格兰）。所有细菌菌株在每升含有16g细菌用胰蛋白胨、10g细菌用酵母提取物和5g NaCl的2XTY培养基上生长。适当时包括抗生素。对于酶制剂，用以下任一种转化的细菌菌株
Materials and MethodsMaterials. Deoxyadenosine 5'-a-[35S] thiotriphosphate and Klenow polymerase were obtained from Amersham. Re-striction enzymes, T4 DNA ligase, and T4 polynucleotide kinase were obtained from New England Biolabs or International Biotechnologies Inc.(IBI). Calf intestinal alkaline phosphatase was from Boehringer-Mannheim or Stratagene. Diisopropyl (/3-cyanoethyl) phosphoramidites, DEAE-Sephacel, and 2/, 5,-ADP-Sepharose were from Pharmacia. Sequenase DNA sequencing kit was from US Biochemicals. Trypanothione and deazatrypanothione (Henderson et al., 1987b) were kind gifts of Dr. G. Henderson (The Rockefeller Univ-ersity, NewYork, NY). All other biochemical reagents were obtained from Sigma Chemical Co. unless otherwise indicated. Vectors and Bacterial Strains. M13mpl8 and M13mpl9 were from Amersham. pKK233-2 and pUC13 were from Pharmacia. pBS+ was purchased from Stratagene. pGPl-2 (Tabor & Richardson, 1985), a plasmid containing the T7 RNA polymerase gene under control of the temperature-sensitive cI857 X repressor, was a gift of Dr. S. Tabor (HarvardMedical School, Boston, MA). Plasmid pCGTR-2 contains the T. congolense trypanothione reductase gene on a 3.3-kb BamHl insert (Shames et al., 1988). Plasmid pSD8SP that contains a tac II promoter and strong ribosome binding site (Shindeling et al., submitted for publication) was a gift from Dr. L. Gold (University of Colorado, Boulder, CO). E. coli strains JM101 and JM105 were from New England Biolabs. E. coli strain SG5 (Greer & Perham, 1986), a glutathione reductase deletion mutant, was a gift of Dr. R. Perham (Cambridge University, Cambridge, England). All bacterial strains were grown on 2XTY media that contained 16 g of Bactotryptone, 10 g of Bacto yeast extract, and 5 g of NaCl per liter. Antibiotics were included when appropriate. For enzyme preparations, bacterial strains transformed with either