Multidrug transporter MexB of Pseudomonas aeruginosa:: overexpression, purification, and initial structural characterization
Multidrug transporter MexB of Pseudomonas aeruginosa:: overexpression, purification, and initial structural characterization
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DOI：
10.1016/j.pep.2004.10.002
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发表时间：
2005-03-01
影响因子：
1.6
通讯作者：
Nakae, T
中科院分区：
文献类型：
--
作者：
Mokhonov, V;Mokhonova, E;Nakae, T
铜绿假单胞菌的多药转运蛋白MexB的结构和功能表征由于天然来源的培养物的约0.1 mg/L的低产率而受到显著限制。为了促进这种医学上重要的转运蛋白的结构研究，我们开发了一个大规模的系统，用于在大肠杆菌细胞中表达基因工程重组体MexB。使用该系统，获得的MexB的最终产量为约10 mg/L的培养物。在十二烷基β-D-麦芽糖苷存在下的优化纯化方案允许分离高度均质的MexB。已对蛋白质在洗涤剂溶液中的寡聚状态进行表征，以验证纯化蛋白质的天然状态已得到保留。发现蛋白质-去污剂复合物的分子量为380-450 kDa。MexB-十二烷基β-D-麦芽糖苷质量比测定为1.8 +/-0.05。考虑到单体MexB的分子量推导出其氨基酸序列（112.8 kDa），我们得出结论，纯化的MexB作为homotrimer在表面活性剂溶液中存在。MexB的圆二色性分析显示α-螺旋结构占主导地位。MexB的高产率、均一性和稳定性使其成为结构和功能表征的良好候选物。(c)2004年爱思唯尔公司All rights reserved.
Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10 mg/L of culture. The optimized purification protocol in the presence of dodecyl P-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450 kDa. The MexB-dodecyl P-D-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the a-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization. (c) 2004 Elsevier Inc. All rights reserved.