Copurification of the lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography
Copurification of the lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography
One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)-carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)6- tagged proteins are isolated from Escherichia coli host cells carrying the lacIq gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacIq-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.
352-360
Owens, R.M.
452d0bdb-1d28-47c1-a7f1-3004df231ea1
Grant, A.
538f8685-0173-4d0a-a87a-cb005a3a9092
Davies, N.
abd1ae3e-1857-4751-9bb7-5e3cb3a972d0
Connor, C.D.
0758d4c1-606a-4ed3-9308-b3c99c41dfe2
1 March 2001
Owens, R.M.
452d0bdb-1d28-47c1-a7f1-3004df231ea1
Grant, A.
538f8685-0173-4d0a-a87a-cb005a3a9092
Davies, N.
abd1ae3e-1857-4751-9bb7-5e3cb3a972d0
Connor, C.D.
0758d4c1-606a-4ed3-9308-b3c99c41dfe2
Owens, R.M., Grant, A., Davies, N. and Connor, C.D.
(2001)
Copurification of the lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography.
Protein Expression and Purification, 21 (2), .
(doi:10.1006/prep.2000.1384).
Abstract
One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)-carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)6- tagged proteins are isolated from Escherichia coli host cells carrying the lacIq gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacIq-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.
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Submitted date: 8 November 2000
Published date: 1 March 2001
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Local EPrints ID: 56628
URI: http://eprints.soton.ac.uk/id/eprint/56628
ISSN: 1046-5928
PURE UUID: 86b99fab-092c-4877-92da-8e6c5f0e1f3a
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Date deposited: 11 Aug 2008
Last modified: 15 Mar 2024 11:02
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Author:
R.M. Owens
Author:
A. Grant
Author:
N. Davies
Author:
C.D. Connor
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