Expression and purification of the central stalk subunits of Na + -translocating V-type ATPase from Enterococcus hirae
Expression and purification of the central stalk subunits of Na + -translocating V-type ATPase from Enterococcus hirae
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海拉肠球菌Na转位V型ATP酶中央柄亚基的表达与纯化
DOI：
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
I. Yamato
中科院分区：
文献类型：
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作者：
K. Hossain;S. Arai;S. Saijo;Y. Kakinuma;T. Murata;I. Yamato
肠球菌（E.液泡膜ATP酶（V-ATP酶）由一个可溶性催化结构域（V1; NtpA 3 -B 3 -D-G）和一个完整的膜结构域（Vo; NtpI-K10）组成，它们之间通过中心柄和外周柄相连。Na +转运V型ATP酶的中心柄。histoprotein由NtpC、NtpD和NtpG亚基组成。本研究的目的是克隆和表达E. HIV-type Na + -ATPase。在这里，我们将合成的DNA片段（对应于ntpC、ntpD和ntpG基因）克隆到质粒载体pET 23 d中。将NtpC、NtpD和NtpG亚基蛋白分别以His标记的可溶性蛋白形式在大肠杆菌BL 21（DE 3）细胞中表达，然后通过Ni Sepharose 6快速流动柱纯化。表达产物经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定。纯化的NtpC、NtpD和NtpG亚基蛋白的量分别测量为14、17和15 mg/l升培养物。关键词：肠球菌，V-ATP酶，中心柄亚基，表达。
Enterococcus hirae (E. hirae) vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V 1 ; NtpA 3 -B 3 -D-G) and an integral membrane domain (V o ; NtpI-K 10 ) connected by a central and peripheral stalks. Central stalk of Na + -translocating V-type ATPase of E. hirae is composed of NtpC, NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na + -ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, into the plasmid vector, pET23d. NtpC, NtpD and NtpG subunit proteins were expressed, separately as His-tagged soluble proteins in Escherichia coli BL21(DE3) cells and then, purified by Ni Sepharose 6 fast flow column. Purification of expressed protein was confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The amount of purified NtpC, NtpD and NtpG subunit proteins were measured as 14, 17 and 15 mg/1 liter culture, respectively. Key words: Enterococcus hirae, V-ATPase, central stalk subunits, expression.
影响因子：
50.3
作者：
Li, SQ;Schmitz, KR;Ferguson, KM
通讯作者：
Ferguson, KM