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1.
Front Mol Biosci ; 10: 1253689, 2023.
Article in English | MEDLINE | ID: mdl-37692063

ABSTRACT

Accurate protein-protein docking remains challenging, especially for artificial biologics not coevolved naturally against their protein targets, like antibodies and other engineered scaffolds. We previously developed ProPOSE, an exhaustive docker with full atomistic details, which delivers cutting-edge performance by allowing side-chain rearrangements upon docking. However, extensive protein backbone flexibility limits its practical applicability as indicated by unbound docking tests. To explore the usefulness of ProPOSE on systems with limited backbone flexibility, here we tested the engineered scaffold DARPin, which is characterized by its relatively rigid protein backbone. A prospective screening campaign was undertaken, in which sequence-diversified DARPins were docked and ranked against a directed epitope on the target protein BCL-W. In this proof-of-concept study, only a relatively small set of 2,213 diverse DARPin interfaces were selected for docking from the huge theoretical library from mutating 18 amino-acid positions. A computational selection protocol was then applied for enrichment of binders based on normalized computed binding scores and frequency of binding modes against the predefined epitope. The top-ranked 18 designed DARPin interfaces were selected for experimental validation. Three designs exhibited binding affinities to BCL-W in the nanomolar range comparable to control interfaces adopted from known DARPin binders. This result is encouraging for future screening and engineering campaigns of DARPins and possibly other similarly rigid scaffolds against targeted protein epitopes. Method limitations are discussed and directions for future refinements are proposed.

2.
MAbs ; 15(1): 2248672, 2023.
Article in English | MEDLINE | ID: mdl-37622732

ABSTRACT

Carbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX's enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody-epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX's IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX's entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody-epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.


Subject(s)
Carbonic Anhydrases , Saccharomyces cerevisiae , Humans , Carbonic Anhydrase IX/chemistry , Carbonic Anhydrase IX/metabolism , Saccharomyces cerevisiae/metabolism , Epitopes , Proteoglycans , Antigens, Neoplasm , Carbonic Anhydrases/chemistry , Magnetic Resonance Spectroscopy
3.
Microbiol Resour Announc ; 12(5): e0134022, 2023 May 17.
Article in English | MEDLINE | ID: mdl-37052479

ABSTRACT

The PacBio whole-genome sequencing of the potential probiotic strain Canan SV-53T recovered from lowbush blueberries demonstrates high homology to Rouxiella badensis but with distinct enough clusters to propose the name Rouxiella badensis subsp. acadiensis. The sequencing also detected the presence of two native plasmids.

4.
J Microbiol Methods ; 204: 106648, 2023 01.
Article in English | MEDLINE | ID: mdl-36470413

ABSTRACT

The efficiency with which E.coli BL21 can be modified using CRISPR-Cas9 genetic engineering is several orders of magnitude lower than that of E. coli W3110. We show that the lack of Lon protease is responsible, and demonstrate that restoration of the Lon protease or knock-out of sulA improves CRISPR-Cas9 engineering efficiency of BL21 to levels comparable to E. coli W3110.


Subject(s)
Escherichia coli Proteins , Protease La , Escherichia coli/genetics , Escherichia coli/metabolism , Protease La/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , ATP-Dependent Proteases/genetics , CRISPR-Cas Systems/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Genetic Engineering
5.
Sci Rep ; 11(1): 4284, 2021 02 19.
Article in English | MEDLINE | ID: mdl-33608571

ABSTRACT

Ligand-activated signaling through the type 1 insulin-like growth factor receptor (IGF1R) is implicated in many physiological processes ranging from normal human growth to cancer proliferation and metastasis. IGF1R has also emerged as a target for receptor-mediated transcytosis, a transport phenomenon that can be exploited to shuttle biotherapeutics across the blood-brain barrier (BBB). We employed differential hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) to characterize the interactions of the IGF1R ectodomain with a recently discovered BBB-crossing single-domain antibody (sdAb), VHH-IR5, in comparison with IGF-1 binding. HDX-MS confirmed that IGF-1 induced global conformational shifts in the L1/FnIII-1/-2 domains and α-CT helix of IGF1R. In contrast, the VHH-IR5 sdAb-mediated changes in conformational dynamics were limited to the α-CT helix and its immediate vicinity (L1 domain). High-resolution NMR spectroscopy titration data and linear peptide scanning demonstrated that VHH-IR5 has high-affinity binding interactions with a peptide sequence around the C-terminal region of the α-CT helix. Taken together, these results define a core linear epitope for VHH-IR5 within the α-CT helix, overlapping the IGF-1 binding site, and suggest a potential role for the α-CT helix in sdAb-mediated transcytosis.


Subject(s)
Blood-Brain Barrier/metabolism , Epitope Mapping , Epitopes , Receptor, IGF Type 1/antagonists & inhibitors , Single-Domain Antibodies/pharmacology , Amino Acid Sequence , Antibody Affinity/immunology , Epitopes/chemistry , Epitopes/immunology , Humans , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Quantitative Structure-Activity Relationship , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/immunology , Receptor, IGF Type 1/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology
6.
Vaccine ; 35(45): 6129-6136, 2017 10 27.
Article in English | MEDLINE | ID: mdl-28951087

ABSTRACT

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide ofHaemophilus influenzaetype b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence ofHaemophilus influenzaetype a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia asa solution to this emerging disease.


Subject(s)
Bacterial Capsules/immunology , Glycoconjugates/immunology , Haemophilus Infections/immunology , Haemophilus Infections/prevention & control , Haemophilus Vaccines/immunology , Haemophilus influenzae/immunology , Polysaccharides, Bacterial/immunology , Alaska , Animals , Canada , Female , Humans , Mice , Mice, Inbred BALB C , Public Health , Rabbits , Serotyping/methods , Vaccination/methods
7.
Nanotoxicology ; 9(4): 502-12, 2015 May.
Article in English | MEDLINE | ID: mdl-25211548

ABSTRACT

The toxicity and toxicogenomics of selected anatase and rutile nanoparticles (NP) and bulk titanium dioxide (TiO2) particles were evaluated in the soil nematode Caenorhabditis elegans. Results indicated that bulk or nano-TiO2 particles were slightly toxic to soil nematode C. elegans, as measured by reproduction EC50 values ranging from 4 to 32 mg/L. Whole-genome microarray results indicated that the regulation of glutathione-S-transferase gst-3, cytochrome P450 cypp33-c11, stress resistance regulator scl-1, oxidoreductase wah-1 and embryonic development pod-2 genes were significantly affected by nano-sized and bulk-TiO2 particles. More specifically, it was determined that anatase particles exerted a greater effect on metabolic pathways, whereas rutile particles had a greater effect on developmental processes. The up-regulation of the pod-2 gene corroborated the phenotypic effect observed in the reproduction test. Our results demonstrated that C. elegans is a good genomic model for nano-TiO2 toxicity assessment.


Subject(s)
Caenorhabditis elegans/drug effects , Genomics , Metal Nanoparticles/toxicity , Titanium/toxicity , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Reverse Transcriptase Polymerase Chain Reaction , Soil
8.
FEMS Yeast Res ; 9(7): 1070-7, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19732157

ABSTRACT

It has come to our attention that approximately 35% of >100 published microarray datasets, where transcript levels were compared between two different strains, exhibit some form of chromosome-specific bias. While some of these arose from the use of strains whose aneuploidies were not known at the time, in a worrisome number of cases the recombinant strains have acquired additional aneuploidies that were not initially present in the parental strain. The aneuploidies often affected a different chromosome than the one harboring the insertion site. The affected strains originated from either CAI-4, RM1000, BWP17 or SN95 and were produced through a variety of strategies. These observations suggest that aneuploidies frequently occur during the production of recombinant strains and have an effect on global transcript profiles outside of the afflicted chromosome(s), thus raising the possibility of unintended phenotypic consequences. Thus, we propose that all Candida albicans mutants and strains should be tested for aneuploidy before being used in further studies. To this end, we describe a new rapid testing method, based on a multiplex quantitative PCR assay, that produces eight bands of distinct sizes from either the left or right arms of each C. albicans chromosome.


Subject(s)
Aneuploidy , Candida albicans/genetics , Chromosomes, Fungal , Molecular Biology/methods , Mycology/methods , Polymerase Chain Reaction/methods , Genetics, Microbial/methods
9.
Appl Environ Microbiol ; 75(19): 6258-67, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19684169

ABSTRACT

High-Arctic soils have low nutrient availability, low moisture content, and very low temperatures and, as such, they pose a particular problem in terms of hydrocarbon bioremediation. An in-depth knowledge of the microbiology involved in this process is likely to be crucial to understand and optimize the factors most influencing bioremediation. Here, we compared two distinct large-scale field bioremediation experiments, located at the Canadian high-Arctic stations of Alert (ex situ approach) and Eureka (in situ approach). Bacterial community structure and function were assessed using microarrays targeting the 16S rRNA genes of bacteria found in cold environments and hydrocarbon degradation genes as well as quantitative reverse transcriptase PCR targeting key functional genes. The results indicated a large difference between sampling sites in terms of both soil microbiology and decontamination rates. A rapid reorganization of the bacterial community structure and functional potential as well as rapid increases in the expression of alkane monooxygenases and polyaromatic hydrocarbon-ring-hydroxylating dioxygenases were observed 1 month after the bioremediation treatment commenced in the Alert soils. In contrast, no clear changes in community structure were observed in Eureka soils, while key gene expression increased after a relatively long lag period (1 year). Such discrepancies are likely caused by differences in bioremediation treatments (i.e., ex situ versus in situ), weathering of the hydrocarbons, indigenous microbial communities, and environmental factors such as soil humidity and temperature. In addition, this study demonstrates the value of molecular tools for the monitoring of polar bacteria and their associated functions during bioremediation.


Subject(s)
Bacteria/genetics , Bacteria/metabolism , Biodiversity , Hydrocarbons/metabolism , Soil Microbiology , Soil Pollutants/metabolism , Arctic Regions , Biodegradation, Environmental , Canada , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Microarray Analysis , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Reverse Transcriptase Polymerase Chain Reaction
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