A two-color bacterial two-hybrid system for selecting sequence-specific DNA-binding proteins via fluorescence activated cell sorting
Name
50575514-MIT.pdf
Description
Full printable version
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13.27 MB
Format
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59341f679b7233d18cc1582dc6a130f9
Author(s)
Miller, Jeffrey Christopher, 1974-
Advisor(s)
Carl O. Pabo.
Alternative Title
2-color bacterial 2-hybrid system for selecting sequence-specific deoxyribonucleic acid-binding proteins via fluorescence activated cell sorting
Date Issued
2002
Publisher
Massachusetts Institute of Technology
Abstract
Proteins that recognize specific DNA sequences play a major role in many biological processes, and the ability to select or design novel DNA binding proteins could have a major impact on many areas of biotechnology and medicine. This thesis begins with background information on zinc finger DNA-binding proteins and on methods to select these proteins, and how they can be used to regulate endogenous human genes. Next, I describe a structural and biochemical study of a DNA-binding protein which demonstrates some of the complexities of the protein-DNA interface, and which highlights difficulties for designing sequence-specific proteins via a simple code. I then develop an experimental system (starting with a pre-existing bacterial two-hybrid selection system) which allows the selection of proteins based on their preference of one DNA site over another. I use this system to attempt to attenuate the affinity of a zinc finger protein without destroying its specificity. Finally, I describe an experiment in which I select a new domain that adds sequence specificity to a pre-existing protein from a library of completely random peptides.
Description
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.
Vita.
Includes bibliographical references.
Subjects
Biology.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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