DNA-support coupling for transcription factor purification. Comparison of aldehyde, cyanogen bromide and N-hydroxysuccinimide chemistries.
Purification of transcription factor IIIA on internal control region DNA coupled to aldehyde-silica is described and compared with purification on cyanogen bromide-activated Sepharose and Bio-Rad Affi-Gel-10. The Affi-Gel support results in mixed-mode chromatography; both ion-exchange and affinity modes contribute. Coupling DNA to aldehyde-silica is advantageous in that it has no ion-exchange properties and performs as well as DNA coupled to CNBr-activated Sepharose. Purification of lac repressor on aldehyde-silica, and CAAT enhancer binding protein on Affi-Gel also shows the advantages of a neutral support and the disadvantages of mixed-mode chromatography for transcription factor purification. Aldehyde-silica couples to alkylamines and to the amines of adenine, guanine, and cytosine nucleoside bases. Reaction occurs with either single- or double-stranded DNA, although it is less efficient with the latter. Overall, the results demonstrate that predominantly neutral coupling chemistries, such as aldehyde or CNBr-mediated coupling, have distinct advantages for transcription factor purification. Since the CNBr chemistry has not yet been applied to silica supports, aldehyde-silica coupling is currently the most attractive method for DNA affinity HPLC.
Chockalingam
Priya Sethu
PS
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Gadgil
Himanshu
H
Jarrett
Harry W
HW
D000447:Aldehydes / Q000737:chemistry*; D001483:Base Sequence; D003488:Cyanogen Bromide / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D013388:Succinimides / Q000737:chemistry*; D014157:Transcription Factors / Q000302:isolation & purification*
D000447:Aldehydes; D017931:DNA Primers; D013388:Succinimides; D014157:Transcription Factors; D004247:DNA; C001426:N-hydroxysuccinimide; D003488:Cyanogen Bromide