The genetically tractable model organism Caenorhabditis elegans was first used to model bacterial virulence in vivo a decade ago. Since then, great strides have been made in identifying the host response pathways that are involved in its defence against infection. Strikingly, C. elegans seems to det

The genetically tractable model organism Caenorhabditis elegans was first used to model bacterial virulence in vivo a decade ago. Since then, great strides have been made in identifying the host response pathways that are involved in its defence against infection. Strikingly, C. elegans seems to detect, and respond to, infection without the involvement of its homologue of Toll-like receptors, in contrast to the well-established role for these proteins in innate immunity in mammals. What, therefore, do we know about host defence mechanisms in C. elegans and what can they tell us about innate immunity in higher organisms?

The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pres

The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pressure in the course of primate evolution. We compared the nucleotide sequences of 16 TLR-related genes, including TLRs (TLR1-10), MYD88, TILAP, TICAM1, TICAM2, MD2, and CD14, among seven primate species. Analysis of the non-synonymous/synonymous substitution ratio revealed the presence of both strictly conserved and rapidly evolving regions in the TLR-related genes. The genomic segments encoding the intracellular Toll/interleukin 1 receptor domains, which exhibited lower rates of non-synonymous substitution, have undergone purifying selection. In contrast, TLR4, which carried a high proportion of non-synonymous substitutions in the part of extracellular domain spanning 200 amino acids, was found to have been the suggestive target of positive Darwinian selection in primate evolution. However, sequence analyses from 25 primate species, including eight hominoids, six Old World monkeys, eight New World monkeys, and three prosimians, showed no evidence that the pressure of positive Darwinian selection has shaped the pattern of sequence variations in TLR4 among New World monkeys and prosimians.

Gene therapy, to delivery of genetic material to a patient for therapeutic benefit, has significant promise for translating basic knowledge of disease mechanism into biomedical treatments. The clinical development of the field has been slowed, however, by the need for improvements in the properties

Gene therapy, to delivery of genetic material to a patient for therapeutic benefit, has significant promise for translating basic knowledge of disease mechanism into biomedical treatments. The clinical development of the field has been slowed, however, by the need for improvements in the properties and capabilities of gene delivery vehicles. Vehicles based on viruses offer the potential for efficient gene delivery, but because viruses did not evolve to serve human therapeutic needs, many of their properties require significant improvement, including their safety, efficiency, and capacity for targeted gene delivery. Since viruses are highly complex biological entities, engineering such properties at the molecular level can be challenging. However, there has been significant progress in developing approaches that mimic the mechanisms by which viruses arose in the first place. In particular, library-based selection, the generation of one diverse genetic library and selection for new properties, and directed evolution, based on the multiple rounds of library generation and selection for iterative improvement of function, have strong potential in engineering novel properties into these complex biomolecular assemblies. This review will discuss progress in the application of peptide display, library selection, and directed evolution technologies toward engineering vectors based on retrovirus, adeno-associated virus, and adenovirus that are capable of targeted delivery to specific cell types. In addition to creating biomedically useful products, these approaches have future potential to yield novel insights into viral structure-function relationships.

Directed evolution uses a combination of powerful search techniques to generate proteins with improved properties. Part of the success is due to the stochastic element of random mutagenesis; improvements can be made without a detailed description of the complex interactions that constitute function

Directed evolution uses a combination of powerful search techniques to generate proteins with improved properties. Part of the success is due to the stochastic element of random mutagenesis; improvements can be made without a detailed description of the complex interactions that constitute function or stability. However, optimization is not a conglomeration of random processes. Rather, it requires both knowledge of the system that is being optimized and a logical series of techniques that best explores the pathways of evolution (Eigen et al., 1988). The weighing of parameters associated with mutation, recombination, and screening to achieve the maximum fitness improvement is the beginning of rational evolutionary design. The optimal mutation rate is strongly influenced by the finite number of mutants that can be screened. A smooth fitness landscape implies that many mutations can be accumulated without disrupting the fitness. This has the effect of lowering the required library size to sample a higher mutation rate. As the sequence ascends the fitness landscape, the optimal mutation rate decreases as the probability of discovering improved mutations also decreases. Highly coupled regions require that many mutations be simultaneously made to generate a positive mutant. Therefore, positive mutations are discovered at uncoupled positions as the fitness of the parent increases. The benefit of recombination is twofold: it combines good mutations and searches more sequence space in a meaningful way. Recombination is most beneficial when the number of mutants that can be screened is limited and the landscape is of an intermediate ruggedness. The structure of schema in proteins leads to the conclusion that many cut points are required. The number of parents and their sequence identity are determined by the balance between exploration and exploitation. Many disparate parents can explore more space, but at the risk of losing information. The required screening effort is related to the number of uphill paths, which decreases more rapidly for rugged landscapes. Noise in the fitness measurements causes a dramatic increase in the required mutant library size, thus implying a smaller optimal mutation rate. Because of strict limitations on the number of mutants that can be screened, there is motivation to optimize the content of the mutant library. By restricting mutations to regions of the gene that are expected to show improvement, a greater return can be made with the same number of mutants. Initial studies with subtilisin E have shown that structurally tolerant positions tend to be where positive activity mutants are made during directed evolution. Mutant fitness information is produced by the screening step that has the potential to provide insight into the structure of the fitness landscape, thus aiding the setting of experimental parameters. By analyzing the mutant fitness distribution and targeting specific regions of the sequence, in vitro evolution can be accelerated. However, when expediting the search, there is a trade-off between rapid improvement and the quality of the long-term solution. The benefit of neutrality has yet to be captured with in vitro protein evolution. Neutral theory predicts the punctuated emergence of novel structure and function, however, with current methods, the required time scale is not feasible. Utilizing neutral evolution to accelerate the discovery of new functional and structural solutions requires a theory that predicts the behavior of mutational pathways between networks. Because the transition from neutral to adaptive evolution requires a multi-mutational switch, increasing the mutation rate decreases the time required for a punctuated change to occur. By limiting the search to the less coupled region of the sequence (smooth portion of the fitness landscape), the required larger mutation rate can be tolerated. Advances in directed evolution will be achieved when the driving forces behind such proce

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure-function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. "Shielding" polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating 'designer' gene delivery vectors with enhanced properties.

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computat

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.

Directed evolution is a term used to describe a variety of related techniques to rapidly evolve peptides and proteins into new forms that exhibit improved properties for specific applications. In this process, molecular biology techniques allow the creation of up to billions of mutants in a single e

Directed evolution is a term used to describe a variety of related techniques to rapidly evolve peptides and proteins into new forms that exhibit improved properties for specific applications. In this process, molecular biology techniques allow the creation of up to billions of mutants in a single experiment, which are then subjected to high-throughput screening to identify those with enhanced activity. Applications of directed evolution to drug and gene delivery have been recently described, including those that improve the effectiveness of therapeutic enzymes, targeting peptides and antibodies, and the effectiveness or tropism of viral vectors for use in gene therapy. This review first introduces fundamental concepts of directed evolution, and then discusses emerging applications in the field of drug and gene delivery.

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 t

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.

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with alte

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then s

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase

Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (Rec(ZF)s) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in approximately 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)s may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.