Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity generating step for directed evolution. A hybrid of maltose binding protein (MBP) and TEM1 ß-lactamase (BLA) in which BLA was circularly permuted (CP) in the Ω-loop and inserted into MBP conferred increased resistance to the antibiotic cefotaxime (CFTX). We wondered if this improved property resulted solely from the circular permutation of BLA and if other CP variants of BLA would also show improved properties. We subjected a library of randomly circularly permuted BLA to selections on increasing concentrations of a variety of ß-lactam antibiotics including CFTX. We identified CP variants that conferred elevated resistance to CFTX. These variants were also circularly permuted in the Ω-loop. In vitro kinetic characterization confirmed that circular permutation improved catalytic activity and altered the substrate specificity.

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