Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase

Biochemistry
Bryan J JonesRomas J Kazlauskas

Abstract

A review of the previous stabilization of α/β-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at http://kazlab.umn.edu .

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Citations

Jun 12, 2018·Chembiochem : a European Journal of Chemical Biology·Alexander SchwengerClemens Richert
May 22, 2020·Frontiers in Bioengineering and Biotechnology·Bernardo J Gomez-FernandezMiguel Alcalde
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Jun 3, 2021·Proteins·Clauber Henrique Souza da CostaJerônimo Lameira
May 22, 2018·ACS Synthetic Biology·Jeffrey C MooreMatthew D Truppo

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