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Structure-guided SCHEMA recombination of distantly related β-lactamase

Meyer, Michelle M. and Hochrein, Lisa and Arnold, Frances H. (2006) Structure-guided SCHEMA recombination of distantly related β-lactamase. Protein Engineering, Design and Selection, 19 (12). pp. 563-570. ISSN 1741-0126. doi:10.1093/protein/gzl045.

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We constructed a library of β-lactamases by recombining three naturally occurring homologs (TEM-1, PSE-4, SED-1) that share 34–42% sequence identity. Most chimeras created by recombining such distantly related proteins are unfolded due to unfavorable side-chain interactions that destabilize the folded structure. To enhance the fraction of properly folded chimeras, we designed the library using SCHEMA, a structure-guided approach to choosing the least disruptive crossover locations. Recombination at seven selected crossover positions generated 6561 chimeric sequences that differ from their closest parent at an average of 66 positions. Of 553 unique characterized chimeras, 111 (20%) retained β-lactamase activity; the library contains hundreds more novel β-lactamases. The functional chimeras share as little as 70% sequence identity with any known sequence and are characterized by low SCHEMA disruption (E) compared to the average nonfunctional chimera. Furthermore, many nonfunctional chimeras with low E are readily rescued by low error-rate random mutagenesis or by the introduction of a known stabilizing mutation (TEM-1 M182T). These results show that structure-guided recombination effectively generates a family of diverse, folded proteins even when the parents exhibit only 34% sequence identity. Furthermore, the fraction of sequences that encode folded and functional proteins can be enhanced by utilizing previously stabilized parental sequences.

Item Type:Article
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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© The Author 2006. Published by Oxford University Press. Received: 20 September 2006; Accepted: 27 September 2006; Published: 06 November 2006. We thank Costas Maranas, Brian Shoichet and Joelle Pelletier for their comments. The sed-1 gene was a gift from S. Petrella and W. Sougakoff. This work was supported by NIH R01 GM068664, an HHMI predoctoral fellowship (to M.M.M.), and a NSF graduate research fellowship (to L.H.).
Funding AgencyGrant Number
NIHR01 GM068664
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Subject Keywords:chimera; directed evolution; mutational robustness; protein design
Issue or Number:12
Record Number:CaltechAUTHORS:20180925-132315308
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Official Citation:Michelle M. Meyer, Lisa Hochrein, Frances H. Arnold; Structure-guided SCHEMA recombination of distantly related β-lactamases, Protein Engineering, Design and Selection, Volume 19, Issue 12, 1 December 2006, Pages 563–570,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89920
Deposited By: Tony Diaz
Deposited On:25 Sep 2018 21:52
Last Modified:16 Nov 2021 00:39

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