RESUMO
Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.
Assuntos
Precursores Enzimáticos , Transglutaminases , Precursores Enzimáticos/genética , Transglutaminases/metabolismoRESUMO
Cytoplasm contains high concentrations of biomacromolecules. Protein behavior under such crowded conditions is reportedly different from that in an aqueous buffer solution, mainly owing to the effect of volume exclusion caused by the presence of macromolecules. Using a crosslinking reaction catalyzed by microbial transglutaminase (MTG) as a model, we herein systematically determined how the substrate size affects enzymatic activity in both dilute and crowded solutions of dextran. We first observed a threefold reduction in MTG-mediated crosslinking of a pair of small peptide substrates in 15 wt% dextran solution. In contrast, when proteinaceous substrates were involved, the crosslinking rates in 15 wt% dextran solutions accelerated markedly to levels comparable with the level in the absence of dextran. Our results provide new insights into the action of enzymes with regard to macromolecular substrates under crowded conditions, of which the potential utility was demonstrated by the formation of highly crosslinked protein polymers.
Assuntos
Aceleração , Dextranos , Dextranos/química , Substâncias MacromolecularesRESUMO
Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both inâ vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.
Assuntos
Biotina , Proteínas , Proteínas/metabolismo , EstreptavidinaRESUMO
There is growing demand for the precise remote control of cellular functions in various fields. Herein, a method for caging mammalian cells by coating with photodegradable protein-polymer hybrid shells to photo-control their functions without genetic engineering is reported. A layer-by-layer assembly of photocleavable synthetic materials through biotin-streptavidin (SA) binding was employed for cell coating. The cell surfaces were first biotinylated with photocleavable biotinylated poly(ethylene glycol)(PEG)-lipid and then coated by repeatedly layering SA and micelles of the PEG-lipid and photocleavable biotinylated four-arm PEG. The cell extension and adhesion were suppressed with the shells and then triggered with the degradation of the shells by light exposure. Macrophage phagocytosis was also stopped by caging with the shells and restarted by light-guided uncaging. This study provides the first proof of principle that cellular functions can be remotely controlled by steric hinderance of cell surfaces with photodegradable materials.
Assuntos
Polietilenoglicóis , Polímeros , Animais , Micelas , EstreptavidinaRESUMO
Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein. The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.
Assuntos
Anfotericina B , Quitinases , Animais , Humanos , Anfotericina B/farmacologia , Anfotericina B/química , Antifúngicos/química , Quitina , Lipossomos , Lipídeos , Mamíferos/metabolismoRESUMO
Protein palmitoylation, a post-translational modification, is universally observed in eukaryotic cells. The localization of palmitoylated proteins to highly dynamic, sphingolipid- and cholesterol-rich microdomains (called lipid rafts) on the plasma membrane has been shown to play an important role in signal transduction in cells. However, this complex biological system is not yet completely understood. Here, we used a combined approach where an artificial lipidated protein was applied to biomimetic model membranes and plasma membranes in cells to illuminate chemical and physiological properties of the rafts. Using cell-sized giant unilamellar vesicles, we demonstrated the selective partitioning of enhanced green fluorescent protein modified with a C-terminal palmitoyl moiety (EGFP-Pal) into the liquid-ordered phase consisting of saturated phospholipids and cholesterol. Using Jurkat T cells treated with an immunostimulant (concanavalin A), we observed the vesicular transport of EGFP-Pal. Further cellular studies with the treatment of methyl ß-cyclodextrin revealed the cholesterol-dependent internalization of EGFP-Pal, which can be explained by a raft-dependent, caveolae-mediated endocytic pathway. The present synthetic approach using artificial and natural membrane systems can be further extended to explore the potential utility of artificially lipidated proteins at biological and artificial interfaces.
Assuntos
Lipoilação , Microdomínios da Membrana , Membrana Celular/química , Colesterol/química , Microdomínios da Membrana/química , Lipossomas Unilamelares/químicaRESUMO
Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.
Assuntos
Bombyx , Vacinas Antimaláricas , Malária Vivax , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície , Bombyx/genética , Dissulfetos , Vacinas Antimaláricas/genética , Malária Vivax/prevenção & controle , Camundongos , Plasmodium vivax/genéticaRESUMO
Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)- and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM2. We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM2 with Y-tag and KY-tag (Y-LysM2-KY) or with a glutamine-containing peptide tag (Q-tag) (LysM2-Q) were constructed. The proteins with Y-tag and KY-tag were efficiently polymerized by HRP reaction through the formation of dityrosine bonds at the tyrosine residues in the peptide tags. The Y-CatD-KY polymer was further treated by MTG to orthogonally graft LysM2-Q to the KY-tag via isopeptide formation between the side chains of the glutamine and lysine residues in the peptide tags to form LysM2-grafted CatD polymer. The LysM2-grafted CatD polymer exhibited significantly higher antifungal activity than the homopolymer of Y-ChiA-KY and the random copolymer of Y-CatD-KY and Y-LysM2-KY, demonstrating that the structural differences of artificial chitinase polymers have a significant impact on the antifungal activity. This strategy of polymerization and grafting reaction of protein can contribute to the further research and development of functional protein polymers for specific applications in various fields in biotechnology.
Assuntos
Antifúngicos/farmacologia , Quitina/química , Quitinases/química , Quitinases/metabolismo , Enzimas/metabolismo , Antifúngicos/síntese química , Enzimas/química , Polímeros , Ligação Proteica , Domínios ProteicosRESUMO
Photoactivatable ligand proteins are potentially useful for light-induced intracellular delivery of therapeutic and diagnostic cargos through receptor-mediated cellular uptake. Here, we report the simple and effective caging of transferrin (Tf), a representative ligand protein with cellular uptake ability, which has been used in the delivery of various cargos. Tf was modified with several biotin molecules through a photocleavable linker, and then the biotinylated Tf (bTf) was conjugated with the biotin-binding protein, streptavidin (SA), to provide steric hindrance to block the interaction with the Tf receptor. Without exposure to light, the cellular uptake of the bTf-SA complex was effectively inhibited. In response to light exposure, the complex was degraded with the release of Tf, leading to cellular uptake of Tf. Similarly, the cellular uptake of Tf-doxorubicin (Dox) conjugates could be suppressed by caging with biotinylation and SA binding, and the intracellular delivery of Dox could be triggered in a light-dependent manner. The intracellularly accumulated Dox decreased the cell viability to 25% because of the cell growth inhibitory effect of Dox. These results provided proof of principle that the caged Tf can be employed as a photoactivatable molecular device for the intracellular delivery of cargos.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Doxorrubicina/administração & dosagem , Transferrina/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Biotinilação , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacocinética , Humanos , Luz , Modelos Moleculares , Neoplasias/tratamento farmacológico , Transferrina/química , Transferrina/farmacocinéticaRESUMO
Synthesis of lipid-protein conjugates is one of the significant techniques in drug delivery systems of proteins; however, the intact conjugation of a lipid and protein is yet challenging due to the hydrophobicity of lipid molecules. In order to facilitate easy handling of the lipid moiety in conjugation, we have focused on a microbial transglutaminase (MTG) that can ligate specific lysine (K) and glutamine (Q) residues in lipopeptides and a protein of interest. As MTG substrates, monolipid- and dilipid-fused amphiphilic short lipopeptide substrates (lipid-G3S-RHK or lipid2-KG3S-RHK) were designed. These amphiphilic lipopeptides and a model protein (enhanced green fluorescent protein, EGFP) fused with LLQG (LQ-EGFP) were both water-soluble, and thus lipid-protein conjugates were efficiently obtained through the MTG reaction with a >80% conversion rate of LQ-EGFP even using cholesterol-G3S-RHK. In vitro cell adhesion and in vivo half-life stability of the successfully obtained lipid-protein conjugates were evaluated, showing that the monocholesterol-G3S-RHK modification of a protein gave the highest cell adhesion efficiency and longest half-life time by formation of a stable albumin/lipid-protein complex.
Assuntos
Lipopeptídeos/metabolismo , Proteínas/metabolismo , Transglutaminases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Meia-Vida , Especificidade por SubstratoRESUMO
Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.
Assuntos
Proteínas de Bactérias , Precursores Enzimáticos , Mutação , Streptomyces/genética , Transglutaminases , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Streptomyces/enzimologia , Transglutaminases/biossíntese , Transglutaminases/química , Transglutaminases/genéticaRESUMO
Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G3 S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid-protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG.
Assuntos
Membrana Celular/metabolismo , Proteínas Ligadas a Lipídeos/química , Transglutaminases/química , Catálise , Linhagem Celular Tumoral , Membrana Celular/química , Colesterol/química , Reagentes de Ligações Cruzadas/química , Ácidos Graxos/química , Glutamina/química , Humanos , Proteínas Ligadas a Lipídeos/toxicidade , Ácido Litocólico/química , Lisina/química , Peptídeos/química , Peptídeos/toxicidade , Propriedades de Superfície , Tocoferóis/químicaRESUMO
Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Animais , Baculoviridae/genética , Sequência de Bases , Bombyx/genética , Extratos Celulares/química , Linhagem Celular , Cromatografia de Afinidade , Expressão Gênica , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Estabilidade Proteica , VirosesRESUMO
DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.
Assuntos
Aminas/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Biocatálise , DNA/metabolismo , Proteínas/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Glutamina/química , Humanos , Lisina/química , Modelos Moleculares , Conformação Proteica , Proteínas/químicaRESUMO
Protein polymers of covalently cross-linked protein monomers are highly attractive biomaterials because each monomer unit possesses distinct protein functions. Protein polymers often show enhancement effects on the function by integrating a large number of molecules into one macromolecule. The cross-linking site of component proteins should be precisely controlled to avoid diminishing the protein function. However, preparing protein polymers that are cross-linked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a site-specifically cross-linked protein polymer that has a hyperbranched polymer-like structure with a high cross-linking degree. A horseradish peroxidase (HRP) reaction was used to achieve the protein polymerization through a peptide tag containing a tyrosine residue (Y-tag). Y-tag sequences were introduced to both N- and C-termini of a model protein, protein G. The dual Y-tagged protein G (Y-pG-Y) was treated with HRP to form a Y-pG-Y polymer possessing average and maximum cross-linking degree of approximately 70-mer and 150-mer, respectively. The Y-pG-Y polymer shows the highest cross-linking degree among the protein polymers reported, which are completely soluble in water and cross-linked via covalent bonding. The Y-pG-Y was cross-linked site-specifically at the Tyr residue in the Y-tag, retaining its function, and the Y-pG-Y polymer showed extremely strong avidity against immunoglobulin G. The reactivities of N- and C-terminal Y-tags were evaluated, and we revealed that the difference in the radical formation rate by HRP was the key for yielding highly cross-linked protein polymers.
Assuntos
Peroxidase do Rábano Silvestre/química , Multimerização Proteica , Tirosina/química , Imunoglobulina G/imunologia , Modelos Moleculares , Peso Molecular , Estrutura Quaternária de ProteínaRESUMO
Several methods have recently been reported for the preparation of redox-sensitive hydrogels using enzymatic reactions, which are useful for encapsulating sensitive materials such as proteins and cells. However, most of the reported hydrogels is difficult to add further function efficiently, limiting the application of the redox-sensitive hydrogels. In this study, peptide sequences of HHHHHHC and GGGGY (Y-tag) were genetically fused to the N- and C-termini of streptavidin (C-SA-Y), respectively, and C-SA-Y was mixed with horseradish peroxidase and thiol-functionalized 4-arm polyethylene glycol to yield a redox-sensitive C-SA-Y immobilized hydrogel (C-SA-Y gel). The C-SA-Y immobilized in the hydrogel retained its affinity for biotin, allowing for the incorporation of proteins and small molecules to hydrogel via biotin. C-SA-Y gel was further prepared within a water-in-oil (w/o) emulsion system to yield a nanosized hydrogel, to which any intracellular and cytotoxic agent can be modified, making it a potential drug delivery carrier.
Assuntos
Biopolímeros/química , Fibroínas/química , Fibronectinas/química , Peroxidase do Rábano Silvestre/química , Hidrogéis/química , Hidrogéis/síntese química , Proteínas Recombinantes de Fusão/química , Estreptavidina/química , Biopolímeros/genética , Reagentes de Ligações Cruzadas/química , Cisteína/química , Escherichia coli/genética , Fibroínas/genética , Fibronectinas/genética , Oxirredução , Plasmídeos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , TemperaturaRESUMO
ß-Annulus peptides bearing Cys at the N-terminal from tomato bushy stunt virus were synthesised using a standard Fmoc-protected solid-phase method, and the peptide was modified with Ni-NTA at the N-terminal. The Ni-NTA-modified ß-annulus peptide self-assembled into virus-like nanocapsules of approximately 40 nm in diameter. The critical aggregation concentration of these nanocapsules in 10 mM Tris-HCl buffer (pH 7.3) at 25 °C was 0.053 µM, which is 470 times lower than that of unmodified ß-annulus peptides. Moreover, size exclusion chromatography of the peptide assembly indicated encapsulation of His-tagged green fluorescent protein in the Ni-NTA-modified artificial viral capsid.
Assuntos
Proteínas do Capsídeo/química , Proteínas de Fluorescência Verde/química , Histidina/química , Níquel/química , Ácido Nitrilotriacético/química , Peptídeos/síntese química , Peptídeos/químicaRESUMO
The fusion protein of an engineered zymogen of microbial transglutaminase (EzMTG) with a protein G variant, EzMTG-pG, enabled the proximity-based, tag-free labeling of Lys65 in the heavy chain of a native IgG antibody (trastuzumab) with a Gln-donor peptidyl substrate functionalized with a fluorescent molecule.
Assuntos
Imunoglobulina G , Lisina , Transglutaminases , Transglutaminases/química , Transglutaminases/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lisina/química , Humanos , Corantes Fluorescentes/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.
Assuntos
Ensaios de Triagem em Larga Escala , Hidrogéis , Animais , Hidrogéis/metabolismo , Hibridomas/metabolismo , Proteínas Recombinantes/metabolismo , Dissulfetos/metabolismo , MamíferosRESUMO
Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production.