Smith, Matthew A. and Bedbrook, Claire N. and Wu, Timothy and Arnold, Frances H. (2013) Hypocrea jecorina Cellobiohydrolase I Stabilizing Mutations Identified Using Noncontiguous Recombination. ACS Synthetic Biology, 2 (12). pp. 690-696. ISSN 2161-5063. https://resolver.caltech.edu/CaltechAUTHORS:20131015-082946657
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Abstract
Noncontiguous recombination (NCR) is a method to identify pieces of structure that can be swapped among homologous proteins to create new, chimeric proteins. These “blocks” are encoded by elements of sequence that are not necessarily contiguous along the polypeptide chain. We used NCR to design a library in which blocks of structure from Hypocrea jecorina cellobiohydrolase I (Cel7A) and its two thermostable homologues from Talaromyces emersonii and Chaetomium thermophilum are shuffled to create 531,438 possible chimeric enzymes. We constructed a maximally informative subset of 35 chimeras to analyze this library and found that the blocks contribute additively to the stability of a chimera. Within two highly stabilizing blocks, we uncovered six single amino acid substitutions that each improve the stability of H. jecorina cellobiohydrolase I by 1−3 °C. The small number of measurements required to find these mutations demonstrates that noncontiguous recombination is an efficient strategy for identifying stabilizing mutations.
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| Additional Information: | © 2013 American Chemical Society. Just Accepted Manuscript May 20, 2013, Received: February 14, 2013. Published: May 20, 2013. The authors thank John McIntosh and Mary Farrow for useful discussions in the preparation of this manuscript. This work was supported by the Institute for Collaborative Biotechnologies through a grant [W911NF-09-D-0001] from the U.S. Army Research and The National Central University, Taiwan, though a Cooperative Agreement for Energy Research Collaboration. M.A.S. is supported by a Resnick Sustainability Institute fellowship. | |||||||||
| Group: | Resnick Sustainability Institute | |||||||||
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| Subject Keywords: | CBHI, Cel7A, cellulase, protein recombination, thermostability | |||||||||
| Issue or Number: | 12 | |||||||||
| Record Number: | CaltechAUTHORS:20131015-082946657 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20131015-082946657 | |||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 41911 | |||||||||
| Collection: | CaltechAUTHORS | |||||||||
| Deposited By: | Joy Painter | |||||||||
| Deposited On: | 15 Oct 2013 20:24 | |||||||||
| Last Modified: | 09 Mar 2020 13:18 |
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