RESUMEN
Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.
Asunto(s)
Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Mapeo de Interacción de Proteínas , Factores de Transcripción , Mapeo de Interacción de Proteínas/métodos , Humanos , Factores de Transcripción/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Unión Proteica , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Endonucleasas , Enzimas MultifuncionalesRESUMEN
Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.
Asunto(s)
Celulasa , Cryptococcus gattii , Escherichia coli , Cryptococcus gattii/genética , Cryptococcus gattii/enzimología , Cryptococcus gattii/química , Celulasa/genética , Celulasa/química , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Celulasa/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/biosíntesis , Expresión Génica , Clonación Molecular , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (Km = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management.
Asunto(s)
Quitina , Quitinasas , Penicillium , Quitinasas/metabolismo , Quitinasas/genética , Quitina/metabolismo , Penicillium/enzimología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Hidrólisis , Ingeniería de Proteínas/métodos , Solubilidad , CinéticaRESUMEN
Importin ß-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin ß binding (IBB) domain, is solubilized by importin ß in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.
Asunto(s)
Regulación hacia Abajo , Señales de Localización Nuclear , Proteína FUS de Unión a ARN , beta Carioferinas , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Humanos , beta Carioferinas/metabolismo , beta Carioferinas/genética , Núcleo Celular/metabolismo , Unión Proteica , Células HEK293 , Agregado de Proteínas , Células HeLa , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Transporte Activo de Núcleo CelularRESUMEN
Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.
Asunto(s)
Estreptavidina , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos/metabolismo , Humanos , Biotinilación , Microscopía Fluorescente/métodos , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismoRESUMEN
Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.
Asunto(s)
Apolipoproteína A-I , Escherichia coli , Proteínas Recombinantes de Fusión , Apolipoproteína A-I/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/genéticaRESUMEN
CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteolisis , Receptores CCR2 , Receptores CCR2/metabolismo , Humanos , Proteolisis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Células HEK293 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Metabolic dysfunction-associated steatohepatitis (MASH) poses challenges for targeted delivery and retention of therapeutic proteins due to excess extracellular matrix (ECM). Here we present a new approach to treat MASH, termed "Fibrosis overexpression and retention (FORT)". In this strategy, we design (1) retinoid-derivative lipid nanoparticle (LNP) to enable enhanced mRNA overexpression in fibrotic regions, and (2) mRNA modifications which facilitate anchoring of therapeutic proteins in ECM. LNPs containing carboxyl-retinoids, rather than alcohol- or ester-retinoids, effectively deliver mRNA with over 10-fold enhancement of protein expression in fibrotic livers. The carboxyl-retinoid rearrangement on the LNP surface improves protein binding and membrane fusion. Therapeutic proteins are then engineered with an endogenous collagen-binding domain. These fusion proteins exhibit increased retention in fibrotic lesions and reduced systemic toxicity. In vivo, fibrosis-targeting LNPs encoding fusion proteins demonstrate superior therapeutic efficacy in three clinically relevant male-animal MASH models. This approach holds promise in fibrotic diseases unsuited for protein injection.
Asunto(s)
Nanopartículas , ARN Mensajero , Animales , Masculino , Nanopartículas/química , Humanos , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Cirrosis Hepática/terapia , Modelos Animales de Enfermedad , Hígado/metabolismo , Hígado/patología , Matriz Extracelular/metabolismo , Ratones Endogámicos C57BL , Lípidos/química , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Fibrosis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , LiposomasRESUMEN
An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins.
Asunto(s)
Proteínas Anticongelantes , Hielo , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Proteínas Anticongelantes/aislamiento & purificación , Proteínas Anticongelantes/genética , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.
Asunto(s)
Inteínas , Proteínas de Unión a Maltosa , Rec A Recombinasas , beta-Galactosidasa , Inteínas/genética , beta-Galactosidasa/genética , beta-Galactosidasa/química , beta-Galactosidasa/aislamiento & purificación , beta-Galactosidasa/metabolismo , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Elastina/química , Elastina/genética , Elastina/aislamiento & purificación , Precipitación Química , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/químicaRESUMEN
This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrapTM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli. This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli. Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.
Asunto(s)
Escherichia coli , Cuerpos de Inclusión , Replegamiento Proteico , Proteínas Recombinantes de Fusión , Anticuerpos de Dominio Único , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificación , Anticuerpos de Dominio Único/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.
Asunto(s)
Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Cadherinas , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insectos , Larva , Mariposas Nocturnas , Proteínas Recombinantes de Fusión , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cadherinas/inmunología , Cadherinas/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/genética , Endotoxinas/inmunología , Endotoxinas/química , Endotoxinas/farmacología , Endotoxinas/metabolismo , Endotoxinas/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacología , Mariposas Nocturnas/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Insectos/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Anticuerpos/inmunología , Anticuerpos/química , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Insecticidas/química , Insecticidas/farmacología , Control Biológico de VectoresRESUMEN
Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas , Nicotiana , Proteínas Recombinantes de Fusión , Nicotiana/metabolismo , Nicotiana/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/genética , Humanos , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni2+-NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni2+, Mg2+, and Na+ inhibited the keratinolytic activity, Cu2+, Ca2+, and Mn2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.
Asunto(s)
Péptido Hidrolasas , Proteínas Recombinantes de Fusión , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Concentración de Iones de Hidrógeno , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genética , Queratinas/química , Queratinas/metabolismo , Estabilidad de Enzimas , Animales , Ingeniería de Proteínas/métodos , Temperatura , Dominios Proteicos , Modelos Moleculares , Plumas/química , Especificidad por SustratoRESUMEN
Association-controllable hemoprotein assemblies were constructed from a fusion protein containing two c-type cytochrome units using 3D domain swapping. The hemoprotein assembly exhibited a dynamic exchange between cyclic and linear structures and could be regulated by carbon monoxide (CO) and imidazole binding.
Asunto(s)
Monóxido de Carbono , Hemoproteínas , Imidazoles , Ligandos , Monóxido de Carbono/química , Imidazoles/química , Hemoproteínas/química , Hemoproteínas/metabolismo , Dominios Proteicos , Unión Proteica , Sitios de Unión , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Modelos MolecularesRESUMEN
Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Progranulinas , Proteolisis , Proteínas Recombinantes de Fusión , Progranulinas/metabolismo , Progranulinas/genética , Humanos , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular Tumoral , Unión Proteica , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/genética , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine ß-sheets, Cys30-Cys67, Cys38-Cys58, Cys53-Cys90, Cys56-Cys66, and Cys81-Cys102 participating in the formation of five pairs of disulfide bonds. In vitro experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 µmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 µmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation.
Asunto(s)
Movimiento Celular , Proliferación Celular , Stichopus , Animales , Stichopus/genética , Stichopus/química , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Humanos , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Clonación Molecular , Secuencia de Aminoácidos , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Células HaCaTRESUMEN
Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: ⢠Chimeric DNA polymerase is generated by rational design method. ⢠Strategies include domain exchange and addition of proteins, domains, and motifs. ⢠Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ingeniería de Proteínas/métodosRESUMEN
Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).
Asunto(s)
Clostridium botulinum , Flagelina , Glicosiltransferasas , Especificidad por Sustrato , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/química , Flagelina/metabolismo , Flagelina/genética , Flagelina/química , Clostridium botulinum/enzimología , Clostridium botulinum/metabolismo , Clostridium botulinum/genética , Glicosilación , Escherichia coli/genética , Escherichia coli/metabolismo , Azúcares Ácidos/metabolismo , Ingeniería de Proteínas , Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Ácidos SiálicosRESUMEN
Many disease resistance genes have been introgressed into wheat from its wild relatives. However, reduced recombination within the introgressed segments hinders the cloning of the introgressed genes. Here, we have cloned the powdery mildew resistance gene Pm13, which is introgressed into wheat from Aegilops longissima, using a method that combines physical mapping with radiation-induced chromosomal aberrations and transcriptome sequencing analysis of ethyl methanesulfonate (EMS)-induced loss-of-function mutants. Pm13 encodes a kinase fusion protein, designated MLKL-K, with an N-terminal domain of mixed lineage kinase domain-like protein (MLKL_NTD domain) and a C-terminal serine/threonine kinase domain bridged by a brace. The resistance function of Pm13 is validated through transient and stable transgenic complementation assays. Transient over-expression analyses in Nicotiana benthamiana leaves and wheat protoplasts reveal that the fragment Brace-Kinase122-476 of MLKL-K is capable of inducing cell death, which is dependent on a functional kinase domain and the three α-helices in the brace region close to the N-terminus of the kinase domain.