Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Directed Molecular Evolution”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed2003

Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.

Amino Acid Sequence↗
PubMed2003

Directed evolution of industrial enzymes: an update.

The use of enzymes in industrial processes can often eliminate the use of high temperatures, organic solvents and extremes of pH, while at the same time offering increased reaction specificity, product purity and reduced environmental impact. The growing use of industrial enzymes is dependent on constant innovation to improve performance and reduce cost. This innovation is driven by a rapidly increasing database of natural enzyme diversity, recombinant DNA and fermentation technologies that allow this diversity to be produced at low cost, and protein modification tools that enable enzymes to be tuned to fit into the industrial marketplace.

6-Phytase↗
PubMed2001

Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries.

The technologies for screening peptide and protein libraries for studies in the fields of directed protein evolution and functional genomics have advanced with astonishing speed. For screening of functional proteins, three technologies are required: (i) the construction of a gene library (genotype), (ii) the establishment of a linkage between each protein (phenotype) and its encoding gene (genotype), and (iii) the selection of desired proteins (phenotype) from the library. This review highlights the genotype-phenotype linkage technologies, which can be classified into three types; that is, cell-type linkage, virus-type linkage, and array-type linkage methods. These methods are summarized, and their advantages and disadvantages are discussed.

Animals↗
PubMed2002

Boron-containing aptamers to ATP.

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.

Adenosine Triphosphate↗
PubMed2002

Directed evolution experiments reveal mutations at cycloartenol synthase residue His477 that dramatically alter catalysis.

[reaction: see text] Cycloartenol synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19. To identify mutants that deprotonate differently, randomly generated mutant cycloartenol synthases were selected in a yeast lanosterol synthase mutant. A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol. The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Delta(7)-lanosterol. These are the most accurate lanosterol synthase and parkeol synthase that have been generated by mutagenesis.

Amino Acid Sequence↗
PubMed2001

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies.

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

AIDS Vaccines↗
PubMed2005

Laboratory evolution of catabolic enzymes and pathways.

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.

Bacteria↗
PubMed2003

Genetic screens and directed evolution for protein solubility.

Overexpressed proteins are often insoluble, and can be recalcitrant to conventional solubilization techniques such as refolding. Directed evolution methods, in which protein diversity libraries are screened for soluble variants, offer an alternative route to obtaining soluble proteins. Recently, several new protein solubility screens have been developed that do not require structural or functional information about the target protein. Soluble protein can be detected in vivo and in vitro by fusion reporter tags. Protein misfolding can be measured in vivo using the bacterial response to protein misfolding. Finally, soluble protein can be monitored by immunological detection. Efficient, well-established strategies for generating and recombining genetic diversity, driven by new screening and selection methods, can furnish correctly folded, soluble protein.

Directed Molecular Evolution↗
PubMed2001

Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate.

We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only approximately 4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and approximately 20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.

Amino Acid Sequence↗
PubMed2004

Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C.

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

Carcinoembryonic Antigen↗
PubMed2001

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

Base Sequence↗
PubMed2005

Directed evolution of human T-cell receptors with picomolar affinities by phage display.

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to approximately 1-100 muM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax(11-19) peptide-HLA-A(*)0201 complex and the NY-ESO-1(157-165) tumor-associated peptide antigen-HLA-A(*)0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

Antibody Affinity↗
PubMed2003

Optimization of protein therapeutics by directed evolution.

Directed evolution is a broadly applicable technology platform that is ideally suited to address the need for protein optimization and to fully exploit the therapeutic potential of biologics. The approach takes advantage of the remarkable structural and functional plasticity of proteins and permits the rapid remodeling of biologics into new entities with improved functions. The ability to ameliorate virtually any characteristic of a protein can translate into significant clinical benefits, including decreased immunogenicity, higher potency, greater efficacy and improved safety profile, and can considerably increase the probability of successfully developing and commercializing biotherapeutics.

Animals↗
PubMed2004

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos.

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.

Amino Acid Substitution↗
PubMed2004

High-activity barley alpha-amylase by directed evolution.

Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations. Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.

Amino Acid Sequence↗
PubMed1997

Role of mutator alleles in adaptive evolution.

Because most newly arising mutations are neutral or deleterious, it has been argued that the mutation rate has evolved to be as low as possible, limited only by the cost of error-avoidance and error-correction mechanisms. But up to one per cent of natural bacterial isolates are 'mutator' clones that have high mutation rates. We consider here whether high mutation rates might play an important role in adaptive evolution. Models of large, asexual, clonal populations adapting to a new environment show that strong mutator genes (such as those that increase mutation rates by 1,000-fold) can accelerate adaptation, even if the mutator gene remains at a very low frequency (for example, 10[-5]). Less potent mutators (10 to 100-fold increase) can become fixed in a fraction of finite populations. The parameters of the model have been set to values typical for Escherichia coli cultures, which behave in a manner similar to the model in long-term adaptation experiments.

Adaptation, Physiological↗
PubMed2005

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

Animals↗
PubMed2001

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Base Sequence↗