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PubMed2001

Combinatorial and Evolution-Based Methods in the Creation of Enantioselective Catalysts.

Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.

Journal Article↗
PubMed2000

Creating hybrid genes by homologous recombination.

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution. In vitro recombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties. In vivo recombination in both E. coli and yeast is greatly enhanced by double-strand breaks; for E. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombination in vivo have distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recently in vivo recombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

Escherichia coli↗
PubMed1999

Selection of functional T cell receptor mutants from a yeast surface-display library.

The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

Animals↗
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↗
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↗
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↗
PubMed2003

Evolution of function in (beta/alpha)8-barrel enzymes.

The (beta/alpha)(8)-barrel is the most common fold in structurally characterized enzymes. Whether the functionally diverse enzymes that share this fold are the products of either divergent or convergent evolution (or both) is an unresolved question that will probably be answered as the sequence databases continue to expand. Recent work has examined natural, designed, and directed evolution of function in several superfamilies of (beta/alpha)(8)-barrel containing enzymes.

Animals↗
PubMed2000

High-throughput screening of enzyme libraries.

Directed evolution is becoming a widely used technique for modifying or enhancing protein performance. Ultimately, the success of directed protein evolution experiments hinges on the efficiency of the methods used to screen libraries for mutants with properties of interest. Although there is still a paucity of general methods for enzyme library screening, in recent years a number of promising strategies have emerged and are increasingly being used to explore challenging issues in protein engineering.

Directed Molecular Evolution↗
PubMed2001

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Bacillus megaterium↗