Novel DNA-Sepharose purification of the FadR transcription factor.
A DNA sequence bound by the FadR transcription factor of Escherichia coli was covalently attached to Sepharose by two different approaches: by chemical coupling or by template-directed enzymatic synthesis using a DNA polymerase. The two kinds of DNA-Sepharose were packed into small columns and used for the purification of the FadR protein; chromatography was without using competitor DNA and the supports contained single-copy, non-repetitive DNA sequences. Comparison showed that the enzymatically prepared support, while having less bound DNA, bound more FadR protein than did the chemically prepared support. This probably results from the lack of detrimental DNA modification by the gentle enzymatic procedure. The chemically prepared support was of lower capacity but yielded purer FadR protein when compared under the same elution conditions. This may be explained by the simpler DNA sequence which could be coupled chemically; less contaminating proteins were bound by the simpler DNA sequence. However, the enzymatically prepared support could also yield comparable purity if the protocol was modified to include additional washes with salt containing buffers. In all cases, FadR was eluted from the DNA using high-salt (0.8 M) mobile phase; ligand-specific elution of FadR using a fatty acyl-coenzyme A thiol ester was ineffective. Affinity chromatography on DNA-Sepharose provided a more rapid, simple purification of FadR than conventional purification techniques and yielded biologically active protein.
DiRusso
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Department of Biochemistry, University of Tennessee, Memphis 38163.
Rogers
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RP
Jarrett
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HW
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D001483:Base Sequence; D002845:Chromatography; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*
D001426:Bacterial Proteins; C057119:FadR protein, Bacteria; D008024:Ligands; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA; D012685:Sepharose; D004259:DNA-Directed DNA Polymerase