RESUMO
Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.
Assuntos
Proteínas Imediatamente Precoces , Imunoprecipitação , Ubiquitina-Proteína Ligases , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Imunoprecipitação/métodos , Humanos , Proteínas Imediatamente Precoces/metabolismo , Ligação Proteica , Proteínas Ubiquitinadas/metabolismo , Herpesvirus Humano 1/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas Virais/metabolismoRESUMO
We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (â¼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.
Assuntos
Lactoferrina , Humanos , Lactoferrina/isolamento & purificação , Lactoferrina/química , Fenantrolinas/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química , Eletroforese em Gel de Poliacrilamida , Dicroísmo Circular , Polietilenoglicóis/química , Ferro/químicaRESUMO
The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.
Assuntos
Parede Celular , Polissacarídeos , Parede Celular/química , Polissacarídeos/análise , Polissacarídeos/química , Oligossacarídeos/análise , Oligossacarídeos/química , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese/métodosRESUMO
Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.
Assuntos
Farinha , Gliadina , Glutens , Triticum , Glutens/análise , Gliadina/análise , Gliadina/química , Triticum/química , Farinha/análise , Humanos , Eletroforese em Gel de PoliacrilamidaRESUMO
Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.
Assuntos
Proteômica , Venenos de Víboras , Viperidae , Irã (Geográfico) , Animais , Venenos de Víboras/química , Espectrometria de Massas em Tandem , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2/análise , Fosfolipases A2/química , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/análise , Cromatografia Líquida de Alta Pressão , Western Blotting , Eletroforese em Gel BidimensionalRESUMO
This study was conducted to formulate a conjugate of soy protein isolate (SPI) and peach gum (PG) with improved functional properties, interacting at mass ratios of 1:1, 1:2, 1:3, 2:1, and 2:3 by Maillard reaction via wet heating method. Conjugation efficiency was confirmed by grafting degree (DG) and browning index (BI). Results indicated that DG increased with increasing concentration of PG, and decreased with increasing pH, whereas no remarkable change was observed with increasing reaction time. The conjugates were optimized at a ratio of 1:3. SDS-PAGE confirmed conjugate formation, Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) verified conjugate secondary structural changes, and scanning electron microscopy (SEM) indicated significant overall structural changes. The functional properties, solubility, emulsifying stability, water holding, foaming, and antioxidant activity were significantly improved. This study revealed the wet heating method as an effective approach to improve the functional properties of soy protein.
Assuntos
Antioxidantes , Temperatura Alta , Reação de Maillard , Solubilidade , Proteínas de Soja , Proteínas de Soja/química , Antioxidantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Gomas Vegetais/química , Emulsões , Microscopia Eletrônica de Varredura , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Eletroforese em Gel de Poliacrilamida , Água/química , Calefação , Manipulação de Alimentos/métodosRESUMO
This study presents and compares two methods for identifying the types of extracellular vesicles (EVs) from different cell lines. Through SDS-PAGE analysis, we discovered that the ratio of CD63 to CD81 in different EVs is consistent and distinct, making it a reliable characteristic for recognizing EVs secreted by cancer cells. However, the electrophoresis and imaging processes may introduce errors in the concentration values, especially at lower concentrations, rendering this method potentially less effective. An alternative approach involves the use of quartz crystal microbalance (QCM) and electroanalytical interdigitated electrode (IDT) biosensors for EV type identification and quantification. The QCM frequency shift caused by EVs is directly proportional to their concentration, while electroanalysis relies on measuring the curvature of the I-V curve as a distinguishing feature, which is also proportional to EV concentration. Linear regression lines for the QCM frequency shift and the electroanalysis curvature of various EV types are plotted separately, enabling the estimation of the corresponding concentration for an unknown EV type on the graphs. By intersecting the results from both biosensors, the unknown EV type can be identified. The biosensor analysis method proves to be an effective means of analyzing both the type and concentration of EVs from different cell lines.
Assuntos
Técnicas Biossensoriais , Vesículas Extracelulares , Técnicas de Microbalança de Cristal de Quartzo , Humanos , Eletroforese em Gel de Poliacrilamida , Linhagem Celular Tumoral , EletrodosRESUMO
SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.
Assuntos
Alérgenos , Insetos Comestíveis , Gryllidae , Imunoglobulina E , Proteínas de Insetos , Animais , Alérgenos/imunologia , Gryllidae/imunologia , Proteínas de Insetos/imunologia , Imunoglobulina E/imunologia , Imunoglobulina E/sangue , Humanos , Insetos Comestíveis/imunologia , Hipersensibilidade Alimentar/imunologia , Reações Cruzadas , Tropomiosina/imunologia , Dípteros/imunologia , Eletroforese em Gel de PoliacrilamidaRESUMO
As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and ß-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and ß-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and ß-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the ß-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and ß-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.
Assuntos
Galinhas , Eletroforese em Gel de Poliacrilamida , Ovomucina , Polissacarídeos , Animais , Glicosilação , Espectrometria de Massa com Cromatografia Líquida , Ovomucina/química , Polissacarídeos/química , Polissacarídeos/análise , Subunidades Proteicas/químicaRESUMO
BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.
Assuntos
Antibacterianos , Proteínas de Bactérias , Brevibacillus , Peso Molecular , Brevibacillus/metabolismo , Brevibacillus/genética , Brevibacillus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mitomicina/farmacologia , Cinética , Insetos/microbiologia , Concentração de Íons de Hidrogênio , Eletroforese em Gel de PoliacrilamidaRESUMO
Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.
Assuntos
Anticorpos , Eletroforese em Gel de Poliacrilamida , Peptídeos , Eletroforese em Gel de Poliacrilamida/métodos , Peptídeos/química , Peptídeos/imunologia , Anticorpos/química , Anticorpos/imunologia , Western Blotting/métodos , Humanos , Calreticulina/química , Calreticulina/imunologia , Calreticulina/metabolismo , Immunoblotting/métodos , Especificidade de Anticorpos , AnimaisRESUMO
The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.
Assuntos
Proteínas Recombinantes , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Fluorescência , Corantes Fluorescentes/química , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , HumanosRESUMO
In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.
Assuntos
Eletroforese em Gel de Poliacrilamida , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Espectrometria de Massas em Tandem , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas em Tandem/métodos , Membrana Celular/metabolismo , Proteômica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas ras/metabolismo , Proteínas ras/genéticaRESUMO
The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3' end 2'-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2'-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2'-O-methylation. We also identified two highly conserved types of 5'-tRNA fragments (tRF) bearing HEN1-independent 2'-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.
Assuntos
MicroRNAs , RNA de Transferência , MicroRNAs/metabolismo , MicroRNAs/genética , MicroRNAs/química , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Análise de Sequência de RNA/métodos , RNA de Plantas/metabolismo , RNA de Plantas/química , RNA de Plantas/genética , Metilação , Arabidopsis/genética , Arabidopsis/metabolismo , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Ácidos Borônicos/químicaRESUMO
Background: The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. Methods: The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Results: Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.
Assuntos
Proteínas de Bactérias , Disenteria Bacilar , Fezes , Shigella flexneri , Humanos , Fezes/microbiologia , Fezes/química , Disenteria Bacilar/diagnóstico , Disenteria Bacilar/imunologia , Disenteria Bacilar/microbiologia , Shigella flexneri/imunologia , Shigella flexneri/isolamento & purificação , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/análise , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/análise , Western Blotting , Eletroforese em Gel de Poliacrilamida , Imunoglobulina A/imunologia , Imunoglobulina A/sangue , Imunoglobulina A/análiseRESUMO
Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.
Assuntos
Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Gonadotropinas , Eletroforese em Gel Bidimensional/métodos , Controle de Qualidade , Isoformas de Proteínas , Ponto Isoelétrico , Glicosilação , Peso Molecular , Espectrometria de Massas/métodosRESUMO
In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.
Assuntos
Fibrinolisina , Animais , Concentração de Íons de Hidrogênio , Fibrinolisina/metabolismo , Fibrinolisina/isolamento & purificação , Poliquetos/enzimologia , Temperatura , Peso Molecular , Estabilidade Enzimática , Metais/farmacologia , Eletroforese em Gel de Poliacrilamida , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Fibrinolíticos/metabolismoRESUMO
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
Assuntos
Lipopolissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Camundongos , Macrófagos/metabolismo , Lipídeo A/química , Lipídeo A/isolamento & purificação , Citocinas/metabolismo , Endopeptidase K/metabolismo , Endopeptidase K/química , Eletroforese em Gel de Poliacrilamida/métodosRESUMO
Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1. Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity. Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.
Assuntos
Alérgenos , Alérgenos/química , Alérgenos/isolamento & purificação , Alérgenos/imunologia , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Sulfato de Amônio/químicaRESUMO
Proteinograms, a semiquantitative analytical method that separates proteins into multiple bands, have not been explored in teleosts for diagnostic or prognostic purposes. This study aimed to establish reference values for proteinograms in the serum of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), two important farmed fish species in the Mediterranean region. Serum proteins were studied using SDS-PAGE, electropherogram, and HPLC-mass spectrometry. SDS-PAGE analysis revealed four major bands of proteins around 11, 25, 70, and 100 kDa in the serum of gilthead seabream and European sea bass. Electropherogram results showed that a protein with a molecular weight of 76.8 kDa was the most abundant protein in the serum of gilthead seabream, while a peak of 75.5 kDa was the most abundant in European sea bass. HPLC-mass spectrometry detected 87 proteins and 119 proteins in the serum of gilthead seabream and European sea bass, respectively, including α1-globulins, α2-globulins, ß-globulins, and γ-globulins. Notably, the albumin sequence was not detected in either of the two species. These results help to characterize the serum protein profile and to establish reference proteinograms for these two fish species. They also provide a basis for the development of novel approaches for the rapid detection of loss of haemostasis due to stress, health disorders or disease in farmed fish.