Persistent Link:
http://hdl.handle.net/10150/244476
Title:
Efforts Towards the Directed Evolution of a Protein Fold
Author:
Merritt, Jonathan Karl
Issue Date:
May-2012
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
More than a thousand different protein folds are known to exist, many of which have been characterized through dedicated research; however, there is little evidence of how these numerous structural forms arose through evolution. One approach to elucidating possible evolutionary pathways of protein structure is to perform directed evolution experiments on ancestrally related protein families which occupy different folds. One approach to the directed evolution of a protein fold is to follow a destroy-and-rebuild scheme where protein stability is knocked out through protein engineering, random mutagenesis is used to resurrect stability, and stable variants are selected through the powerful method of phage display coupled to proteolysis. The Cordes lab has previously identified a case suitable for such an investigation in the Cro protein family where two small DNA binding proteins, Xfaso 1 and P 6, occupy different folds yet retain high sequence identity. Previous work has identified deletion mutations which destroy the stability of the ancestral α-helical fold, but are also known to be compatible with the descendant β-sheet fold. Here, error prone PCR is presented as a possible method of resurrecting stability through random mutagenesis in Cro protein variants destabilized through these deletions. A protocol has been optimized for the production of mutant libraries of these genes Through EP-PCR. Test clones of these libraries into a prototype pCANTAB B phagemid vector for later phage display investigations have been successful, and sequencing has revealed that the implemented protocol yields an acceptable nucleotide substitution rate of 2.9%.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Chemistry and Biochemistry
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleEfforts Towards the Directed Evolution of a Protein Folden_US
dc.creatorMerritt, Jonathan Karlen_US
dc.contributor.authorMerritt, Jonathan Karlen_US
dc.date.issued2012-05-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractMore than a thousand different protein folds are known to exist, many of which have been characterized through dedicated research; however, there is little evidence of how these numerous structural forms arose through evolution. One approach to elucidating possible evolutionary pathways of protein structure is to perform directed evolution experiments on ancestrally related protein families which occupy different folds. One approach to the directed evolution of a protein fold is to follow a destroy-and-rebuild scheme where protein stability is knocked out through protein engineering, random mutagenesis is used to resurrect stability, and stable variants are selected through the powerful method of phage display coupled to proteolysis. The Cordes lab has previously identified a case suitable for such an investigation in the Cro protein family where two small DNA binding proteins, Xfaso 1 and P 6, occupy different folds yet retain high sequence identity. Previous work has identified deletion mutations which destroy the stability of the ancestral α-helical fold, but are also known to be compatible with the descendant β-sheet fold. Here, error prone PCR is presented as a possible method of resurrecting stability through random mutagenesis in Cro protein variants destabilized through these deletions. A protocol has been optimized for the production of mutant libraries of these genes Through EP-PCR. Test clones of these libraries into a prototype pCANTAB B phagemid vector for later phage display investigations have been successful, and sequencing has revealed that the implemented protocol yields an acceptable nucleotide substitution rate of 2.9%.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineChemistry and Biochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
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