Elsevier

Analytical Biochemistry

Volume 390, Issue 2, 15 July 2009, Pages 221-223
Analytical Biochemistry

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Thiol protection in membrane protein purifications: A study with phage holins

https://doi.org/10.1016/j.ab.2009.04.031Get rights and content

Abstract

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′-dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′-dithiodipyridine protection protocol was also successfully used for another unrelated holin, S2168, and should be generally useful for the purification of membrane proteins.

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Acknowledgments

We thank the members of the Young lab, past and present, for their support, especially Ting Pang who generously provided the plasmid pETS2168G48Chis, and Daisy Wilbert for her clerical assistance. This work was supported by PHS grant GM27099 to R.Y. and the Program for Membrane Structure and Function, a Program of Excellence grant from the Office of the Vice President for Research at Texas A&M University.

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