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 lacI(q) 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-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.
Division of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, SO16 7PX, United Kingdom.
R M
RM
Owens
A
A
Grant
N
N
Davies
C D
CD
O'Connor
D001224:Aspartic Acid / Q000031:analogs & derivatives*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019921:Biotinylation; D002846:Chromatography, Affinity; D003035:Cobalt / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine; D007763:Lac Operon / Q000235:genetics; D056968:Lac Repressors; D013058:Mass Spectrometry; D008958:Models, Molecular; D008970:Molecular Weight; D009875:Operator Regions, Genetic / Q000235:genetics; D010449:Peptide Mapping; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C070802:carboxymethylaspartic acid agarose; C033223:polyhistidine; D001224:Aspartic Acid; D003035:Cobalt; D006639:Histidine; D012685:Sepharose; D020558:GTP Phosphohydrolases; C408370:typA protein, E coli