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Published December 2006 | Supplemental Material
Journal Article Open

Structure-guided SCHEMA recombination of distantly related β-lactamase

Abstract

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.

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.).

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Created:
August 22, 2023
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October 18, 2023