title: Improving a circularly permuted TEM-1 beta-lactamase by directed evolution.
abstract: Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme.
authors.affiliation: Instituto de Biotecnología, UNAM, Apdo. Postal 510-3 Cuernavaca, Morelos 62250, México. joel@ibt.unam.mx
authors.forename: Joel
authors.initials: J
authors.lastname: Osuna
authors.forename: Alejandra
authors.initials: A
authors.lastname: Pérez-Blancas
authors.forename: Xavier
authors.initials: X
authors.lastname: Soberón
mesh_terms: D019020:Directed Molecular Evolution; D004926:Escherichia coli; D014644:Genetic Variation; D066298:In Vitro Techniques; D008958:Models, Molecular; D010456:Peptides, Cyclic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D010456:Peptides, Cyclic; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1