RESUMO
We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.
Assuntos
Desenho de Fármacos , Epitopos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Proteínas/química , Ligantes , Peso Molecular , Peptídeos Cíclicos/química , Proteínas/antagonistas & inibidoresRESUMO
Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values.
Assuntos
Cromatografia de Fase Reversa , Extração em Fase Sólida , Espectrometria de Massas/métodos , Cromatografia de Fase Reversa/métodos , Peso Molecular , Anticorpos Monoclonais/químicaRESUMO
The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation, and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that selectively forms a covalent linkage to the target protein. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide, TOP1, that weakly binds to the target protein, the Src homology 3 (SH3) domain of human Abelson tyrosine kinase (Abl), was fused to green fluorescent protein (GFP) and l-DOPA was site-specifically incorporated into the peptide region (TOP1-DOPA-GFP). TOP1-DOPA-GFP produced from Escherichia coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.
Assuntos
Western Blotting/métodos , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-abl/análise , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Levodopa/genética , Levodopa/metabolismo , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
Come together right now with L-DOPA: Chemical cross-linking is widely used to study protein-protein interactions. However, many cross-linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site-specific protein cross-linking was achieved using genetically incorporated 3,4-dihydroxy-L-phenylalanine.
Assuntos
Códon de Terminação/genética , Reagentes de Ligações Cruzadas/química , Di-Hidroxifenilalanina/química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Códon de Terminação/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas Mutantes/metabolismo , Engenharia de Proteínas , RNA Mensageiro/metabolismo , Tirosina-tRNA Ligase/metabolismoRESUMO
A mammalian two-hybrid system (termed as trM2H) was developed to detect protein-protein interactions in vivo, based on the reconstitution of the functions the of tetracycline repressor (TetR). The system is sensitive enough to detect protein-protein interactions with K(d) up to 55microM in mammalian cells, and the system can be regulated by small molecules. This system can be used as an efficient genetic selection system to map protein-protein interactions in mammalian cells.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Animais , Humanos , Métodos , Ligação Proteica , Projetos de Pesquisa , TetraciclinaRESUMO
Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.
Assuntos
Mutação Puntual , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Epitopos/química , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60(o)C.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Soropositividade para HIV/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos Anti-Idiotípicos/imunologia , Química Click/métodos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , Soropositividade para HIV/diagnóstico , Soropositividade para HIV/virologia , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica/imunologia , Estabilidade Proteica , Razão Sinal-Ruído , TemperaturaRESUMO
In this work, focused near-infrared (NIR) femtosecond laser pulses were used to transiently perforate the cellular membrane of targeted human embryonic kidney (HEK) cells and the uptake of extrinsic molecules into the targeted cells was observed. Various cellular responses to the laser treatments were closely analyzed to optimize several experimental parameters such as laser power, exposure time and location of laser irradiation using a membrane impermeable fluorescent dye. The optimized parameters were used to investigate the entry of a plasmid DNA encoding green fluorescent protein (GFP) into the target cells. Since laser beam with higher-than-threshold energy level will disintegrate cells, we used Matlab simulations to characterize the laser irradiance and free electron distribution caused by the femtosecond-optoporation process. The simulation results showed that the free electron distribution is much narrower than the laser irradiance, which implies that the transient perforation can even be smaller than the size of the laser focal volume. Femtosecond laser-assisted optoporation when combined with lab-on-a-chip devices can be useful in single cell-based high-throughput screening.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Técnicas de Transferência de Genes/instrumentação , Lasers , Permeabilidade da Membrana Celular , Sobrevivência Celular , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Células HEK293 , Humanos , PorosidadeRESUMO
A previously engineered Methanocaldococcus jannaschii tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Archaea/enzimologia , RNA de Transferência/genética , Aminoacil-tRNA Sintetases/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Humanos , Mamíferos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência/químicaRESUMO
Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization. Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography were used to detect the dimer-monomer equilibrium of SrtA mutants. Circular dichroism spectrum experiments were performed to study the conformational change of each SrtA mutant. An enzyme activity assay confirmed that all the SrtA mutants were active in vitro. Our results not only are important for understanding the SrtA protein self-associating mechanism but also provided the necessary starting materials for the study of sortase A pathway in vivo, which may have significant implications for discovering microbial physiology and give a potential target for novel Gram-positive antibiotics.
Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Staphylococcus aureus/enzimologia , Sequência de Bases , Cromatografia em Gel , Dicroísmo Circular , Primers do DNA , Dimerização , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.