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

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, S(21)68, and should be generally useful for the purification of membrane proteins.