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...

Full description

Saved in:
Bibliographic Details
Published in:Biochimica et biophysica acta 2000-12, Vol.1524 (2), p.149-154
Main Authors: Piatibratov, Mikhail, Hou, Shaobin, Brooun, Alexei, Yang, Jinsheng, Chen, Huaiyang, Alam, Maqsudul
Format: Article
Language:English
Subjects:
Archaeal Proteins - biosynthesis
Archaeal Proteins - genetics
Archaeal Proteins - isolation & purification
Buffers
Cations, Divalent
Chromatography, Affinity - methods
Cobalt
Escherichia coli - metabolism
Gene Expression
Halobacterium
Halobacterium - genetics
Halobacterium - metabolism
Hemeproteins - biosynthesis
Hemeproteins - genetics
Hemeproteins - isolation & purification
Histidine
Metal chelate chromatography
Oxygen sensing transducer
Perfusion chromatography
Polyhistidine tag
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0304-4165
0006-3002
1872-8006
DOI:10.1016/S0304-4165(00)00151-3