...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of bacteriology >Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.
【24h】

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

机译：大肠杆菌中algE的过表达：亚细胞定位，纯化和离子通道特性。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.
机译：铜绿假单胞菌的藻酸盐生产（粘液）菌株具有54 kDa的外膜（OM）蛋白（AlgE），该蛋白在非粘液细菌中缺失。来自粘液状铜绿假单胞菌CF3 / M1的algE基因的编码区被亚克隆到表达载体pT7-7中，并在大肠杆菌中表达。重组AlgE的表达水平比铜绿假单胞菌中的天然蛋白高7倍。重组AlgE主要在OM中发现。可以在细胞质膜（CM）中通过免疫学方法检测到推定的AlgE前体蛋白（56 kDa）。通过用N-羟基琥珀酰亚胺-生物素标记赖氨酸残基来指示AlgE在大肠杆菌OM中的表面暴露。二级结构分析表明，AlgE通过18条跨膜的β链锚固在OM中，可能形成了β桶。纯化了重组AlgE，等电聚焦显示pI为4.4。重组AlgE自发结合到平面脂质双层中，形成离子通道，在1 M KCl中的单通道电导为0.76 nS，平均寿命为0.7 ms。在存在其他盐的情况下进行单通道电流测量以及在盐梯度中进行逆向电势测量表明，AlgE通道具有强烈的阴离子选择性。对于氯离子，计算出弱的结合常数（Km = 0.75 M），这表明AlgE可能构成对另一种特定阴离子（例如，藻酸盐的前体）聚甘露糖醛酸具有特异性的离子通道。与此想法一致，当添加GDP-甘露糖醛酸时，通道的开态概率降低。当施加高于+85 mV的膜电压时，AlgE通道失活。 AlgE的电生理特性（包括其整流特性）与典型孔蛋白有很大的不同。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号