Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins

Methods in Enzymology
Max J L J FürstMarco W Fraaije

Abstract

Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools. Accordingly, algorithms to predict the differences in folding energy of a mutant compared to the wild-type, ΔΔGfold, are used in the highly successful FRESCO workflow. The resulting single mutant prediction library consists typically of a few hundred amino acid exchanges, and after combining the most successful hits we so far obtained stabilized mutants which exhibited increases in apparent melting temperature of 20-35°C and showed vastly increased half-lives, as well as resistance to cosolvents. Here, we report a detailed protocol to generate these mutant libraries experimentally, covering the entire workflow from primer design, through mutagenesis, prot...Continue Reading

Citations

Jan 22, 2020·Chembiochem : a European Journal of Chemical Biology·Hesam ArabnejadDick B Janssen
Dec 11, 2019·International Journal of Molecular Sciences·Nikola LončarMarco W Fraaije
Aug 14, 2020·Chembiochem : a European Journal of Chemical Biology·Chao GuoGerrit J Poelarends

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