Abstract
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, cassette 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. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.
Highlights
Directed evolution is a powerful technique used to improve protein properties
An iterative process involving random circular permutations of bla and random insertions of bla into the gene for maltose binding protein yielded hybrid proteins in which BLA enzyme activity was modulated by maltose [19]
MBP317347, was identified from Library 7 in which random circular permutations of bla were inserted in place of the codon 317 in the gene for maltose binding protein (MBP)
Summary
Directed evolution is a powerful technique used to improve protein properties. Directed evolution involves the generation of a protein library and subsequent rounds of selection or screening to identify improved protein variants. Generation of diverse protein libraries containing improved variants is important for successful application of directed evolution techniques. Many studies have demonstrated the use of random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches as effective diversity generating methods for directed evolution. Circular permutation is an atypical method for diversity generation, since it can be used to change the linear order of the primary sequence, but not the identity of the amino acids in the sequence. Circular permutation can alter the folding kinetics of a protein and create variants that could retain comparable wild-type functionality [2,3,4,5]
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