ABSTRACT
The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.
Subject(s)
Antibodies, Viral/immunology , Drug Resistance, Multiple/immunology , Drug Resistance, Viral/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Mutation/genetics , Neuraminidase/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Viral/chemistry , Antibody Affinity/immunology , Antigens, Viral/metabolism , Body Fluids/virology , DNA Mutational Analysis , Dogs , Epitopes/chemistry , Epitopes/immunology , Humans , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/enzymology , Madin Darby Canine Kidney Cells , Molecular Docking Simulation , Optical Imaging , Protein Binding , Spectrum Analysis, RamanABSTRACT
Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.
Subject(s)
Bacterial Proteins/metabolism , Ligands , Polymers/metabolism , Animals , Bacterial Proteins/chemistry , Biotin/chemistry , Biotin/metabolism , Biotinylation , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Mice , Polymers/chemistry , Protein Binding , Rabbits , Surface Plasmon Resonance , Surface PropertiesABSTRACT
Copper is indispensable in most aerobic organisms although it is toxic if unregulated as illustrated in many neurodegenerative diseases. To elucidate the mechanisms underlying copper release from cells, a membrane-targeting reporter which can compete with extracellular copper-binding molecules is highly desirable. However, engineering a reporter protein to provide both high sensitivity and selectivity for copper(ii) has been challenging, likely due to a lack of proper copper(ii)-chelating strategies within proteins. Here, we report a new genetically encoded fluorescent copper(ii) reporter by employing a copper-binding tripeptide derived from human serum albumin (HSA), which is one of the major copper-binding proteins in extracellular environments. Optimized insertion of the tripeptide into the green fluorescent protein leads to rapid fluorescence quenching (up to >85% change) upon copper-binding, while other metal ions have no effect. Furthermore, the high binding affinity of the reporter enables reliable copper detection even in the presence of competing biomolecules such as HSA and amyloid beta peptides. We also demonstrate that our reporter proteins can be used to visualize dynamic copper fluctuations on living HeLa cell surfaces.
Subject(s)
Azides/chemistry , Escherichia coli Proteins/chemistry , Ligases/chemistry , Molecular Probes/chemistry , Protein Engineering , Protein Interaction Mapping/methods , Adenosine Triphosphate/chemistry , Binding Sites , Cell Line , Escherichia coli Proteins/genetics , HeLa Cells , Humans , Ligases/genetics , Mutation , Peptides/chemistry , Photochemistry , Thioctic Acid/chemistry , Ultraviolet RaysSubject(s)
Biotin/chemistry , Carbon-Nitrogen Ligases/chemistry , Adenosine Triphosphate/chemistry , Azides/chemistry , Chromatography, High Pressure Liquid , Cross-Linking Reagents/chemistry , Crystallography, X-Ray/methods , Models, Chemical , Mutagenesis , Phosphines/chemistry , Pyrococcus horikoshii/enzymology , Saccharomyces cerevisiae/enzymology , Species Specificity , Substrate Specificity , TemperatureABSTRACT
Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.
Subject(s)
Biotin/metabolism , Ligases/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Peptide Library , Quantum Dots , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs , Cell Survival , Color , Kinetics , Models, Biological , Molecular Sequence Data , Saccharomyces cerevisiae/cytology , Substrate SpecificityABSTRACT
Wilkinson's complex has been found to catalyze the one-pot transformation of aldoximes to the corresponding amides with high selectivity and efficiency under essentially neutral conditions.
