Library Of Chimeras Research Articles

Host-derived chimeric peptides clear the causative bacteria and augment host innate immunity during infection: A case study of HLB in citrus and fire blight in apple.

Bacterial diseases cause severe losses in the production and revenue of many fruit crops, including citrus and apple. Huanglongbing (HLB) in citrus and fire blight in apple are two deadly diseases without any cure. In this article, we introduce a novel therapy for HLB and fire blight by enhancing the innate immunity of the host plants. Specifically, we constructed in silico a library of chimeras containing two different host peptides with observed or predicted antibacterial activity. Subsequently, we performed bactericidal and toxicity tests in vitro to select a few non-toxic chimeras with high antibacterial activity. Finally, we conducted ex planta studies to show that not only do the chimeras clear the causative bacteria from citrus leaves with HLB and from apple leaves with fire blight but they also augment the host's innate immunity during infection. This platform technology can be extended to design host-derived chimeras against multiple pathogenic bacteria that cause diseases in plants and animals of agricultural importance and in humans.

Exploring Performance Parameters of Artificial Allosteric Protein Switches

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera’s activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen–deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.

A Minimal Protein Region Required for the Chemical Activation of the Mechanosensitive Channel Piezo1

The homologous Piezo1 and Piezo2 mechanosensitive ion channels are central to many important mechanotransduction processes in both normal and pathological states. Selectively targeting each Piezo homolog with pharmacological agents represents a promising avenue to probe their individual biological contribution and to treat diseases associated with Piezo channel dysfunctions. The small molecule Yoda1, which activates Piezo1 but does not affect Piezo2, is the only known molecular modulator exhibiting selectivity among mammalian Piezo channels. To date, the molecular basis for this selectivity is unknown. Here, a library of chimeras was created by replacing various protein regions from mouse Piezo1 by their homologous counterpart from mouse Piezo2. The characterization of these chimeras by calcium imaging and pressure-clamp electrophysiology led to the identification of a minimal Piezo1 region responsible for the chemical activation of Piezo1 by Yoda1. This work paves the way to elucidate the mechanisms underlying chemical activation of Piezo channels and will help expand the list of selective small molecule modulators targeting the small Piezo channel family.

Determining the Molecular Basis for Differential Hyperoxidation Sensitivity in Peroxiredoxin 1 & 2

Oxidative stress, a cellular condition in which the levels of reactive oxygen species (ROS; e.g., hydrogen peroxide, H2O2) are too high, causes damage to macromolecules and has been linked to several pathologies including cancer, diabetes, Alzheimer's disease, and cardiovascular disease. One way cells maintain homeostatic levels of ROS is to produce a variety of peroxiredoxins (Prxs). Prxs are redox‐sensitive, antioxidant enzymes that remove H2O2. However, Prxs can be inactivated by hyperoxidation of the active site cysteine (Cys‐SPH) residue to Cys sulfinic acid (Cys‐SPO2H). Interestingly, Prx1 is more resistant to hyperoxidation than Prx2, despite the fact that they share 77% sequence identity and are both located in the cytoplasm. The molecular basis for this variability between isoforms in their susceptibilities to inactivation is unknown, but it is important to study since Prx inactivation is implicated in oxidative stress‐associated diseases. We hypothesize that residues unique to hPrx1 cause it to be more resistant to hyperoxidation. We have successfully cloned, expressed, and purified a library of chimeras that target groups of the most dissimilar residues between the two isoforms. We have determined the hyperoxidation sensitivity of the wild type hPrx1 and hPrx2 as well as each chimera using a variety of kinetic assays. By analyzing the changes these chimeras cause in hyperoxidation sensitivity, we have identified two novel, resistance‐conferring motifs.Sources of research support: AHA MAA: 14PRE19970007, NIGMS/NIH: R01 GM072866

ITCHY: Incremental Truncation for the Creation of Hybrid enzYmes.

Incremental Truncation for the Creation of Hybrid enzYmes (ITCHY) is a directed evolution technique for randomly recombining two genes. The chief advantage of ITCHY is that there is no requirement for the two genes to share any sequence similarity. This distinguishes ITCHY from directed evolution methods that are based on homologous recombination, such as DNA shuffling. In ITCHY, Escherichia coli exonuclease III is used to incrementally truncate one of the parental genes from its 3' end and the other from its 5' end. Ligation of the randomly truncated gene fragments yields a combinatorial library of chimeras. In this chapter, we provide detailed protocols for constructing libraries using both the user-friendly thio-ITCHY method and also time-dependent incremental truncation. We illustrate the protocols with the data that we obtained when we recombined two alcohol dehydrogenase genes that only share 47 % sequence identity.

Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

AbstractRecombination of distantly related and nonrelated genes is difficult using traditional PCR‐based techniques, and truncation‐based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments. Curr. Protoc. Protein Sci. 61:26.2.1‐26.2.20. © 2010 by John Wiley & Sons, Inc.

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.

Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

Combinatorial recombination of gene fragments to construct a library of chimeras.

Recombination of distantly related and nonrelated genes is difficult using traditional PCR-based techniques, and truncation-based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments.

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers.

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.

On the structural and functional modularity of glycinamide ribonucleotide formyltransferases.

Glycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate. Despite the low amino acid-sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo-functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their k(cat) and K(M) values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are "modular encoded" and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.

Analysis of shuffled libraries by oligonucleotide probe hybridization.

In vitro recombination is often used to generate genetic diversity for directed evolution. Recombination techniques that rely on fragment hybridization yield libraries with preferred crossover positions and, in some cases, a bias toward incorporation of one parent over the others. This limits the diversity, and thus the utility of the library. These biases vary depending on the technique used for recombination, the distribution of sequence similarities within the parental genes, and the efficiency by which the different parental genes are PCR amplified. To assess the diversity generated in a library of chimeras, sequences of a large number of chimeras are required. While DNA sequencing can yield this information, sequencing these genes is prohibitively expensive, especially when the genes being recombined are large. Oligonucleotide probe hybridization, in contrast, offers a cost-effective approach for obtaining information about library biases that allows for optimization of shuffling procedures. When coupled with functional information, this technique can provide information about the relationship between sequence and function (,).

DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.

Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.