Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer

A Co2+-affinity, fast;flow perfusion chromatography method to purify a poly histidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been deve loped. The method relies upon a six-histidine affinity tag fused to the C-t erminus and N-terminus of HemAT-Hs for expression in the native host, an ex tremely halophilic Archaeon Halobacterium salinarum, and in the heterologou s host Escherichia coil, respectively. The His-tagged HemAT-Hs can be purif ied rapidly using either low or high ionic strength buffers. Purified His-t agged HemAT-Hs in high or low salt buffers demonstrated no difference in sp ectral characteristics and retained reversible oxygen binding capacity. Thi s fast-flow Co2+-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophys ical and structural studies. (C) 2000 Elsevier Science B.V. All rights rese rved.