Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit.
Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit.
复制标题
大肠杆菌亚硫酸盐还原酶的质子核磁共振：未连接的血红素蛋白亚基。
DOI：
10.1021/bi00062a017
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Siegel,LM
中科院分区：
文献类型：
--
作者：
Kaufman,J;Spicer,LD;Siegel,LM
材料和方法。大肠杆菌K12在最小培养基上批量生长（90-100公斤）（Siegel等人，1973;Grain Processing Corp, Muscatine， IA），并在77 K下保存在2.5公斤的蛋糕中直到使用。大肠杆菌nadph -亚硫酸盐还原酶通过已发表的程序（Siegel et al ., 1973; Siegel & Davis, 1974）从该细胞膏中纯化，并进行了以下细微修改：省略了琼脂糖柱的最后一步。在3 M尿素-10 mM KP±（pH 7.7）中室温孵育30 min，然后在50 mM KP±4 M尿素-100µEDTA （pH 7.7）中deae -纤维素层析，将第二步硫酸铵沉淀后的全酶与血红蛋白亚基分离。将SiR-HP进一步纯化，应用于在100 mM KP±pH 7.7 -1.0 M硫酸铵中平衡的phenyl-Sepharose CL4B （Pharmacia）柱上，然后在SBE中逐级梯度为0.5,0.3和0.1 M硫酸铵。分离的SiR-HP按纯度指数（v^ so/^ as?）分为两组。·纯度指数小于1.75的蛋白质用于核磁共振研究，而纯度指数大于1.75的蛋白质仅用于血红素提取。如果只需要siroheme，则省略最后的列步骤。分离和SBE透析后，用Amicon过滤浓缩至110µ，保存在液氮中待用。使用€591= 18.1 mM-1 cm-1分光光度法测定浓度（Siegel et al, 1982）。后来的研究使用了从全酶或血红蛋白亚基的质粒过量生产者获得的亚硫酸盐还原酶。全酶过量产生者由pT7T3质粒组成，在天然启动子控制下含有cysl、cy¿J和cysG （Wu etal, 1991）。质粒在一株鼠伤寒沙门菌中生长，这种沙门菌是胱氨酸的，因此只有大肠杆菌亚硫酸盐还原酶存在。细胞在37℃的最低盐培养基（Medium E; Vogel & Bonner, 1956）中在New Brunswick发酵罐中生长，工作体积为174 L.典型产量为每次运行500-800 g湿细胞膏，每g细胞膏中添加5-15 mg亚硫酸盐还原酶全酶。改进了全酶的分离方法（Siegel et al ., 1973）
MATERIALS AND METHODSE. coli K12 was grown in bulk (90-100 kg) on minimal media (Siegel et al, 1973; Grain Processing Corp, Muscatine, IA) and stored at 2.5-kg cakes at 77 K until used. E. coli NADPH-sulfite reductase was purified from this cell paste by published procedures (Siegel et al, 1973; Siegel & Davis, 1974) with the following minor modifications: The final agarose column step was omitted. The hemeprotein subunit was dissociated from the holoenzyme present after the second ammonium sulfate precipitation step by room temperature incubation in 3 M urea-10 mM KP¡(pH 7.7) for 30 min followed by DEAE-cellulose chromatography in 50 mM KP¡-4 M urea-100 µ EDTA (pH 7.7). The SiR-HP was further purified by application to a column of phenyl-Sepharose CL4B (Pharmacia) equilibrated in 100 mM KP¡(pH 7.7)—1.0 M ammonium sulfate followed by a step gradient of 0.5, 0.3, and 0.1 M ammonium sulfate in SBE. The isolated SiR-HP was divided into two pools by the purity index (v^ so/^ as?)· Protein with a purity index less than 1.75 was used for NMR studies while that with an index greater than 1.75 was used solely for heme extraction. In cases where only siroheme was desired, the final column step was omitted. Following isolation and dialysis vs SBE, the protein was concentrated by Amicon filtration to 110 µ and stored in liquid nitrogen until used. Concentrations were determined spectrophotometrically using an€ 591= 18.1 mM-1 cm-1 (Siegel et al, 1982). Later studies used sulfite reductase obtained from plasmid overproducers of the holoenzyme or the hemeprotein subunit. The holoenzyme overproducer consisted of a pT7T3 plasmid with cysl, cy¿ J, and cysG under native promoter control (Wu etal, 1991). The plasmid was grown in a strain of Salmonella typhimurium that was cysl-so that only the E. coli sulfite reductase was present. Cells were grown on minimal salts media (Medium E; Vogel & Bonner, 1956) at 37 C in a New Brunswick fermentor with a working volume of 174 L. Typical yields were 500-800 g of wet cell paste per run with 5-15 mg of sulfite reductase holoenzyme/g of cell paste. The isolation procedure for holoenzyme (Siegel et al, 1973) was modified