RESUMEN

Outer membrane vesicles (OMVs), non-replicating spherical liposomes derived from Gram-negative bacteria, are a promising vaccine platform and multifunctional delivery systems. Their ability to be modified via genetic engineering for the incorporation and display of heterologous proteins enhances their functionality. In this study, we demonstrated a bio-ligation approach to display single-chain variable fragments (scFv) on the OMV surface using the SpyTag/SpyCatcher system. SpyTag-fused scFv, expressed by mammalian cells, bound to OMVs with SpyCatcher-fused Lpp'OmpA after a simple incubation. Biophysical analysis indicated that the conjugated OMVs maintained their physicochemical properties. We used an scFv targeting mucin 1 protein (MUC1) for specific cell targeting. Confocal microscopy revealed that conjugated OMVs specifically bound to and were internalized by MUC1-presenting cells, but not by MUC1-deficient cells. In conclusion, this rapid and efficient bio-ligation system facilitates the display of functional scFv on OMV surfaces, offering a promising approach for targeted delivery to MUC1-expressing cancer cells.

RESUMEN

In this study, a γPFD-SpyCatcher hydrogel scaffold with the capacity for spontaneous assembly was established. With a maximum loading capacity of a 1:1 molar ratio with SpyTag-enzymes, the immobilized proteins can not only rapidly provide pure enzymes but also exhibit improved thermal and pH stability. The results of the transmission electron microscopic analysis and the traits they present indicated that SpyCatcher promotes the aggregation of γPFD and the formation of hydrogels. In the cell-free pyruvate synthesis system, the γPFD-SpyCatcher coimmobilized SpyTag-hexokinase (HK), SpyTag-phosphofructokinase (PFK) and SpyTag-pyruvate kinase (PK) were employed, and the production of pyruvate increased by 43, 78 and 47% respectively. In in vitro experiments, the oxidative deamination activity of glutamate dehydrogenase (GDH) coimmobilized with γPFD-SpyCatcher was 38% higher than that of purified enzymes. These findings indicate that the γPFD-SpyCatcher-based hydrogels play an important role in breaking the barrier of regulatory enzymes and will provide more strategies for the development of synthetic biology.

Asunto(s)

Enzimas Inmovilizadas , Hidrogeles , Hidrogeles/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glutamato Deshidrogenasa/metabolismo , Glutamato Deshidrogenasa/química , Estabilidad de Enzimas , Biocatálisis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Concentración de Iones de Hidrógeno , Ácido Pirúvico/metabolismo , Ácido Pirúvico/química

RESUMEN

Non-ribosomal peptide synthesis produces a wide range of bioactive peptide natural products and is reliant on a modular architecture based on repeating catalytic domains able to generate diverse peptide sequences. In this chapter we detail an in vitro biochemical assay to explore the substrate specificity of condensation domains, which are responsible for peptide elongation, from the biosynthetic machinery that produces from the siderophore fuscachelin. This assay removes the requirement to utilise the specificity of adjacent adenylation domains and allows the acceptance of a wide range of synthetic substrates to be explored.

Asunto(s)

Sideróforos , Especificidad por Sustrato , Sideróforos/química , Sideróforos/biosíntesis , Péptido Sintasas/metabolismo , Péptido Sintasas/química , Péptido Sintasas/genética , Péptidos/química , Péptidos/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Dominio Catalítico

RESUMEN

Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag-TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag-TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1H NMR and 13C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems.

Asunto(s)

Cromatografía de Afinidad , Inhibidores Enzimáticos , Monofenol Monooxigenasa , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cromatografía de Afinidad/métodos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética

RESUMEN

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

Asunto(s)

Hidroliasas , Rhodococcus , Rhodococcus/enzimología , Rhodococcus/genética , Hidroliasas/metabolismo , Hidroliasas/genética , Hidroliasas/química , Estabilidad de Enzimas , Estrés Fisiológico , Acrilamida/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética

RESUMEN

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pollos , Inmunoglobulinas , SARS-CoV-2 , Animales , SARS-CoV-2/inmunología , Inmunoglobulinas/inmunología , Pollos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Conejos , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Humanos , Pruebas de Neutralización

RESUMEN

There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.

Asunto(s)

Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Complejos Multienzimáticos , Nanopartículas , Infecciones por Orthomyxoviridae , Proteínas de la Matriz Viral , Animales , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Nanopartículas/química , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Ratones , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Complejos Multienzimáticos/inmunología , Complejos Multienzimáticos/metabolismo , Femenino , Ratones Endogámicos BALB C , Anticuerpos Antivirales/inmunología , Citocinas/metabolismo , Protección Cruzada/inmunología , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/virología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Viroporinas

RESUMEN

Intercellular membrane-membrane interfaces are compartments with specialized functions and unique biophysical properties that are essential in numerous cellular processes including cell signaling, development, and immunity. Using synthetic biology to engineer or to create novel cellular functions in the intercellular regions has led to an increasing need for a platform that allows generation of functionalized intercellular membrane-membrane interfaces. Here, we present a synthetic biology platform to engineer functional membrane-membrane interfaces using a pair of dimerizing proteins in both cell-free and cellular environments. We envisage this platform to be a helpful tool for synthetic biologists who wish to engineer novel intercellular signaling and communication systems.

Asunto(s)

Transducción de Señal , Biología Sintética , Animales , Membranas , Biofisica , Dimerización , Mamíferos

RESUMEN

Background: Porcine deltacoronavirus (PDCoV), a novel swine enteropathogenic coronavirus, challenges the global swine industry. Currently, there are no approaches preventing swine from PDCoV infection. Methods: A new PDCoV strain named JS2211 was isolated. Next, the dimer receptor binding domain of PDCoV spike protein (RBD-dimer) was expressed using the prokaryotic expression system, and a novel nanoparticle containing RBD-dimer and ferritin (SC-Fe) was constructed using the SpyTag/SpyCatcher system. Finally, the immunoprotection of RBD-Fe nanoparticles was evaluated in mice. Results: The novel PDCoV strain was located in the clade of the late Chinese isolate strains and close to the United States strains. The RBD-Fe nanoparticles were successfully established. Immune responses of the homologous prime-boost regime showed that RBD-Fe nanoparticles efficiently elicited specific humoral and cellular immune responses in mice. Notably, high level PDCoV RBD-specific IgG and neutralizing antibody (NA) could be detected, and the histopathological results showed that PDCoV infection was dramatically reduced in mice immunized with RBD-Fe nanoparticles. Conclusion: This study effectively developed a candidate nanoparticle with receptor binding domain of PDCoV spike protein that offers protection against PDCoV infection in mice.

Asunto(s)

Nanovacunas , Glicoproteína de la Espiga del Coronavirus , Porcinos , Animales , Ratones , Deltacoronavirus , Inmunidad , SARS-CoV-2

RESUMEN

African swine fever is an acute and highly contagious infectious disease with a mortality rate of up to 100%. The lack of commercial vaccines and drugs is a serious economic threat to the global pig industry. Cell-mediated immunity plays an essential role in protection against viral infection. We previously reported the rational design of a T-cell-activating thermostable scaffold (RPT) for antigen delivery and improved cellular immunity. We conjugated antigens P30, P54, P72, CD2 V, and CP312R to RPT, using a SpyCatcher/SpyTag covalent attachment strategy to construct nanovaccines (multiantigens-RPT). Multiantigens-RPT exhibited significantly higher thermal, storage, and freeze-thaw stability. The specific antibodies IgG and IgG2a of the multiantigen-RPT-immunized were higher than the antigens cocktail-immunized by approximately 10-100 times. ELISpot demonstrated that more IFN-γ-secreting cells were produced by the multiantigen-RPT-immunized than by the antigens cocktail-immunized. Delivery of the multiantigen nanovaccine by a T-cell-activating scaffold induced strong humoral and cellular immune responses in mice and pigs and is a potentially useful candidate vaccine for the African swine fever virus.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Ratones , Fiebre Porcina Africana/prevención & control , Linfocitos T , Nanovacunas , Adyuvantes Inmunológicos

RESUMEN

Enzyme immobilization is an effective method for improving the stability and reusability. However, linking at random sites on the enzyme results in low catalytic efficiency due to blockage of the active site or conformational changes. Therefore, controlling the orientation of enzymes on the carrier has been developed. Here, the site-specific mutation and the SpyTag/SpyCatcher systems were used to prepare a site-directed immobilized enzyme. The thermal stability of the immobilized enzyme was better than that of the free enzyme, and ≥80 % of the catalytic activity was retained after 30 days of storage. Furthermore, the Michaelis constant (Km) and the turnover number (kcat) of the immobilized enzyme were 5.23-fold lower and 6.11-fold higher than those of the free enzyme, respectively, which appeared to be related to changes in secondary structure after immobilization. These findings provide a new and effective option for enzyme-directed immobilization.

Asunto(s)

Enzimas Inmovilizadas , Nanopartículas , Enzimas Inmovilizadas/metabolismo , Estabilidad de Enzimas , Catálisis , Concentración de Iones de Hidrógeno

RESUMEN

It is of great significance to study the effect of multi-enzyme aggregation behavior at the interface on the formation of multi-enzyme complexes and their co-catalytic characteristics, which is helpful for us to design and construct immobilized multi-enzyme complex systems for in vitro synthetic biology. Here, a magnetic microsphere with chelated Ni2+, was prepared to explore the self-assembly characteristics of PduQ-SpyTag (P-T) and Nox-SpyCatcher (NC) on its surface, based on the mixed interaction mode consisting the affinity of His-tag/Ni2+ and covalent binding of SpyTag/SpyCatcher. After studying the effect of saturated or unsaturated adsorption of P-T on the covalent binding between P-T and NC at the interface, a possible multienzyme interaction mechanism for the affinity-assisted covalent self-assembly on the Ni2+ chelating surface was proposed. The time evolution of NADH showed that the immobilized P-T/N-C complex formed by this method and the free P-T/N-C complex exhibited similar synergistic catalytic properties, and presented higher catalytic efficiency than the simple mixing of P-T and NC. The optimal catalytic conditions, stability and reusability of the immobilized multi-enzyme complexes prepared in this study were also discussed by comparing them with free enzymes. In this study, we demonstrate a simple and effective strategy for self-assembling SpyTag/SpyCatcher fusion proteins on the surface of magnetic beads, which is inspiring for the construction of more cascade enzyme systems at the interface. It provides a new method for facilitating the rapid construction of immobilized multi-enzyme complexes in vitro from the crude cell lysis.

Asunto(s)

Enzimas Inmovilizadas , Complejos Multienzimáticos , Microesferas , Catálisis , Complejos Multienzimáticos/química , Fenómenos Magnéticos

RESUMEN

We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.

Asunto(s)

Proteolisis , Péptidos , Procesamiento Proteico-Postraduccional

RESUMEN

BACKGROUND: Safe and effective vaccines are crucial for the control and eventual elimination of malaria. Novel approaches to optimize and improve vaccine efficacy are urgently required. Nanoparticle-based delivery platforms are considered potent and powerful tools for vaccine development. METHODS: In this study, we developed a transmission-blocking vaccine against malaria by conjugating the ookinete surface antigen PSOP25 to the Acinetobacter phage coat protein AP205, forming virus-like particles (VLPs) using the SpyTag/SpyCatcher adaptor system. The combination of AP205-2*SpyTag with PSOP25-SpyCatcher resulted in the formation of AP205-PSOP25 complexes (VLP-PSOP25). The antibody titers and avidity of serum from each immunization group were assessed by ELISA. Western blot and IFA were performed to confirm the specific reactivity of the elicit antisera to the native PSOP25 in Plasmodium berghei ookinetes. Both in vitro and in vivo assays were conducted to evaluate the transmission-blocking activity of VLP-PSOP25 vaccine. RESULTS: Immunization of mice with VLP-PSOP25 could induced higher levels of high-affinity antibodies than the recombinant PSOP25 (rPSOP25) alone or mixtures of untagged AP205 and rPSOP25 but was comparable to rPSOP25 formulated with alum. Additionally, the VLP-PSOP25 vaccine enhanced Th1-type immune response with remarkably increased levels of IgG2a subclass. The antiserum generated by VLP-PSOP25 specifically recognizes the native PSOP25 antigen in P. berghei ookinetes. Importantly, antisera generated by inoculation with the VLP-PSOP25 could inhibit ookinete development in vitro and reduce the prevalence of infected mosquitoes or oocyst intensity in direct mosquito feeding assays. CONCLUSIONS: Antisera elicited by immunization with the VLP-PSOP25 vaccine confer moderate transmission-reducing activity and transmission-blocking activity. Our results support the utilization of the AP205-SpyTag/SpyCatcher platform for next-generation TBVs development.

Asunto(s)

Vacunas contra la Malaria , Malaria , Animales , Ratones , Proteínas Protozoarias/metabolismo , Plasmodium berghei , Formación de Anticuerpos , Malaria/prevención & control , Sueros Inmunes , Anticuerpos Antiprotozoarios , Ratones Endogámicos BALB C

RESUMEN

African swine fever (ASF) is caused by the African swine fever virus (ASFV) and is a highly contagious, acute, febrile disease that has high morbidity and mortality rates in domestic and wild swine. However, a safe and effective vaccine against ASF remains unavailable as single antigens fail to provide sufficient protection. Therefore, a combination of multiple antigens with an efficient delivery system might be an alternative strategy. Herein, a de novo-designed antigen with multiple T-cell epitopes (TEPs) of ASFV was conjugated for surface display on self-assembled nanoparticles (NPs) of Aquifex aeolicus lumazine synthase (AaLS) and Quasibacillus thermotolerans encapsulin (QT) through the SpyCatcher/SpyTag system to construct nanovaccines (TEP-Spy-NPs). TEP-Spy-NPs exhibited significantly more thermal, storage, and freeze-thaw stability in comparison to TEP monomers. TEP-Spy-NPs were highly immunogenic and induced strong polyclonal antibody responses in mice and pigs. The specific antibody titers against the TEP of the TEP-Spy-AaLS and TEP-Spy-QT groups were significantly higher than those of the TEP monomer immune group after the second booster immunization. The antibody titer against TEP of the TEP-Spy-QT group was approximately twice that of the TEP-Spy-AaLS group in mice. ELISpot analysis demonstrated that more IFN-γ- and IL-2-secreting splenic lymphocytes were produced by TEP-Spy-AaLS- and TEP-Spy-QT-immunized mice than by TEP monomer-immunized mice. TEP-Spy-NPs elicited stronger cellular immunity and in vivo immunity in immunized pigs than did TEP monomers. Thus, the TEP nanovaccine successfully induced strong humoral and cellular immune responses in mice and pigs, and TEP-Spy-NPs have the potential as candidate vaccines for ASFV.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Virus de la Fiebre Porcina Clásica , Nanopartículas , Animales , Porcinos , Ratones , Fiebre Porcina Africana/prevención & control , Formación de Anticuerpos , Linfocitos T

RESUMEN

Protein engineering allows for the programming of specific building blocks to form functional and novel materials with customisable physical properties suitable for tailored engineering applications. We have successfully designed and programmed engineered proteins to form covalent molecular networks with defined physical characteristics. Our hydrogel design incorporates the SpyTag (ST) peptide and SpyCatcher (SC) protein that spontaneously form covalent crosslinks upon mixing. This genetically encodable chemistry allowed us to easily incorporate two stiff and rod-like recombinant proteins in the hydrogels and modulate the resulting viscoelastic properties. We demonstrated how differences in the composition of the microscopic building blocks change the macroscopic viscoelastic properties of the hydrogels. We specifically investigated how the identity of the protein pairs, the molar ratio of ST:SC, and the concentration of the proteins influence the viscoelastic response of the hydrogels. By showing tuneable changes in protein hydrogel rheology, we increased the capabilities of synthetic biology to create novel materials, allowing engineering biology to interface with soft matter, tissue engineering, and material science.

RESUMEN

Presenting exogenous antigens on virus-like particles (VLPs) through "plug-and-display" decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on "plug-and-display" decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

Asunto(s)

Hepatitis B , Vacunas contra la Influenza , Gripe Humana , Vacunas de Partículas Similares a Virus , Humanos , Animales , Ratones , Vacunas de Partículas Similares a Virus/genética , Vacunas Sintéticas/genética , Vacunas contra la Influenza/genética , Ratones Endogámicos BALB C , Antígenos del Núcleo de la Hepatitis B/genética

RESUMEN

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Asunto(s)

Hidrogeles , Análisis por Matrices de Proteínas , Proteínas/genética , Materiales Biocompatibles/química , Secuencia de Aminoácidos

RESUMEN

At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.

Asunto(s)

Bacterias , Microorganismos Modificados Genéticamente , Animales , Ratones , Escherichia coli/genética

RESUMEN

Plant viral nanoparticles (VNPs) have become an attractive platform for the development of novel nanotools in the last years because of their safety, inexpensive production, and straightforward functionalization. Turnip mosaic virus (TuMV) is one example of a plant-based VNP used as a nanobiotechnological platform either as virions or as virus-like particles (VLPs). Their functionalization mainly consists of coating their surface with the molecules of interest via chemical conjugation or genetic fusion. However, because of their limitations, these two methods sometimes result in non-viable constructs. In this paper, we applied the SpyTag/SpyCatcher technology as an alternative for the functionalization of TuMV VLPs with peptides and proteins. We chose as molecules of interest the green fluorescent protein (GFP) because of its good traceability, as well as the vasoactive intestinal peptide (VIP), given the previous unsuccessful attempts to functionalize TuMV VNPs by other methods. The successful conjugation of VLPs to GFP and VIP using SpyTag/SpyCatcher was confirmed through Western blot and electron microscopy. Moreover, the isopeptide bond between SpyTag and SpyCatcher occurred in vivo in co-agroinfiltrated Nicotiana benthamiana plants. These results demonstrated that SpyTag/SpyCatcher improves TuMV functionalization compared with previous approaches, thus implying the expansion of the application of the technology to elongated flexuous VNPs.

Asunto(s)

Nanopartículas , Potyvirus , Western Blotting , Proteínas Fluorescentes Verdes