RESUMEN
Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Albúmina Sérica Bovina , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Células CHO , Ligandos , Albúmina Sérica Bovina/química , Porosidad , Cromatografía de Afinidad/métodos , Proteína Estafilocócica A/química , Transferrina/química , Transferrina/aislamiento & purificación , Pepsina A/química , Pepsina A/metabolismo , Proteínas/aislamiento & purificación , Proteínas/química , Resinas Sintéticas/química , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Transferrin (TRF), a glycoprotein involved in cellular iron uptake, is a potential target for the diagnosis and treatment of several diseases and cancers. Therefore, the identification and isolation of TRF is clinically important. In this work, we prepared magnetic molecularly imprinted polymers (EMIP) based on metal chelation using norepinephrine and 3-aminophenylboronic acid as functional monomers. The obtained EMIP shows excellent recognition of TRF with the adsorption capacity of 94.2 mg/g and imprinting factor of 3.50. In addition, EMIP was characterized by high specificity, good adsorption performance and stability, and was successfully used for the analysis of TRF in human serum. The present study provides a reliable scheme for targeted epitope imprinting of polymers with metal chelating and dual-functional monomers, showing great potential for biosample analysis.
Asunto(s)
Ácidos Borónicos , Polímeros Impresos Molecularmente , Transferrina , Transferrina/química , Transferrina/aislamiento & purificación , Humanos , Adsorción , Polímeros Impresos Molecularmente/química , Ácidos Borónicos/química , Impresión Molecular , Epítopos/química , Polímeros/química , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans' role in their distinct physiological functions.
Asunto(s)
Inmunoglobulina G , Procesamiento Proteico-Postraduccional , Transferrina , Humanos , Glicosilación , Ensayos Analíticos de Alto Rendimiento , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Transferrina/química , Transferrina/aislamiento & purificación , Polisacáridos/análisisRESUMEN
Transferrin is an iron binding glycoprotein actively involved in the growth and maintenance of cell cycle. The transferrin receptors expression is increased on growing cancer/tumor cells for absorption of iron through transferrin and participation in biological activity. In this study, a novel method for the purification of transferrin by using magnetic nanoparticles (MNP) is developed and compared with reported method. Magnetic nanoparticles were synthesized by co-precipitation method under hydrothermal conditions in the presence of ammonium hydroxide. MNP were characterized by FTIR, VSM, DLS, TEM, and SEM. Purified transferrin was characterized by SDS-PAGE, MALDI-TOF, ELISA, Western blot, and its activity was further confirmed by iron binding assay and receptor binding assays. Purified transferrin was also conjugated with cysteine capped gold nanoparticles (GNP) and characterized by UV-Vis spectra, TEM, DLS, and fluorescent spectrophotometry. Transferrin conjugated cysteine capped GNP used as a targeted fluorescent probe on gastric cancer, tumor tissue and MDA-MB 231 cancer cells to confirm transferrin receptor binding activity and application as diagnostic probe. The purified transferrin showed stability and activity up to 36 months. The results indicated that the synthesized superamagnetic MNP are good for the purification of transferrin. A good yield of transferrin was purified by this method, good quality and showed active biological activity. GNP conjugated transferrin has a potential to be used in cancer diagnosis as targeted diagnosis probe in vivo and in vitro. Experiments are underway for utilizing transferrin as carrier for targeting drug delivery.
Asunto(s)
Cisteína/química , Oro/química , Nanopartículas de Magnetita/química , Transferrina/aislamiento & purificación , Línea Celular Tumoral , Humanos , Sondas MolecularesRESUMEN
With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High-performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow-through poly(styrene-co-divinylbenzene) microspheres characteristic of the binary pores, i.e. flow-through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene-co-divinylbenzene) microspheres were suitable as the reversed-phase stationary phase for separation of proteins. For the high permeability of the poly(styrene-co-divinylbenzene) microspheres packed column, fast separation of the studied six proteins in â¼2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0-99.4%. The proposed column had good pH stability of 1-13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio-sample separation. The flow-through poly(styrene-co-divinylbenzene) microspheres were promising for fast separation of large molecules.
Asunto(s)
Cromatografía de Fase Inversa , Microesferas , Poliestirenos/química , Animales , Bovinos , Citocromos c/química , Citocromos c/aislamiento & purificación , Lactoglobulinas/química , Lactoglobulinas/aislamiento & purificación , Muramidasa/química , Muramidasa/aislamiento & purificación , Muramidasa/metabolismo , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/aislamiento & purificación , Ribonucleasa Pancreática/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/aislamiento & purificación , Porcinos , Transferrina/química , Transferrina/aislamiento & purificación , Tripsina/química , Tripsina/aislamiento & purificación , Tripsina/metabolismoRESUMEN
Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a 'bottom-up' method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.
Asunto(s)
Carbono , Electroforesis Capilar , Puntos Cuánticos , Transferrina/química , Transferrina/aislamiento & purificación , Tampones (Química) , Carbono/química , Electrólitos , Electroforesis Capilar/métodos , Fluorometría , Concentración de Iones de Hidrógeno , Metaloproteínas/química , Metaloproteínas/aislamiento & purificación , TemperaturaRESUMEN
Immunoaffinity chromatography has a huge interest in the biomedical and biotechnological fields, in particular for one-step isolation, purification and removal of analyte compounds. In this study, uniform-sized microcryogels, a new type of cryogels, were synthesized using 2-hydroxyetyhl methacrylate and epoxy-group-containing monomer, glycidyl methacrylate for purification of a plasma protein, transferrin. Immunoaffinity microcryogels containing anti-Tf antibodies were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, optical microscopy, scanning electron microscopy, density measurements and swelling tests. Adsorption studies in aqueous media were carried out in order to examine the effects of medium pH, initial concentration of analyte and contact time. It was found that the optimum pH was 6.0 and the maximum adsorption capacity of immunoaffinity microcryogels at this pH value found to be 9.82â¯mg/g. The KL constant for Langmuir isotherm was calculated as 2.65â¯mL/mg. The maximum adsorption capacity obtained from experimental studies is also very close to the calculated Langmuir adsorption capacity (11.27â¯mg/g). Langmuir adsorption isotherms and pseudo-second-order kinetic models are consistent with the adsorption process, which means that the adsorption is single layered and chemically controlled. The purity of the eluted hsTf from plasma was about 84% with yield about 82%. After the tenth use of the same microcryogels, the maximum hsTf adsorption capacity decreased by about 20%. The results indicated that the immunoaffinity microcryogels having anti-Tf antibody ligands could be a safe and cost-friendly method for purification of transferrin.
Asunto(s)
Cromatografía de Afinidad/métodos , Criogeles/química , Transferrina/aislamiento & purificación , Adsorción , Anticuerpos Inmovilizados/metabolismo , Humanos , Cinética , Transferrina/análisis , Transferrina/química , Transferrina/metabolismoRESUMEN
The fully-mechanized Multicommutated Flow Analysis (MCFA) system for determination of transferrin in human serum has been developed. The analytical methodology concerns immunoprecipitation measurements with the use of LED-based nephelometric flow-through detectors with polymeric light guides. To improve the mechanization degree of the MCFA system, the Do-It-Yourself electronic module for mobile phone control has been developed. The design and structure of an Android software (build using open source application) controlling the operation of presented flow system has been presented. To bypass the problem of nonlinearity of calibration curve caused by the nature of antigen-antibody interactions, the on-line dilution module has been integrated into the presented manifold. Under optimized conditions, the developed flow analysis system is characterized by relatively low limit of detection and quantitation (2.0 and 4.9â¯mgâ¯L-1, respectively), good precision (RSD <â¯4%), low reagent and sample consumption per one measurement (16⯵L of undiluted reagent and 20⯵L of undiluted sample without further on-line dilution, respectively) and relatively high throughput (approximately 35 signal recordings per hour). The developed MCFA system enabled to analyze 16 serum samples with the transferrin concentration from 90 to almost 350â¯mgâ¯dL-1 with statistical agreement to the reference method.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas Sanguíneas/aislamiento & purificación , Inmunoquímica/métodos , Transferrina/aislamiento & purificación , Proteínas Sanguíneas/química , Electrónica , Humanos , Nefelometría y Turbidimetría , Transferrina/químicaRESUMEN
Biomedical applications and biomarkers for early clinical diagnostics and the treatment of diseases demand efficient and selective enrichment platforms for glycoproteins. Thus, we herein report a facile and general approach for the preparation of boronic acid-functionalized magnetic nanoparticles for the selective enrichment of glycoproteins. More specifically, Fe3O4 magnetic nanoparticles were initially prepared via a solvothermal reaction, and core-shell-structured Fe3O4@SiO2 nanoparticles were obtained according to a sol-gel process. Subsequently, the Fe3O4@SiO2 surfaces were modified using 4-formylphenylboronic acid to allow the formation of strong yet reversible covalent bonds between boronic acid (BA) and the cis-1,2-diol groups of glycoproteins. The morphology and structure of the Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2-BA nanoparticles were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and vibrating sample magnetometry, thereby confirming their successful preparation. The obtained BA-modified Fe3O4@SiO2 magnetic nanoparticles were applied in the attempted enrichment of two glycoproteins (ovalbumin (OVA) and transferrin (TRF)) and two non-glycoproteins (bovine serum albumin (BSA) and cytochrome c (Cyt C)). The results confirmed a significant difference in affinity between glycoproteins and non-glycoproteins. In addition, the recognition capability of the Fe3O4@SiO2-BA nanoparticles was demonstrated by the selective enrichment of glycoproteins from a complex system containing both glycoproteins (i.e., TRF) and non-glycoproteins (i.e., Cyt C).
Asunto(s)
Benzaldehídos/química , Ácidos Borónicos/química , Nanopartículas de Magnetita/química , Ovalbúmina/química , Transferrina/química , Adsorción , Animales , Bovinos , Dispersión Dinámica de Luz , Óxido Ferrosoférrico/química , Ovalbúmina/aislamiento & purificación , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/aislamiento & purificación , Dióxido de Silicio/química , Espectroscopía Infrarroja por Transformada de Fourier , Transferrina/aislamiento & purificaciónRESUMEN
Poly(glycidyl methacrylate-ethyleneglycol dimethacrylate) (Poly(GMA-EDMA)) monolith functionalized with cobalt phthalocyanine tetracarboxylic acid (CoPcTc) was prepared. The monolith was used for transferrin (Tf) glycopeptide enrichment. By taking advantage of hydrogen bonds between isoindole subunits of phthalocyanine and glycans and coordination interaction between cobalt and glycopeptides, the monolithic material was efficient and selective. After enrichment of transferrin through the functionalized monolith, 17 glycopeptides were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. When the concentration of transferrin was reduced to 8.8×10-10mol/L, three glycopeptides could still be obtained. The present method has great potential for trace sample analysis.
Asunto(s)
Glicopéptidos/aislamiento & purificación , Indoles/química , Compuestos Organometálicos/química , Polímeros , Transferrina/aislamiento & purificación , Compuestos Epoxi , Isoindoles , Metacrilatos , MetilmetacrilatosRESUMEN
Transferrin (TrF) is a very important human body glycoprotein and a clinical biomarker which controls the body's iron ion channels and iron ion balance. Any change in TrF concentration and isoform also reflects the emergence of some diseases. In this work, we prepared magnetic molecularly imprinted nanoparticles (deep eutectic solvent-molecular imprinting polymers [DES-MIPs]) with a deep eutectic solvent (DES) as a functional monomer to separate TrF in human serum. The DES dosage for MIP, pH value, and time for adsorption have been optimized, and these materials show special adsorption properties for TrF. The maximum adsorption capacity (Qmax) and dissociation constant KL of the MIP by the Langmuir adsorption curve (R2 = 0.9949) were 37.5 mg/g and 0.015 g/L, respectively. The imprinting factor of the MIP is 3.50 with relative standard deviation (5.63%). In summary, the use of DES as a functional monomer in molecular imprinting technology provides a novel, efficient, and biocompatible method for the isolation and purification of proteins. Graphical abstract á .
Asunto(s)
Nanopartículas de Magnetita/química , Impresión Molecular/métodos , Polímeros/química , Extracción en Fase Sólida/métodos , Transferrina/aislamiento & purificación , Adsorción , Humanos , Solventes/química , Transferrina/análisisRESUMEN
In the present study, the narrow-dispersed penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EDMA)) microspheres were successfully synthesized based on the sacrificial support method. The poly(MAA-co-EDMA) microspheres mirrored the porous structure of the sacrificial support, i.e. penetrable silica, characteristic of copious mesopores and throughpores. In addition, they possessed large surface area, adjustable hydrophobicity and the cation-exchange ability. Owing to their multi functionalities, they were applied as chromatographic stationary phase to separate proteins in different separation modes, including reversed phase, hydrophobic interaction and weak cation exchange. Moreover, thanks to their throughpores, fast separation at low column backpressure could be achieved in these three modes. Both protein recovery and column stability were satisfactory. The penetrable poly(MAA-co-EDMA) microspheres were potential stationary phase matrix for fast protein separation.
Asunto(s)
Citocromos c/aislamiento & purificación , Microesferas , Ácidos Polimetacrílicos/síntesis química , Ribonucleasa Pancreática/aislamiento & purificación , Transferrina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Citocromos c/química , Citocromos c/metabolismo , Tamaño de la Partícula , Ácidos Polimetacrílicos/química , Porosidad , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismo , Dióxido de Silicio/química , Propiedades de Superficie , Transferrina/químicaRESUMEN
BACKGROUND: Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. METHODS: The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. RESULTS: Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. CONCLUSION: The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Trastornos Congénitos de Glicosilación/diagnóstico , Electroforesis Capilar/métodos , Transferrina/análisis , Femenino , Humanos , Masculino , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Transferrina/química , Transferrina/aislamiento & purificaciónRESUMEN
A microscale procedure for the isolation of transferrin directly from human serum (hTf) is described in this study. The protocol is based on three precipitation steps without application of chromatography. It lasts 90min with the initial sample volume of 250µL. The yield of the isolated hTf is 58%, which is considerable in biochemical terms. The purity of the isolated hTf is 97%, as assessed by three methods: electrophoresis followed by protein staining, immunoblotting and HPLC. Immunoblotting with antibodies against other major serum proteins indicated that isolated hTf does not contain albumin, immunoglobulin G or alpha-2-macroglobulin. Lectin dot-blot demonstrated that isolated hTf preserved its glycan moieties. Fluorescent emission spectroscopy of the isolated hTf has shown no changes in tertiary structure. Isolated hTf was approximately 26% saturated with iron ion, which is comparable to physiological value (although a degree of saturation decreases to some extent during isolation procedure). Finally, co-immunoprecipitation experiment confirmed that isolated hTf retained its ligand characteristics crucial for the ligand-receptor type of interaction with the hTf receptor. To conclude, the procedure described in this work, is time and cost-effective, allows multiple sample handling and provides high-purity hTf isolate with preserved structural and functional properties.
Asunto(s)
Inmunoprecipitación/métodos , Suero/química , Transferrina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Glicosilación , Humanos , Receptores de Transferrina/metabolismo , Transferrina/análisis , Transferrina/metabolismoRESUMEN
Transferrin (TF) is recognized as a multifunctional protein and has been implicated in antioxidative, antimicrobial protection, growth, differentiation and cytoprotection effects. An efficient, original three-step isolation procedure for TF consisting in hydrophobic interaction chromatography, gel filtration and preparative electrophoresis was developed. Rainbow trout TF was found to be N-glycosylated (not O-glycosylated) and phosphorylated at all serine, threonine, and tyrosine residues. The protein consists of several proteoforms with an average molecular weight of 76.9kDa and isoelectric point ranging from 5.2 to 5.7. Rainbow trout TF has two functional iron-binding sites and appears to be quite distinct from carp TF regarding glycosylation and iron-binding properties. The highest gene expression of TF was detected in liver and testis, the lowest was detected in head kidney, spleen and efferent ducts. For the first time TF was identified in the semen of several salmonid species. TF was localized within testis, mainly in spermatozoa, Sertoli, Leydig cells, as well as in both columnar secretory and basal cells within the efferent duct. This work contributes to the existing knowledge information indicating significant variations in TF structure within teleost fish. The results obtained in this study provide valuable data on the TF from trout seminal plasma and the physiological role of this protein in the reproductive tract of salmonids. The results are important for our understanding of the role of TF in the antioxidant protection and resistance to pathogenic infections of reproductive cells. The protective role of TF against environmental pollution with heavy metals, especially during prolonged storage of spermatozoa in the spermatic duct, as well as regulation of spermatogenesis and providing Fe for developing germ cells is also postulated.
Asunto(s)
Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/metabolismo , Oncorhynchus mykiss/metabolismo , Semen/metabolismo , Testículo/metabolismo , Transferrina/aislamiento & purificación , Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas de Peces/genética , Glicosilación , Técnicas para Inmunoenzimas , Hierro/metabolismo , Masculino , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/crecimiento & desarrollo , Fosforilación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/crecimiento & desarrollo , Transferrina/genéticaRESUMEN
Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 µM to 10 µM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 µM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 µM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.
Asunto(s)
Manduca/inmunología , Transferrina/fisiología , Animales , Líquido Extracelular/metabolismo , Hierro/metabolismo , Pruebas de Sensibilidad Microbiana , Transferrina/química , Transferrina/aislamiento & purificaciónRESUMEN
We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding.
Asunto(s)
Transferrina/aislamiento & purificación , Acetonitrilos , Resinas de Intercambio Aniónico , Cromatografía por Intercambio Iónico/métodos , Cromatografía Liquida , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Pliegue de Proteína , Isoformas de Proteínas/aislamiento & purificaciónRESUMEN
Immunoaffinity adsorbent for transferrin (Tf) purification was prepared by immobilizing anti-transferrin (Anti-Tf) antibody on magnetic monosizepoly(glycidyl methacrylate) beads, which were synthesized by dispersion polymerization technique in the presence of Fe3 O4 nanopowder and obtained with an average size of 2.0 µm. The magnetic poly(glycidyl methacrylate) (mPGMA) beads were characterized by Fourier transform infrared spectroscopy, swelling tests, scanning electron microscopy, electron spin resonance spectroscopy, thermogravimetric analysis and zeta sizing analysis. The density and swelling ratio of the beads were 1.08 g/cm(3) and 52%, respectively. Anti-Tf molecules were covalently coupled through epoxy groups of mPGMA. Optimum binding of anti-Tf was 2.0 mg/g. Optimum Tf binding from aqueous Tf solutions was determined as 1.65 mg/g at pH 6.0 and initial Tf concentration of 1.0 mg/mL. There was no remarkable loss in the Tf adsorption capacity of immunoaffinity beads after five adsorption-desorption cycles. Tf adsorption from artificial plasma was also investigated and the purity of the Tf molecules was shown with gel electrophoresis studies.
Asunto(s)
Separación Inmunomagnética , Transferrina/aislamiento & purificación , Cromatografía de Afinidad/métodos , Espectroscopía de Resonancia por Spin del Electrón , Microscopía Electrónica de Rastreo , Espectroscopía Infrarroja por Transformada de Fourier , TermogravimetríaRESUMEN
It was found that recoveries of proteins depend on trifluoroacetic acid concentration in the mobile phase and showed maximum in the range of 0.01-0.1 v/v%. Transferrin and lysozyme were used to evaluate the recoveries of proteins from dedicated reversed-phase columns. Different types of reversed-phase columns were evaluated, such as core shell type materials (Aeris Widepore with C4, C8 and C18 modification) as well as fully porous hybrid particles (Waters BEH, modified with C4 and C18 alkyl chains). Recoveries ranged between 60.7-95.2% for transferrin and 72.1-99.8% for lysozyme. Based on the data presented, at least two different adsorption effects, the well-known hydrophobic and silanophilic/polar interaction might influence the recovery. In addition to this, conformational effects due to ion pairing with the acidic mobile phase additive might change them.
Asunto(s)
Cromatografía de Fase Inversa/métodos , Muramidasa/aislamiento & purificación , Transferrina/aislamiento & purificación , Ácido Trifluoroacético/química , Adsorción , Animales , Pollos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , PorosidadRESUMEN
In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein-ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein-ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE-UV are applied for interactive evaluation. By increasing injection time, larger amount protein-ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein-ssDNA complex in low pH CE separation is discussed.