Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer.
A Co(2+)-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been developed. The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT-Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively. The His-tagged HemAT-Hs can be purified rapidly using either low or high ionic strength buffers. Purified His-tagged HemAT-Hs in high or low salt buffers demonstrated no difference in spectral characteristics and retained reversible oxygen binding capacity. This fast-flow Co(2+)-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophysical and structural studies.
Piatibratov
M
M
Department of Microbiology, Snyder Hall 207, 2538 The Mall, University of Hawaii, Honolulu, HI 96822, USA.
Hou
S
S
Brooun
A
A
Yang
J
J
Chen
H
H
Alam
M
M
D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002021:Buffers; D002413:Cations, Divalent; D002846:Chromatography, Affinity / Q000379:methods; D003035:Cobalt; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006217:Halobacterium / Q000235:genetics / Q000378:metabolism*; D006420:Hemeproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine; D008565:Membrane Proteins
D019843:Archaeal Proteins; D002021:Buffers; D002413:Cations, Divalent; C404452:HEMAT protein, Halobacterium salinarum; D006420:Hemeproteins; D008565:Membrane Proteins; D003035:Cobalt; D006639:Histidine