Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli.
A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D012097:Repressor Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014157:Transcription Factors / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein