Combining computational and experimental screening for rapid optimization of protein properties

We present a combined computational and experimental method for the rapid optimization of proteins. Using β-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space. By eliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of ≈200,000 mutants. These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime. In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance. To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM β-lactamases, the most prevalent type of Gram-negative β-lactamases. This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.