A rapid method to capture and screen for transcription factors by SELDI mass spectrometry.
A novel method to screen for transcription factors binding to promoter DNA sequences has been developed using DNA chip surfaces and mass spectrometry. This technique was demonstrated with Escherichia coli lac repressor, LacI. The consensus promoter binding sequence for LacI and a scrambled version of the same DNA sequence were prepared on two affinity chip surfaces. Total E. coli protein lysate was applied to the two surfaces. A 38.2 kDa protein, as detected by SELDI-MS, was captured on the chip surface containing the binding sequence for LacI but not on the surface containing the scrambled sequence. The protein was identified following one-step, small-scale affinity capture and peptide mapping. Subsequent database searches identified the 38.2 kDa protein as the lac repressor of E. coli. We discuss application of DNA chip affinity capture to characterize transcription factors and to screen for differences in cellular regulatory networks.
Forde
Cameron E
CE
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550, USA.
Gonzales
Arlene D
AD
Smessaert
Jean M
JM
Murphy
Gloria A
GA
Shields
Sharon J
SJ
Fitch
J Patrick
JP
McCutchen-Maloney
Sandra L
SL
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D020411:Oligonucleotide Array Sequence Analysis; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D014157:Transcription Factors