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PubMed2004

Directed evolution of soluble single-chain human class II MHC molecules.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present antigenic peptides to T cells. Expression of these molecules in soluble form has met limited success, presumably due to their large size, heterodimeric structure and the presence of multiple disulfide bonds. Here we have used directed evolution and yeast surface display to engineer soluble single-chain human lymphocyte antigen (HLA) class II MHC DR1 molecules without covalently attached peptides (scDR1alphabeta). Specifically, a library of mutant scDR1alphabeta molecules was generated by random mutagenesis and screened by fluorescence activated cell sorting (FACS) with DR-specific conformation-sensitive antibodies, yielding three well-expressed and properly folded scDR1alphabeta variants displayed on the yeast cell surface. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo indicated three amino acid residues in the beta1 domain are important for the improved protein folding yield. Further, molecular modeling studies suggested these mutations might increase the protein folding efficiency by improving the packing of a hydrophobic core in the alpha1beta1 domain of DR1. The scDR1alphabeta mutants displayed on the yeast cell surface are remarkably stable and bind specifically to DR-specific peptide HA(306-318) with high sensitivity and rapid kinetics in flow cytometric assays. Moreover, since the expression, stability and peptide-binding properties of these mutants can be directly assayed on the yeast cell surface using immuno-fluorescence labeling and flow cytometry, time-consuming purification and refolding steps of recombinant DR1 molecules are eliminated. Therefore, these scDR1alphabeta molecules will provide a powerful technology platform for further design of DR1 molecules with improved peptide-binding specificity and affinity for therapeutic and diagnostic applications. The methods described here should be generally applicable to other class II MHC molecules and also class I MHC molecules for their functional expression, characterization and engineering.

Directed Molecular Evolution↗

PubMed2006

Emergent robustness in competition between autocatalytic chemical networks.

The origin of auto-catalytic networks has been proposed as an initial step in pre-biotic evolution. It is possible to derive simple models where auto-catalytic networks naturally arise from simple chemical mixtures. In order for such a system to develop, there needs to be some degree of stability, what is characterised as ;robustness'. We demonstrate that competing systems generate this robustness as they create a distributed network of catalytic pathways.

Computer Simulation↗

PubMed2003

Assembly of designed oligonucleotides as an efficient method for gene recombination: a new tool in directed evolution.

A new and practical method for gene recombination with formation of libraries of mutant genes is presented. The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information. High recombination frequency with formation of full-length products is achieved by controlled overlapping of the designed oligomers. This process (ADO) minimizes self-hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes. ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB). In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso-diacetate.

Amino Acid Sequence↗

PubMed2004

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

Amino Acid Motifs↗

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↗

PubMed2002

Evolutionary dynamics of viral attenuation.

The genetic trajectory leading to viral attenuation was studied in a canine parvovirus (CPV) strain grown on dog kidney cells for 115 transfers. Consensus sequences of viral populations at passages 0, 3, 30, 50, 80, and 115 were obtained from PCR products covering 86% of the genome; clones from each of the 80th and 115th passages were also sequenced, covering 69% of the genome. Sixteen changes were fixed in the 115th-passage virus sample. Levels of polymorphism were strikingly different over time, in part because of a plaque-cloning step at passage 112 that reduced variation: passage 80 had 19 variants common among the clones, but passage 115 had only a single common variant. Several mutations increased in the culture at the same time, with most reaching fixation only after the 80th passage. The pattern of evolution was consistent with recombination and not with separate selective sweeps of individual mutations. Thirteen of the changes observed were identical to or at the same positions as changes observed in other isolates of CPV or feline panleukopenia virus.

Adaptation, Physiological↗

PubMed2006

Mutant clouds and occupation of sequence space in plant RNA viruses.

In nature, RNA viruses of plants often must adapt to ever-changing environments in the form of frequent host switches. This would favor a highly diverse population for transmission. However, most viruses that have been studied have been viruses of monocultural crops. In crop viruses, the mutation frequency of individual viral quasispecies varies greatly, both in experiment evolution studies and in populations of viruses within single field plants. There is some correlation between host range and mutation frequency in experimental evolution studies, but few viruses have been examined at the individual quasispecies level. Many questions about the nature of plant RNA virus populations and factors that affect the effective population sizes, such as genetic bottlenecks and postive and negative selection, have only begun to be studied. Many more analyses are required before generalized patterns can be determined.

Adaptation, Physiological↗

PubMed2002

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents.

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

Amino Acid Substitution↗

PubMed2004

Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA(Pro) or tRNA(1,2)(Lys). These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA(1,2)(Lys)-using virus.

Amino Acid Substitution↗

PubMed2005

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals.

Directed Molecular Evolution↗

PubMed2006

Experimental evolution of resistance to an antimicrobial peptide.

A novel class of antibiotics based on the antimicrobial properties of immune peptides of multicellular organisms is attracting increasing interest as a major weapon against resistant microbes. It has been claimed that cationic antimicrobial peptides exploit fundamental features of the bacterial cell so that resistance is much less likely to evolve than in the case of conventional antibiotics. Population models of the evolutionary genetics of resistance have cast doubt on this claim. We document the experimental evolution of resistance to a cationic antimicrobial peptide through continued selection in the laboratory. In this selection experiment, 22/24 lineages of Escherichia coli and Pseudomonas fluorescens independently evolved heritable mechanisms of resistance to pexiganan, an analogue of magainin, when propagated in medium supplemented with this antimicrobial peptide for 600-700 generations.

Animals↗

PubMed2005

Improved solubility of TEV protease by directed evolution.

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Amino Acid Substitution↗

PubMed2000

Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

Aldose-Ketose Isomerases↗

PubMed2005

Screening mutant libraries of fungal laccases in the presence of organic solvents.

Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/microL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 microM CuSO4 where expression levels (approximately 14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.

Acetonitriles↗

PubMed1998

A method for directed evolution and functional cloning of enzymes.

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Amino Acid Sequence↗

PubMed2005

New genotype-phenotype linkages for directed evolution of functional proteins.

The key practical consideration in directed evolution of functional biomolecules is the linkage of genotype and phenotype. In vitro selections offer the potential to select from libraries with up to 10(10)-10(14) members, with fewer constraints than current cell-based selections. New approaches such as mRNA display, ribosome display and in vitro compartmentalisation have complementary areas of application in selections for binding or catalysis.

Directed Molecular Evolution↗

PubMed1996

An enzyme-linked oligonucleotide assay.

The recent development of in vitro methods to select high-affinity ligands by combinatorial chemistry methodologies promises unique and theoretically unlimited supplies of novel therapeutic and diagnostic reagents. One such combinatorial chemistry process, systematic evolution of ligands by exponential enrichment (SELEX), allows rapid identification, from large random sequence pools, of the few oligonucleotide sequences that bind to a desired target molecule with high affinity and specificity. We describe an enzyme-linked sandwich assay that uses a SELEX-derived oligonucleotide. This assay demonstrates that these oligonucleotides can be effective and useful analytical reagents.

Animals↗

PubMed2006

RNA aptamers: from basic science towards therapy.

The SELEX technique (systematic evolution of ligands by exponential enrichment) provides a powerful tool for the in vitro selection of nucleic acid ligands (aptamers) from combinatorial oligonucleotide libraries against a target molecule. In the beginning of the technique's use, RNA molecules were identified that bind to proteins that naturally interact with nucleic acids or to small organic molecules. In the following years, the use of the SELEX technique was extended to isolate oligonucleotide ligands (aptamers) for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do. The in vitro selection of oligonucleotides with enzymatic activity, denominated aptazymes, allows the direct transduction of molecular recognition to catalysis. Recently, the use of in vitro selection methods to isolate protein inhibitors has been extended to complex targets, such as membrane-bound receptors, and even entire cells. RNA aptamers have also been expressed in living cells. These aptamers, also called intramers, can be used to dissect intracellular signal transduction pathways. The utility of RNA aptamers for in vivo experiments, as well as for diagnostic and therapeutic purposes, is considerably enhanced by chemical modifications, such as substitutions of the 2'-OH groups of the ribose backbone in order to provide resistance against enzymatic degradation in biological fluids. In an alternative approach, Spiegelmers are identified through in vitro selection of an unmodified D-RNA molecule against a mirror-image (i.e. a D-peptide) of a selection target, followed by synthesis of the unnatural nuclease-resistant L-configuration of the RNA aptamer that recognizes the natural configuration of its selection target (i.e. a L-peptide). Recently, nuclease-resistant inhibitory RNA aptamers have been developed against a great variety of targets implicated in disease. Some results have already been obtained in animal models and in clinical trials.

Animals↗