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Non-contiguous protein recombination

Citation

Smith, Matthew Alexander (2013) Non-contiguous protein recombination. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:04102013-164733450

Abstract

Swapping sequence elements among related proteins can produce chimeric proteins with novel behaviors and improved properties such as enhanced stability. Although homologous mutations are much more conservative than random mutations, chimeras of distantly-related proteins have a low probability of retaining fold and function. Here, I introduce a new tool for protein recombination that identifies structural blocks that can be swapped among homologous proteins with minimal disruption. This non-contiguous recombination approach enables design of chimeras and libraries of chimeras with less disruption than can be achieved by swapping blocks of sequence. Less disruption means that one can generate libraries with higher fractions of functional enzymes and enables recombination of more distant homologs.

Using this new tool I design and construct many functional chimeric cellulases. I illustrate the structurally conservative nature of this recombination by creating a functional prokaryotic-eukaryotic chimera and solving its structure. I also show how non-contiguous recombination can be used to efficiently identify stabilizing mutations that have been incorporated into homologs in nature.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:protein engineering; chimeragenesis; protein recombination; directed evolution; graph partitioning
Degree Grantor:California Institute of Technology
Division:Engineering and Applied Science
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Group:Resnick Sustainability Institute
Thesis Committee:
  • Tirrell, David A. (chair)
  • Marcus, Rudolph A.
  • Phillips, Robert B.
  • Arnold, Frances Hamilton
Defense Date:7 March 2013
Record Number:CaltechTHESIS:04102013-164733450
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:04102013-164733450
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1111/febs.12032DOIUNSPECIFIED
http://dx.doi.org/10.1002/pro.2202DOIUNSPECIFIED
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7607
Collection:CaltechTHESIS
Deposited By: Matthew Smith
Deposited On:07 May 2013 21:34
Last Modified:07 Oct 2014 17:58

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