Purification and characterization of 6xHis-tagged Bm3R1 repressor of Bacillus megaterium.
The bm3R1 gene-encoded repressor controls the expression of cytochrome P450BM-3 gene as well as its own expression in Bacillus megaterium. We have developed an efficient system for overexpression and purification of Bm3R1 protein by nickel ion affinity chromatography. Adding six histidine residues at either N-terminus or C-terminus of Bm3R1 repressor caused no loss of its operator DNA binding ability. We have investigated the interaction between Bm3R1(His)6 and its operator DNA by equilibrium and kinetic methods. The results showed that the apparent dissociation constant is around 1.8 x 10(-9) M and the half-life of Bm3R1(His)6-operator complex is about 50 minutes. We have also found by gel filtration chromatography that Bm3R1(His)6 may exist in low oligomeric state(s) in solution, which is competent for binding to its operator DNA.
Shaw
G C
GC
Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, R.O.C.
Sun
C H
CH
Chiang
A
A
D001410:Bacillus megaterium / Q000737:chemistry*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000302:isolation & purification*; D014157:Transcription Factors
D001426:Bacterial Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C073493:Bm3R1 protein, Bacillus megaterium; D006639:Histidine