Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol.

ABSTRACT: The 'Appelmans protocol' is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three Pseudomonas phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive Pseudomona aeruginosa strains. After 30 rounds of selection, phages were isolated from the evolved cocktail with greatly increased host range. Control experiments with individual phages showed little host-range expansion, and genomic analysis of one of the broad-host-range output phages showed its recombinatorial origin, suggesting that the protocol works predominantly via recombination between phages. The Appelmans protocol may be useful for evolving therapeutic phage cocktails as required from well-defined precursor phages.

SUBMITTER: Burrowes BH

PROVIDER: S-EPMC6466182 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol.

Burrowes Ben H BH Molineux Ian J IJ Fralick Joe A JA

Viruses 20190311 3

The 'Appelmans protocol' is used by Eastern European researchers to generate therapeutic phages with novel lytic host ranges. Phage cocktails are iteratively grown on a suite of mostly refractory bacterial isolates until the evolved cocktail can lyse the phage-resistant strains. To study this process, we developed a modified protocol using a cocktail of three <i>Pseudomonas</i> phages and a suite of eight phage-resistant (including a common laboratory strain) and two phage-sensitive <i>Pseudomon ...[more]

PMID: 30862096

Similar Datasets

Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer.

Project description:Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19-9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.

| S-EPMC7601599 | biostudies-literature

Directed evolution of streptavidin variants using in vitro compartmentalization.

Project description:We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.

| S-EPMC2615486 | biostudies-literature

Directed -in vitro- evolution of Precambrian and extant Rubiscos.

Project description:Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere's carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.

| S-EPMC5883036 | biostudies-literature

Discovery of tumoricidal DNA oligonucleotides by response-directed in vitro evolution.

Project description:Drug discovery is challenged by ineffectiveness of drugs against variable and evolving diseases, and adverse effects due to poor selectivity. We describe a robust platform which potentially addresses these limitations. The platform enables rapid discovery of DNA oligonucleotides evolved in vitro for exerting specific and selective biological responses in target cells. The process operates without a priori target knowledge (mutations, biomarkers, etc). We report the discovery of oligonucleotides with direct, selective cytotoxicity towards cell lines, as well as patient-derived solid and hematological tumors. A specific oligonucleotide termed E8, induced selective apoptosis in triple-negative breast cancer (TNBC) cells. Polyethylene glycol-modified E8 exhibited favorable biodistribution in animals, persisting in tumors up to 48-hours after injection. E8 inhibited tumors by 50% within 10 days of treatment in patient-derived xenograft mice, and was effective in ex vivo organ cultures from chemotherapy-resistant TNBC patients. These findings highlight a drug discovery model which is target-tailored and on-demand.

| S-EPMC6962221 | biostudies-literature

H9 hESC directed differentiation following Novocell protocol

Project description:Human embryonic stem cells (WiCell H9 passage 52) were differentiated following the Novocell protocol (D'Amour et al. Nature Biotechnology, 2005). The two biological replicates (Trial 15 and Trial 16) were analyzed by Real-Time PCR utilizing TaqMan Low Density Array Cards that have been customized for a set of 46 genes specific for endoderm and pancreatic gene expression.

2014-07-24 | E-MTAB-2786 | biostudies-arrayexpress

Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes.

Project description:To enable discovery of serum antibodies indicative of disease and simultaneously develop reagents suitable for diagnosis, in vitro directed evolution was applied to identify consensus peptides recognized by patients' serum antibodies. Bacterial cell-displayed peptide libraries were quantitatively screened for binders to serum antibodies from patients with celiac disease (CD), using cell-sorting instrumentation to identify two distinct consensus epitope families specific to CD patients (PEQ and (E)/DxFV(Y)/FQ). Evolution of the (E)/DxFV(Y)/FQ consensus epitope identified a celiac-specific epitope, distinct from the two CD hallmark antigens tissue transglutaminase-2 and deamidated gliadin, exhibiting 71% sensitivity and 99% specificity (n = 231). Expansion of the first-generation PEQ consensus epitope via in vitro evolution yielded octapeptides QPEQAFPE and PFPEQxFP that identified ?- and ?-gliadins, and their deamidated forms, as immunodominant B-cell epitopes in wheat and related cereal proteins. The evolved octapeptides, but not first-generation peptides, discriminated one-way blinded CD and non-CD sera (n = 78) with exceptional accuracy, yielding 100% sensitivity and 98% specificity. Because this method, termed antibody diagnostics via evolution of peptides, does not require prior knowledge of pathobiology, it may be broadly useful for de novo discovery of antibody biomarkers and reagents for their detection.

| S-EPMC3845167 | biostudies-literature

Directed evolution of angiotensin-converting enzyme 2 peptidase activity profiles for therapeutic applications.

Project description:Angiotensin-converting enzyme 2 (ACE2) has been investigated for its ability to beneficially modulate the angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases. Its broad substrate scope and diverse physiological roles, however, limit its potential as a therapeutic agent. In this work, we address this limitation by establishing a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants that possess both wild-type or greater Ang-II hydrolytic activity and improved specificity toward Ang-II relative to the off-target peptide substrate Apelin-13. To obtain these results, we screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371, and Y510) that can be mutated to enhance ACE2's activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wild-type ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat ), a sixfold diminished catalytic efficiency (kcat /Km ) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in the directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes as much or more Ang-II than wild-type ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts.

| S-EPMC10019445 | biostudies-literature

Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential.

Project description:BACKGROUND: Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. RESULTS: Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. CONCLUSIONS: Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

| S-EPMC3118219 | biostudies-literature

Directed polymerase evolution.

Project description:Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.

| S-EPMC3982152 | biostudies-literature

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction.

Project description:Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).

| S-EPMC6834295 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data