circular permutation in the ω-loop of tem-1 β-lactamase results in improved activity and altered substrate specificity圆形排列的ω-loop tem-1β-lactamase改善活动的结果和底物特异性改变.pdfVIP

Circular Permutation in the V-Loop of TEM-1 b- Lactamase Results in Improved Activity and Altered Substrate Specificity ¤ Gurkan Guntas , Manu Kanwar, Marc Ostermeier* Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America 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 b- lactamase to selections on increasing concentrations of a variety of b-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 V-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. Citation: Guntas G, Kanwar M, Ostermeier M (2012) Circular Permutation in the V-Loop of TEM-1 b-Lactamase Results in Improved Activity and Altered Substrate Specificity. PLoS ONE 7(4): e35998. doi:10.1371/journal.pone.0035998 Editor: Mark Isalan, Center for Genomic Regulation, Spain Received February 24, 2012; Accepted March 27, 2012; Published April 1