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Results for “Directed Molecular Evolution”

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At least 73 records · Page 4Linked to original sources
PubMed1998

Phage display selection can differentiate insecticidal activity of soybean cystatins.

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200-1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.

Amino Acid Sequence↗
PubMed2002

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells.

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.

Abatacept↗
PubMed2000

Models of experimental evolution: the role of genetic chance and selective necessity.

We present a theoretical framework within which to analyze the results of experimental evolution. Rapidly evolving organisms such as viruses, bacteria, and protozoa can be induced to adapt to laboratory conditions on very short human time scales. Artificial adaptive radiation is characterized by a list of common observations; we offer a framework in which many of these repeated questions and patterns can be characterized analytically. We allow for stochasticity by including rare mutations and bottleneck effects, demonstrating how these increase variability in the evolutionary trajectory. When the product Np, the population size times the per locus error rate, is small, the rate of evolution is limited by the chance occurrence of beneficial mutations; when Np is large and selective pressure is strong, the rate-limiting step is the waiting time while existing beneficial mutations sweep through the population. We derive the rate of divergence (substitution rate) and rate of fitness increase for the case when Np is large and illustrate our approach with an application to an experimental data set. A minimal assumption of independent additive fitness contributions provides a good fit to the experimental evolution of the bacteriophage phiX174.

Animals↗
PubMedDate not supplied

[Transgenics and controlled evolution].

Mutation events are responsible for the generation of genetic variability in the populations enabling the occurrence of natural selection which favors the better-adapted types. The exploitation of this variability, though carried out empirically, dates from ten thousand years ago with the domestication of the first cultivated crops. With the advent of genetics, rational selection procedures were adopted with a view of the genetic breeding of plants, animals and microorganisms which might be of interest to men. Recently, new DNA manipulation techniques came up enabling the transference of genes between organisms, cutting across barriers which hindered crossing between the vegetable, animal, protist and fungus kingdoms. The generation of genetically modified organisms, or transgenics, has aroused a heated and controversial debate in various sectors of our society. Yet we must be cautious before generalizing the use of transgenics since each one should be analyzed at a time for its particular advantages and drawbacks, and for its contribution to the improvement of life quality. This paper also considers recent methods of mutation and in vitro genic recombination.

Biological Evolution↗
PubMed1991

Directionality of evolution at molecular and organismic levels.

The molecular evolution theories of Eigen and Kimura are compared and their difference is explained. In terms of Eigen's theory for the evolution of macromolecules, the selection of genotypes occurs directly. The physical meaning of the neutral theory is the degeneracy of the correlation between a phenotype and a genotype at the molecular level. A model theory of evolution on a fitness landscape is proposed. The theory shows that the constraints of selection determined by the structure and dynamics of previous evolution stages increases its rate strongly.

Biological Evolution↗
PubMed2005

Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis.

VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.

Evolution, Molecular↗
PubMed2003

Synthetic gene libraries: in search of the optimal diversity.

Directed evolution has proven to be an effective method for evolving proteins with desired properties. A key step is the creation of suitably diverse gene libraries. Two new methods for creating such libraries make sole use of synthesized oligonucleotides and allow researchers to tailor the diversity of a library with greater precision and create libraries with greater diversity than was previously possible. Such increased diversity appears to accelerate directed evolution.

Cloning, Molecular↗
PubMed1988

Directional mutation pressure and neutral molecular evolution.

A quantitative theory of directional mutation pressure proposed in 1962 explained the wide variation of DNA base composition observed among different bacteria and its small heterogeneity within individual bacterial species. The theory was based on the assumption that the effect of mutation on a genome is not random but has a directionality toward higher or lower guanine-plus-cytosine content of DNA, and this pressure generates directional changes more in neutral parts of the genome than in functionally significant parts. Now that DNA sequence data are available, the theory allows the estimation of the extent of neutrality of directional mutation pressure against selection. Newly defined parameters were used in the analysis, and two apparently universal constants were discovered. Analysis of DNA sequence has revealed that practically all organisms are subject to directional mutation pressure. The theory also offers plausible explanations for the large heterogeneity in guanine-plus-cytosine content among different parts of the vertebrate genome.

Animals↗
PubMed2005

Directed evolution of (betaalpha)(8)-barrel enzymes.

Natural molecular evolution supplies us with manifold examples of protein engineering. The imitation of these natural processes in the design of new enzymes has led to surprising and insightful results. Well-suited for design by evolutionary methods are enzymes with the common and versatile (betaalpha)(8)-barrel fold. Studies of enzyme stability, folding and design as well as the evolution of (betaalpha)(8)-barrel enzymes are discussed.

Animals↗
PubMed2001

Improved biocatalysts by directed evolution and rational protein design.

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.

Catalysis↗
PubMed1997

The advantage of sex in evolving yeast populations.

Sex is a general feature of the life cycle of eukaryotes. It is not universal, however, as many organisms seem to lack sex entirely. The widespread occurrence of sex is puzzling, both because meiotic recombination can disrupt co-adapted combinations of genes, and because it halves the potential rate of reproduction in organisms with strongly differentiated male and female gametes. Most attempts to explain the maintenance of sexuality invoke differences between parents and sexual offspring. These differences may be advantageous in novel or changing environments if new gene combinations are favoured from time to time. Sex would then serve to concentrate beneficial mutations that have arisen independently into the same line of descent. But in a stable environment sex might serve to concentrate deleterious mutations, so that they will be more effectively purged from the population by selection. We have studied the effect of sex on mean fitness in experimental populations of the budding yeast Saccharomyces cerevisiae. Our results show that sex increases mean fitness in an environment to which the populations were well adapted, but not in an environment to which new adaptation occurred, supporting the hypothesis that the advantage of sexuality lay in the removal of deleterious mutations.

Adaptation, Physiological↗
PubMed2006

The diversity challenge in directed protein evolution.

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships. This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and cost-effectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle). We hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.

Base Composition↗
PubMed2001

Industrial biocatalysis.

The number of industrial processes for the synthesis of fine and commodity chemicals, pharmaceutical and agrochemical intermediates and drug substances utilizing biological catalysts continues to grow. The combination of new molecular biology techniques, such as directed evolution and pathway engineering, with new and efficient high-throughput screening methods is poised to bolster this field and further advance the contribution of biocatalysis to the chemical and the pharmaceutical industries.

Catalysis↗
PubMed2003

A comparison of directed evolution approaches using the beta-glucuronidase model system.

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.

Directed Molecular Evolution↗
PubMed2002

Generating segmental mutations in haloalkane dehalogenase: a novel part in the directed evolution toolbox.

Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3' or 5' side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments.

Amino Acid Sequence↗
PubMed2004

Learning from directed evolution: theoretical investigations into cooperative mutations in lipase enantioselectivity.

Molecular modeling with classical force-fields has been used to study the reactant complex and the tetrahedral intermediate in lipase-catalyzed ester hydrolysis in 20 enzyme/substrate combinations. The R and S enantiomers of alpha-methyldecanoic acid ester served as substrates for the wild-type lipase from Pseudomonas aeruginosa and nine selected mutants. After suitable preparation of initial structures from an available wild-type crystal structure, each system was subjected to 1 ns CHARMM force-field molecular dynamics simulations. The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the "hot spots" at residues 155 and 162. The replacement S155F enhances S enantiopreference through a steric relay involving Leu162. The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83. The simulations provide insight into remote and cooperative effects of mutations.

Catalysis↗
PubMed2004

Directed evolution of ligand dependence: small-molecule-activated protein splicing.

Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.

Amebicides↗