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1.
Biomacromolecules ; 23(9): 3688-3697, 2022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-35977087

RESUMO

In this study, functional twin liposomes (TLs) were designed by linking avidin-anchored single liposomes and biotin-anchored single liposomes via avidin-biotin interactions. Here, we first punched a hole on the liposome surface using the liposome magnetoporation method to prepare functional single liposomes, which were used for safely encapsulating quercetin (QER, as a model prodrug) or laccase (LAC, as a bioactive enzyme) inside the liposomes without the use of organic solvents; the pores were then plugged by pH-sensitive glycol chitosan grafted with 3-diethylaminopropylamine (GDEAP) and avidin (or biotin). As a result, single liposomes with QER and biotin-GDEAP were efficiently coupled with other liposomes with LAC and avidin-GDEAP. We demonstrated that the TLs could accelerate QER and LAC release at acidic pH (6.8), improving the LAC-mediated oxidization of QER and significantly elevating tumor cell death, suggesting that this strategy can be used as an efficient method for the programmed action of prodrugs.


Assuntos
Avidina , Pró-Fármacos , Avidina/metabolismo , Biotina , Concentração de Íons de Hidrogênio , Lacase , Lipossomos , Pró-Fármacos/farmacologia , Quercetina/farmacologia
2.
Methods ; 197: 54-62, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-33677061

RESUMO

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.


Assuntos
Aptâmeros de Nucleotídeos , Avidina , Aptâmeros de Nucleotídeos/metabolismo , Avidina/química , Avidina/metabolismo , Biotina/química , DNA , Humanos , Microscopia de Força Atômica/métodos
3.
Hum Cell ; 35(1): 163-178, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34643933

RESUMO

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme-inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators.


Assuntos
Motivos de Aminoácidos , Arginina/metabolismo , Membrana Celular/metabolismo , Espaço Intracelular/metabolismo , Lisina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Avidina/metabolismo , Biotinilação , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Humanos , Domínios Proteicos , Proteólise , Serina Endopeptidases/metabolismo
4.
ACS Biomater Sci Eng ; 7(12): 5611-5621, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34767332

RESUMO

Biotin-avidin interactions have been explored for decades as a technique to functionalize biomaterials, as well as for in vivo targeting, but whether changes in these interactions can be leveraged for immunomodulation remain unknown. The goal of this study was to investigate how biotin density and avidin variant can be used to deliver the immunomodulatory cytokine, interleukin 4 (IL4), from a porous gelatin scaffold, Gelfoam, to primary human macrophages in vitro. Here, we demonstrate that the degree of scaffold biotinylation controlled the binding of two different avidin variants, streptavidin and CaptAvidin. Biotinylated scaffolds were also loaded with streptavidin and biotinylated IL4 under flow, suggesting a potential use for targeting this biomaterial in vivo. While biotin-avidin interactions did not appear to influence the protein release in this system, increasing degrees of biotinylation did lead to increased M2-like polarization of primary human macrophages over time in vitro, highlighting the capability to leverage biotin-avidin interactions to modulate the macrophage phenotype. These results demonstrate a versatile and modular strategy to impart immunomodulatory activity to biomaterials.


Assuntos
Avidina , Biotina , Avidina/metabolismo , Materiais Biocompatíveis , Biotina/metabolismo , Biotinilação , Humanos , Imunomodulação
5.
J Biol Chem ; 295(32): 11174-11183, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32554809

RESUMO

Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.


Assuntos
Avidina/metabolismo , Produtos do Gene rev/metabolismo , Proteínas de Membrana/genética , Animais , Biotinilação , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Peptídeos/metabolismo , Fosforilação , Células RAW 264.7 , Transdução de Sinais
6.
Biotechnol Prog ; 36(5): e3031, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463160

RESUMO

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.


Assuntos
Avidina , Proteínas Recombinantes , Animais , Avidina/química , Avidina/isolamento & purificação , Avidina/metabolismo , Galinhas , Dissulfetos/química , Corpos de Inclusão/química , Oxirredução , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
7.
Methods Mol Biol ; 2127: 151-165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32112321

RESUMO

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.


Assuntos
Avidina/metabolismo , Biotina/metabolismo , Biotinilação/métodos , Proteínas de Membrana/metabolismo , Anticorpos de Domínio Único/isolamento & purificação , Sequência de Aminoácidos , Avidina/química , Biotina/química , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Clonagem Molecular/métodos , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo
8.
Methods Enzymol ; 633: 21-28, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32046847

RESUMO

Chicken avidin and bacterial streptavidin are workhorses in biotechnology. We have used avidin as a scaffold protein to develop avidin variants with novel ligand-binding affinity, so-called antidins. This article covers the strategy applied in the development of antidins. Using a phage display developed for avidin, immobilized ligands were used to select binders from a phage pool displaying avidin variants with randomized sequence in the protein loops. Antidins binding various ligands with nanomolar affinity were obtained. Antidins have already been demonstrated to be suitable for a diagnostic assay measuring serum progesterone levels and they offer a promising alternative to antibodies for the recognition of small molecules.


Assuntos
Avidina/química , Biotina/química , Hidrocortisona/análise , Progesterona/análise , Engenharia de Proteínas/métodos , Estreptavidina/química , Animais , Avidina/genética , Avidina/metabolismo , Sítios de Ligação , Biotina/metabolismo , Galinhas , Biblioteca Gênica , Hidrocortisona/metabolismo , Ligantes , Biblioteca de Peptídeos , Progesterona/metabolismo , Ligação Proteica , Estreptavidina/genética , Estreptavidina/metabolismo , Streptomyces
9.
J Mol Model ; 25(12): 361, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31773283

RESUMO

Biotin is well known to be bound with exceptional strength by the avidin class of proteins. This ability comes from a match between the biotin-binding pocket of the protein and the structural elements of biotin, including its ureido and thiolane rings. Here we investigate the solvation shell of biotin in water as revealed by classical force field molecular dynamics with GAFF force field. Snapshots from the classical molecular dynamics were then used to generate microsolvated structures. Details of hydrogen bonding patterns present in these microsolvated structures were studied by symmetry-adapted perturbation theory (SAPT). Interaction energy values for small models of biotin hydrated by 5 or 6 water molecules show that the cooperativity constitutes 15-22% of the total interaction energy and corresponds roughly to formation of one additional hydrogen bond to biotin. The SAPT analysis shows the differences underlying hydrogen bonds of similar strength (with oxygen or sulfur atoms as the hydrogen bond acceptors, and with nitrogen atom playing a dual role of the donor and acceptor).


Assuntos
Avidina/metabolismo , Biotina/metabolismo , Solventes/química , Água/química , Avidina/química , Sítios de Ligação , Biotina/química , Ligação de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade
10.
Nat Commun ; 10(1): 4347, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554812

RESUMO

Spatiotemporal control over engineered tissues is highly desirable for various biomedical applications as it emulates the dynamic behavior of natural tissues. Current spatiotemporal biomaterial functionalization approaches are based on cytotoxic, technically challenging, or non-scalable chemistries, which has hampered their widespread usage. Here we report a strategy to spatiotemporally functionalize (bio)materials based on competitive supramolecular complexation of avidin and biotin analogs. Specifically, an injectable hydrogel is orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contra-directional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method's universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials.


Assuntos
Avidina/química , Materiais Biocompatíveis/química , Biotina/química , Substâncias Macromoleculares/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Avidina/metabolismo , Materiais Biocompatíveis/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Biotinilação/métodos , Linhagem Celular , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Nanopartículas/química , Peptídeos/química , Peptídeos/metabolismo , Análise Espaço-Temporal , Engenharia Tecidual/métodos
11.
Chem Asian J ; 14(17): 2953-2957, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31321878

RESUMO

This paper describes the synthesis of protein microtube motors having a urease interior surface and highlights their nonbubble-propelled behavior driven by enzymatic reaction (urea→NH3 and CO2 ). The precursor microtubes were prepared by layer-by-layer assembly using a track-etched microporous polycarbonate membrane. Immobilization of a urease on the internal wall was accomplished using avidin-biotin interaction. The tubules swam smoothly in an aqueous media containing a physiological concentration of urea. Each tubule was rotating laterally while moving forward. It is remarkable that the microtubes were digested completely by proteases, demonstrating perfect biodegradability.


Assuntos
Avidina/química , Biotina/química , Urease/metabolismo , Avidina/metabolismo , Biotina/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cimento de Policarboxilato/química , Porosidade , Ureia/química , Ureia/metabolismo , Urease/química
12.
Adv Healthc Mater ; 8(17): e1900665, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31318180

RESUMO

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.


Assuntos
Neutrófilos/metabolismo , Toxinas Biológicas/farmacologia , ADP Ribose Transferases/metabolismo , Avidina/metabolismo , Biotinilação , Toxinas Botulínicas/metabolismo , Linhagem Celular , Citosol/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/química
13.
Clin Lab ; 65(6)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31232038

RESUMO

BACKGROUND: GLP-1 as an incretin, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Besides the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Thus, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment. METHODS: In this work, we reported an electrochemical sense array based on the aptamer and biotin-avidin system for the detection of glucagon-like peptide-1 (GLP-1). The sense array employed a "stem-loop" conformation ap-tamer which was immobilized on the electrode of the 16-unit gold array via pre-labeled thiol group (-SH). Pre-labeled biotin serves as an affinity tag for the binding of avidin-horseradish peroxidase (avidin-HRP). The stem-loop structure of the aptamer kept the biotin from being approached by a bulky avidin-HRP by means of the steric hindrance. After the interaction of the target (GLP-1) and the aptamer, the aptamer would undergo a significant conformational change to force biotin away from the electrode, giving the avidin-HRP easy access to the labeled biotin. The HRP in the substrate could sensitively transduce the concentration of GLP-1 into the electrical signals, which were then measured by electrochemical technology of cyclic voltammetry and amperometric i-t curve. RESULTS: Under the optimal experimental conditions, the proposed sense array for GLP-1 had a good linear relationship from 0.1 pmol/L to 20 pmol/L with a detection limit of 0.05 pmol/L and can be used to accurately detect the GLP-1 in serum. CONCLUSIONS: The experimental results show that GLP-1 could be selectively detected by the electrochemical sense array, indicating that the proposed sense array based on the biotin-avidin system and the stem-loop aptamer has great potential in the detection of GLP-1.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Avidina/metabolismo , Técnicas Biossensoriais/métodos , Biotina/metabolismo , Técnicas Eletroquímicas/métodos , Peptídeo 1 Semelhante ao Glucagon/sangue , Aptâmeros de Nucleotídeos/química , Diabetes Mellitus Tipo 2/sangue , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Ligação Proteica , Reprodutibilidade dos Testes
14.
Nanoscale ; 11(19): 9436-9443, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31038504

RESUMO

Ionic concentration-polarization (CP)-based biomolecule preconcentration is an established method for enhancing the detection sensitivity of a target biomolecule immunoassay. However, its main drawback lies in its inability to directly control the spatial overlap between the preconcentrated plug of biomolecules and the surface immobilized antibodies. To overcome this, we simultaneously preconcentrated freely suspended, surface functionalized nanoparticles and target molecules along the edge of a depletion layer, thus, increasing the binding kinetics and avoiding the need to tune their relative locations to ensure their spatial overlap. After the desired incubation time, the nanoparticles were dielectrophoretically trapped for postprocessing analysis of the binding signal. This novel combination of CP-based preconcentration and dielectrophoresis (DEP) was demonstrated through binding of avidin and biotin-conjugated particles as a model bead-based immunoassay, wherein increased detection sensitivity was demonstrated compared to an immunoassay without CP-based preconcentration. The DEP trapping of the beads following binding is important not only for an enhanced detection signal due to the preconcentration of the beads at the electrode edges but also for controlling their location for future applications integrating localized sensors. In addition, DEP may be important also as a preprocessing step for controlling the number of beads participating in the immunoassay.


Assuntos
Imunoensaio/métodos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Nanopartículas/química , Imagem com Lapso de Tempo
15.
Arch Biochem Biophys ; 665: 87-95, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30831071

RESUMO

In sedimentation velocity experiments, we have been able to detect hybrid Rhizobium etli pyruvate carboxylase tetramers formed between subunits that contain covalently bound biotin and mutant subunits that do not. This was performed by forming complexes of the tetramers with the biotin-binding protein avidin. In addition, we have shown that it is possible to form hybrid tetramers of pyruvate carboxylase subunits from two different organisms (bacteria - Rhizobium etli and fungi - Aspergillus nidulans). In hybrid tetramers containing mutant subunits that are not fully catalytically active and fully catalytically active subunits, the catalytic and regulatory properties of these hybrid tetramers are modified compared to homotetramers of the fully active pyruvate carboxylase subunits. Our data indicates that the model of catalysis involving half-of-the-sites activity in which there is obligatory alternation of pyruvate carboxylating activity between pairs of subunits either face of the tetramer, does not occur in the hybrid tetramers. Our results are also discussed in relation to recent findings that there are multiple pathways of biotin carboxylation and decarboxylation between subunits in pyruvate carboxylase tetramers.


Assuntos
Biopolímeros/metabolismo , Piruvato Carboxilase/metabolismo , Termodinâmica , Regulação Alostérica , Avidina/metabolismo , Biopolímeros/química , Catálise , Cinética , Piruvato Carboxilase/química , Ultracentrifugação
16.
PLoS One ; 14(2): e0212339, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30785944

RESUMO

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.


Assuntos
Avidina/metabolismo , Biotina/metabolismo , Progesterona/sangue , Progesterona/metabolismo , Animais , Avidina/química , Sítios de Ligação , Bioensaio , Biotina/química , Cães , Ligantes , Modelos Moleculares , Progesterona/química , Ligação Proteica
17.
Cancer Res Treat ; 51(3): 861-875, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30282451

RESUMO

PURPOSE: This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. MATERIALS AND METHODS: Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1-overexpressing and KIM-1-low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1-overexpressing and KIM1-low expressing tumors in mice was examined by whole-body fluorescence imaging. RESULTS: A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1-overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1-overexpressing A498 renal tumor compared to KIM1-low expressing HepG2 liver tumor in mice. CONCLUSION: The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.


Assuntos
Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Neoplasias Renais/metabolismo , Imagem Molecular/métodos , Peptídeos/metabolismo , Regulação para Cima , Células A549 , Animais , Avidina/metabolismo , Biotina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Camundongos , Transplante de Neoplasias , Imagem Óptica , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação
18.
Adv Biosyst ; 3(3): e1800288, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-32627405

RESUMO

With high brightness and photostability, quantum dots (QDs) are potent probes for long-term imaging of dynamic cell surface proteins, but practical methods to covalently label QDs to target proteins for stable imaging are largely lacking. Here, a small covalent-bond forming protein (Covalent-avidin)/peptide pair is introduced, which provides a recombinant protein-based rapid and covalent QD labeling strategy. Covalent-avidin is constructed by optimized fusion of circular permuted monomeric avidin to SpyCatcher, which forms an isopeptide bond with the SpyTag peptide. Covalent-avidin-conjugated QDs allow for strong and irreversible QD labeling to the biotinylated SpyTag-fused adrenergic receptor on live cells in 2 min. In addition, QDs with only a minimum number of conjugated Covalent-avidin show more stable receptor labeling than commercially available streptavidin-conjugated QDs, also with minimal unwanted clustering of labeled receptors. Monomeric Covalent-avidin will be a valuable protein linker for diverse other nanolabeling structures with beneficial properties such as covalent linkages and facile valency control.


Assuntos
Avidina , Técnicas Citológicas/métodos , Proteínas de Membrana , Pontos Quânticos , Animais , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Células Cultivadas , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
19.
J Am Chem Soc ; 140(49): 16925-16928, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30484642

RESUMO

A bionanocomposite with artificial binding pockets for a DNA repair enzyme has been developed by in situ assembly of an affinity protein with a surrounding contact surface of polydopamine on the surface of silica coated magnetic nanoparticles via molecular imprinting reactions. The obtained nanoparticles exhibited antibody-like binding behavior toward the target enzyme with highly specific and efficient inhibition effect. Moreover, the binding and inhibition could be flexibly tuned by the addition of metal ions such as Mn2+ and Mg2+, which provided a convenient tool to regulate enzyme activity with artificially engineered nanoinhibitors.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Inibidores Enzimáticos/química , Nanopartículas de Magnetita/química , Nanocompostos/química , Avidina/química , Avidina/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Indóis/química , Ligantes , Magnésio/química , Manganês/química , Impressão Molecular/métodos , Polímeros/química , Ligação Proteica , Dióxido de Silício/química
20.
FEBS J ; 285(24): 4617-4630, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30369031

RESUMO

The subfamily of bacterial dimeric avidins is being extended through the discovery of additional members originating from diverse sources. All of these newly discovered dimeric avidin forms exhibit high affinity towards biotin, despite their lack of critical Trp in the classical tetrameric forms. The common feature of forming cylinder-like multimers (hexamers and octamers) seems to be more than a random occurrence, which generally characterizes their apo forms in the crystalline state and also in some cases in solution. Afifavidin from the Gram-negative α-proteobacterium Afifella pfennigii is the fourth member of the subfamily of dimers, which, in the intact apo form, also congregates into octamers both in the solution and in the crystalline state, whereby the C-terminal extended segments stretch into the biotin-binding sites of adjacent non-canonical monomers. The intact apo afifavidin molecule self-assembles into toroid-shaped nanostructures that dissociate into the inherent dimers upon binding biotin. On removal of the C-terminal regions, the short-form of afifavidin forms dimers both in the solution and in the crystalline states. The high affinity of the dimeric forms of afifavidin towards biotin is maintained, due to the conserved disulfide bridge between L3,4 and L5,6 and the presence of Phe50 in L3,4 that compensate for the lack of the critical Trp in the tetrameric avidins. These cyclic multimeric-avidin assemblies may be exploited in the future to further diversify biotin-based nanotechnology or to serve as building blocks in the construction of bio-inspired materials. DATABASE: Structural data are available in the PDB databases under the accession numbers: 6HDV, 6HDS, 6HDT.


Assuntos
Alphaproteobacteria/metabolismo , Avidina/química , Avidina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Homologia de Sequência
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