William Himanshu S. Gadgil

Document Type: Doctoral Dissertation
Name: William Himanshu S. Gadgil
Email address: gethim007@hotmail.com
Title: Advances in DNA Affinity Chromatography
Degree: Ph.D.
Program: Biochemistry
Research Advisor: Harry W. Jarrett, Ph.D.
Advisor's email: hjarrett@utmem.edu
Committee Members: William L. Taylor, Ph.D.
Satoru K. Nishimoto, Ph.D.
Roderick T. Hori, Ph.D.
Edwards A. Park, Ph.D.
 
 
Keywords: DNA affinity chromatography, transcription factor,
purification
Availability: Release the entire work for world access.

Abstract

Different aspects of DNA affinity chromatography such as DNA complexity heparin elution, the Bi-column method and the oligonucluotide trapping method were studied. The complexity (length) of a DNA sequence attached to an affinity chromatography column affects column retention, and the purity of transcription factors obtained. T18: A18 tailed DNA affinity columns were better suited for purification of most of the transcription factors than either the discrete or concatemeric DNA affinity columns. A novel method using heparin for eluting transcription factors from DNA Sepharose columns was characterized. The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration. The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA. These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution. Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction of lac repressor was significantly purer than that eluted with salt and comparable to that obtained by elution with the specific ligand IPTG, a lactose analog. A novel Bi-column method was developed in which lac repressor is eluted from the Op1-Sepharose with a low heparin concentration and trapped on a Op1T18-Sepharose column because of its higher affinity for the lac repressor protein. Elution of the latter column with buffer containing a high salt concentration gives significantly purer transcription factor than the conventionally used single column methods and removes residual heparin. Highly pure CAAT enhancer binding protein(C/EBP) and the B3 transcription factor are also obtained by using variants of this Bi-column method. A new oligonucleotide trapping method in which a short oligonucleotide coupled to Sepharose is used to trap a complex of the transcription factor and its corresponding specific DNA sequence was developed. Highly purified transcription factor B3 was obtained using the oligonucleotide trapping method.

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Revised 24 Nov.  2003