RESUMO
DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.
Assuntos
Complexo de Endopeptidases do Proteassoma , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Serina Endopeptidases/genética , Subtilisinas/genética , Estabilidade Enzimática , Biblioteca Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/classificação , Peptídeo Hidrolases/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Seleção Genética , Serina Endopeptidases/metabolismo , Subtilisinas/isolamento & purificação , Subtilisinas/metabolismoRESUMO
BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.
Assuntos
Evolução Molecular Direcionada , Hidrolases/química , Hidrolases/genética , Proteínas/química , Triazinas/química , Aminoidrolases , Escherichia coli/química , Escherichia coli/genética , Hidrolases/metabolismo , Mutagênese Sítio-Dirigida , Proteínas/genética , Proteínas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Triazinas/metabolismoRESUMO
The accuracies with which pharmacists and technicians checked medications in a unit dose distribution system were compared. The study was conducted at three large hospitals in Washington State. From August through October 1991, technicians filled unit dose medication drawers and pharmacists verified the accuracy of each fill (pharmacist verification period). From November 1991 through January 1992, technicians who had undergone special training verified the accuracy with which medication drawers were filled by other technicians (technician verification period). For each study period, two error rates were estimated: the frequency with which the pharmacists or technicians identified dispensing errors and the frequency of verification errors identified by the investigators in a final independent check. A total of 143,952 unit doses were dispensed during the pharmacist verification period, of which 49,718 were randomly analyzed for accuracy. A total of 151,721 doses were dispensed during the technician intervention period, of which 55,470 were assessed. The mean +/- S.E. daily rates of dispensing-error identification by pharmacists (0.0125 +/- 0.0069%) and technicians (0.0119 +/- 0.0001) did not differ significantly. While pharmacists overlooked more errors (107) than technicians (50), the percentage of such missed errors classified as potentially serious did not differ significantly between the groups (25.2% versus 32.0%, respectively). Pharmacy technicians who underwent special training were able to verify medications in a unit dose distribution system without compromising the accuracy of dispensing.
Assuntos
Erros de Medicação , Farmacêuticos/normas , Técnicos em Farmácia/normas , Educação em Farmácia , Sistemas de Medicação , Serviço de Farmácia Hospitalar/organização & administração , WashingtonRESUMO
Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.