Purification of a Tat leader peptide by co-expression with its chaperone

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• 作者：Charles M. Stevens ; Mark Paetzel ; ^{mpaetzel@sfu.ca}
• 关键词：Leader peptide ; Signal peptide ; Twin arginine translocase ; Dimethyl sulfoxide reductase ; REMP ; Chaperone
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：July, 2012
• 卷：84
• 期：1
• 页码：167-172
• 文件大小：630 K

摘要

We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA_L), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA_L/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA_L and DmsD. A construct with DmsA_L fused to the N-terminus of DmsD (DmsA_L-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA_L-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.