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Results for “directed evolution”

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At least 505 records · Page 28Linked to original sources
PubMed1998

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis.

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.

Animals↗
PubMed2004

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering.

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.

Animals↗
PubMed2004

Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

Adipates↗
PubMed2003

Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3.

Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers. The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.

Alcohols↗
PubMed2006

Testing the directionality of evolution: the case of chydorid crustaceans.

Although trends are of central interest to evolutionary biology, it is only recently that methodological advances have allowed rigorous statistical tests of putative trends in the evolution of discrete traits. Oligomerization is one such proposed trend that may have profoundly influenced evolutionary pathways in many types of animals, especially arthropods. It is a general hypothesis that repeated structures (such as appendage segments and spines) tend to evolve primarily through loss. Although largely untested, this principle of loss is commonly invoked in morphological studies of crustaceans for drawing conclusions about the systematic placements of taxa and about their phylogeny. We present a statistical evaluation of this hypothesis using a molecular phylogeny and character matrix for a family of crustaceans, the Chydoridae, analysed using maximum likelihood methods. We find that a unidirectional (loss-only) model of character evolution is a very poor fit to the data, but that there is evidence of a trend towards loss, with loss rates of structures being perhaps twice the rates of gain. Thus, our results caution against assuming loss a priori, in the absence of appropriate tests for the characters under consideration. However, oligomerization, considered as a tendency but not a rule, may indeed have had ramifications for the types of functional and ecological shifts that have been more common during evolutionary diversification.

Cladocera↗
PubMed2000

Pulse oximetry: evolution and directions.

Pulse oximetry monitoring is a relatively recent technique that has been embraced enthusiastically by medical, nursing and paramedical personnel in many clinical situations. Few people, however, have any idea of how it provides the reading of arterial blood saturation. This review performs several functions: it discusses the haemoglobin molecular structure and explains its relation to spectrophotometry, describes the history and principles of pulse oximetry and the nature of the equipment used, and reviews its common uses. It also explores the principles and proposed uses of its lesser known corollary, photoplethysmography.

Hemoglobins↗
PubMed1996

Evolution of directional preferences in the supplementary eye field during acquisition of conditional oculomotor associations.

We assessed the preferred directions (PDs) of supplementary eye field (SEF) neurons during conditional visuomotor learning. Monkeys learned to select one of four saccadic eye movements in response to a foveal instruction stimulus (IS). ISs were either familiar or novel. Each familiar IS reliably evoked one saccade: 7 degrees left, right, up, or down form the central fixation point. Novel ISs initially triggered virtually random responses among those four possibilities, but the monkeys ultimately learned to select the instructed saccade. As reported previously, activity rates on novel IS trials significantly changed during learning. Some of these cells (learning-dependent) also have significant modulation on familiar IS trials, but others (learning-selective) lack such activity. Of the former, the familiar IS activity can be either directionally selective or omnidirectional. For most neurons, PDs were apparent during all phases of learning, but they were rarely constant. Only infrequently did a neuron's PD for novel ISs closely match that for familiar ISs throughout the learning process. In directional learning-dependent cells, the PD usually reoriented near the end of learning to resemble that for familiar IS trials. In omnidirectional cells, initially evident PDs dissipated with learning, even as the cell became more strongly modulated. Learning-selective cells typically began with significant PDs, but became unmodulated as learning progressed. Our findings show a pervasive lability in SEF PDs that may reflect a flexible and rapid remapping between inputs and responses within the premotor cortical network.

Animals↗
PubMed2006

Improving evolution.

By combining elements of protein engineering and directed evolution, researchers open the door to creating enzymes with diverse catalytic functions in a protein scaffold of their choice.

Animals↗
PubMed2001

DNA shuffling and vaccines.

One challenge of biotechnology is to find ways to optimize enzymes, cytokines, vaccines or transgenes in new contexts that are typically not found in nature. The approach of DNA shuffling is a test-tube process that takes advantage of recombination to generate libraries of chimeric genes, which can then be screened to identify the encoded proteins improved in one or more functions. DNA shuffling of two or more genes that are structurally similar and homologous in function is particularly efficacious in generating large libraries of functionally novel molecules. Other directed evolution methods, such as those involving directed or random mutagenesis, have several limitations compared to the DNA shuffling recombination process. A wide variety of genes have been submitted to DNA shuffling, and significant improvements in various functional parameters have been obtained. Several examples of the application of DNA shuffling to vaccine development, therapeutics and gene therapy are discussed here.

Allergens↗