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Title: A system for the generation and identification of mutant DNA-binding proteins
Author: Webster, Scott
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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GCN4 is a transcriptional activatory protein found in S. cerevisiae and is a member of the basic region leucine zipper (bZip) family of DNA-binding proteins. The full length protein is 281 residues in length and consists of an activatory region and a DNA-binding region. The DNA-binding region of the protein consists of two separate regions; a leucine zipper through which he protein dimerises, and a basic region which contacts target DNA. Various studies have shown that it is possible to mutate residues in the basic region and create proteins with changed specificity. The aim of this project was to use λZAPII technology to create a large library of mutants and investigate the effects of single and double mutants in the basic region and linker segment of GCN4. The DNA-binding (bZip) region of GCN4 was extracted from a plasmid containing the entire GCN4 gene and cloned into the lacZ gene of the bacteriophage vector λZAPII. The recombinant vector was infected into a strain of E. coli which allowed the bZip gene to be expressed as a fusion protein with β-galactosidase. In vitro screening experiments were carried out and any 'positive' clones which bound to a DNA target identified. The DNA sequence of each positive clone was then determined. These experiments demonstrated that the λZAPII system was an excellent vehicle for the expression and identification of a DNA library and that identical conditions could be used to screen a large library of mutants. A phagemid containing the bZip gene was excised from λZAPII in vivo and infected into E. coli. These cells were used to overexpress the β-galactosidase-bZip fusion protein. The fusion protein was purified and shown to bind with high affinity to a specific DNA target.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available