RESUMEN
Activin A is a member of the transforming growth factor-beta (TGF-ß) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-ß-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.
Asunto(s)
Activinas/genética , Escherichia coli/genética , Periplasma/metabolismo , Señales de Clasificación de Proteína , Activinas/química , Activinas/aislamiento & purificación , Cromatografía de Afinidad , Humanos , Células K562 , Estructura Secundaria de Proteína , alfa-Amilasas/metabolismoRESUMEN
Therapeutic proteins are a continuously growing class of pharmaceuticals and comprise several drug candidates with potential performance-enhancing properties. In particular, activin receptor competitors, such as the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc), have the potential for being misused as doping agents in sports as they were found to inhibit negative regulators of late-stage erythropoiesis. Within this study, ammonium sulfate precipitation, immunoaffinity purification, tryptic digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to develop an assay for the combined detection of Sotatercept and Luspatercept in doping control serum samples. The assay was optimized, comprehensively characterized, and found to be fit-for-purpose for application to sports drug testing. It complements existing tests for ActRII-Fc fusion proteins and expands the range of available detection methods for novel protein therapeutics.
Asunto(s)
Activinas/sangre , Cromatografía Líquida de Alta Presión/métodos , Fragmentos Fc de Inmunoglobulinas/sangre , Inmunoprecipitación/métodos , Proteínas Recombinantes de Fusión/sangre , Espectrometría de Masas en Tándem/métodos , Receptores de Activinas Tipo II , Activinas/análisis , Activinas/aislamiento & purificación , Precipitación Química , Humanos , Fragmentos Fc de Inmunoglobulinas/análisis , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Límite de Detección , Sustancias para Mejorar el Rendimiento/sangre , Proteolisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Detección de Abuso de Sustancias/métodos , Tripsina/químicaRESUMEN
We recently published two protocols for the detection of Sotatercept (ACE-011, ACVR2A-Fc) and Luspatercept (ACE-536, ACVR2B-Fc) in human serum. Both methods used covalently immobilized antibodies on agarose beads for immunoprecipitation and SAR-PAGE/Western blotting for detection. Disadvantages were the relatively high amount of antibody required per sample (10 µg) and the need of a secondary antibody for the final detection. The updated protocols overcome these limitations by antigen-antibody complex formation in solution followed by capture of the complex with anti-antibody-coated magnetic beads. They also omit the secondary antibody incubation step by usage of biotinylated primary antibodies, which can be directly incubated with streptavidin-HRP. Thus, the new protocols are faster, simpler, and cheaper and offer comparable sensitivities.
Asunto(s)
Activinas/sangre , Fragmentos Fc de Inmunoglobulinas/sangre , Proteínas Recombinantes de Fusión/sangre , Receptores de Activinas Tipo II , Activinas/aislamiento & purificación , Anticuerpos Inmovilizados/química , Biotinilación , Western Blotting/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoprecipitación/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
AB204 is an Activin/BMP2 chimera, which has been found to exhibit a higher activity than Bone Morphogenetic Protein 2 (BMP2) in osteogenic activity. To prepare AB204 for its preclinical studies, AB204 has been characterized in various formulation buffers. We observed that AB204 purified by ion-exchange chromatography has low water solubility (2.0 mg/ml), whereas it has high water solubility (higher than 10.0 mg/ml) when purified by reverse-phase chromatography. Analysis of the purification procedures reveals that the buffer composition at the lyophilization step determines the solubility. Lyophilization from sodium acetate buffer at pH 4.5 resulted in formation of sodium hydroxide, which caused low solubility of AB204 by pH increase upon reconstitution in water. However, lyophilization from buffers, containing acetic acid or trifluoroacetic acid (TFA) rendered AB204 to be highly soluble. During the course of these analyses, we found a simple procedure to further reduce residual amount of TFA in the purified AB204.
Asunto(s)
Activinas/genética , Activinas/aislamiento & purificación , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/aislamiento & purificación , Activinas/química , Animales , Proteína Morfogenética Ósea 2/química , Línea Celular , Cromatografía por Intercambio Iónico , Cromatografía de Fase Inversa , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , SolubilidadAsunto(s)
Factor de Crecimiento Transformador beta , Activinas/aislamiento & purificación , Activinas/metabolismo , Congresos como Asunto , Folistatina/metabolismo , Humanos , Inhibinas/aislamiento & purificación , Inhibinas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/aislamiento & purificación , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/química , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Factor 5 de Diferenciación de Crecimiento/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores de Activinas/química , Receptores de Activinas/genética , Receptores de Activinas/aislamiento & purificación , Receptores de Activinas/metabolismo , Activinas/química , Activinas/genética , Activinas/aislamiento & purificación , Activinas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Técnicas Biosensibles , Receptores de Proteínas Morfogenéticas Óseas/genética , Receptores de Proteínas Morfogenéticas Óseas/aislamiento & purificación , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/aislamiento & purificación , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Ácidos Cólicos/química , Detergentes/química , Factor 5 de Diferenciación de Crecimiento/química , Factor 5 de Diferenciación de Crecimiento/genética , Factor 5 de Diferenciación de Crecimiento/aislamiento & purificación , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Cinética , Ligandos , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Isoformas de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The effects of arginine on protein binding and elution in hydrophobic interaction chromatography (HIC) were examined using recombinant human interleukin-6 (IL-6) and activin-A. Binding of IL-6 in the presence of ammonium sulfate (AS) was tested using low- and high-substituted phenyl-sepharose. While inclusion of arginine during loading of IL-6 resulted in incomplete binding to the low-substituted phenyl-sepharose, binding was complete to the high-substituted phenyl-sepharose. Arginine facilitated elution of IL-6 from both columns. These results demonstrate that arginine weakens hydrophobic interactions between IL-6 and the phenyl-sepharose. More drastic results were obtained using activin-A, which showed undetectable recovery from phenyl-sepharose. Although no apparent elution of activin-A was observed from butyl-sepharose in aqueous buffer alone, the addition of arginine to the buffer resulted in partial elution recovery and, together with ethanol, resulted in greatly improved recovery of the protein. Two arginine derivatives, acetylarginine and agmatine, were also effective. These results show that arginine improves protein elution in HIC.
Asunto(s)
Activinas/aislamiento & purificación , Arginina/química , Cromatografía Liquida/métodos , Interleucina-6/aislamiento & purificación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/aislamiento & purificación , Sefarosa/análogos & derivadosRESUMEN
Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.
Asunto(s)
Activinas/aislamiento & purificación , Activinas/metabolismo , Folistatina/metabolismo , Subunidades beta de Inhibinas/aislamiento & purificación , Subunidades beta de Inhibinas/metabolismo , Activinas/genética , Animales , Bovinos , Células Cultivadas , Cricetinae , Folistatina/genética , Subunidades beta de Inhibinas/genética , Inhibinas/genética , Inhibinas/aislamiento & purificación , Inhibinas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Regulatory factors other than erythropoietin (Epo) dependence, that control mammalian erythroid terminal differentiation, are currently uncertain. Here we report the existence of erythroid differentiation factors in erythroid cytoplasm. Purification of these factors from cultured Friend virus anaemia (FVA)-infected mouse splenic erythroblasts was carried out using isoelectrophoresis and high performance of liquid chromatography techniques. We have identified intracellular erythroid differentiation denucleation factors (EDDFs) that were able to mediate the events of post-Epo-dependent erythroblast terminal differentiation. Purified EDDF proteins bound specifically to the enhancer HS2 sequence of the globin gene activated the expression of haemoglobin in mouse erythroleukaemia and K562 erythroleukaemic cells and promoted them to differentiate into mature erythrocytes. EDDF proteins began to emerge at the pro-early erythroblast stages upon exposure to Epo in culture, and increased dramatically in early erythroblast stage. The dynamic of EDDF expression and its action on the key events of erythroblast differentiation and denucleation appeared to be closely consistent with its spatiotemporal distribution. These results suggest that EDDFs are the critical intracellular regulatory factors that may act as the successive regulators to Epo, responsible for the final stages of erythroid terminal differentiation.
Asunto(s)
Activinas/metabolismo , Eritrocitos/citología , Células Precursoras Eritroides/citología , Eritropoyesis/fisiología , Eritropoyetina/fisiología , Subunidades beta de Inhibinas/metabolismo , Activinas/aislamiento & purificación , Animales , Diferenciación Celular , Fusión Celular , Línea Celular Transformada , Células Cultivadas , Citoplasma/metabolismo , Elementos de Facilitación Genéticos , Eritroblastos/citología , Eritroblastos/metabolismo , Eritroblastos/virología , Femenino , Virus de la Leucemia Murina de Friend , Globinas/genética , Globinas/metabolismo , Humanos , Subunidades beta de Inhibinas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Bazo/citologíaRESUMEN
A major problem in gel permeation chromatography (GPC) or size exclusion chromatography is non-specific binding of applied proteins to the column matrix (stationary phase). We have tested an aqueous arginine solution as the GPC mobile phase on silica-based and polymer-based columns, using mouse monoclonal antibody and recombinant human activin, interleukin-6, basic fibroblast growth factor, and interferon-gamma as model proteins. We observed that addition of arginine to the mobile phase improves separation of the proteins and their soluble aggregates from the GPC columns, which suggests that arginine is an effective additive for the GPC mobile phase.
Asunto(s)
Arginina/química , Cromatografía en Gel/métodos , Activinas/aislamiento & purificación , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Humanos , Interferón gamma/aislamiento & purificación , Interleucina-6/aislamiento & purificación , Ratones , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The role of regulation of erythroid differentiation denucleation factor(EDDF) on mammalian erythroid differentiation and myeloma cell malignancy as well as cloning of their stage related genes were serially studied. Through a series of cybrid and hybridization experiments between mammalian erythroid cells and erythroleukemia or non-erythroid myeloma cells, we have demonstrated a novel family of erythroid regulators(EDDFs) in the mammalian differentiating erythroblasts which with an active peak occurred concomitantly with marked decreases in DNA, RNA and the nuclear anchoring vimentin-IF, but increased in hemoglobin synthesis in cytoplasm prior to the denucleation process during terminal differentiation. The results of cell fusion experiments verified that the supplement of regulators(EDDFs) was critical to the recovery of the originally lost features of terminal differentiation and the reversion of malignant phenotype of tumor cells. Here we showed that the erythroid regulator family EDDFs were essential regulators for the sequential expression of stage related genes of erythroid terminal differentiation, and for the redifferentiation of tumor cells to express the originally inactive globe genes, repressed the oncogenes, and vimentin-IF system, thus initiated nuclear condensation and denucleation. The EDDF gene family consisted of MEDDF, HEDDF-1, HEDRF-1, HEDRF-2 and HCNBP-1 were cloned. All were novel cDNA sequences that have been searched and registered in GenBank. They expressed varying in a stage specific manner, and acted on corresponding genes of terminal differentiation.
Asunto(s)
Activinas/genética , Activinas/fisiología , Eritroblastos/citología , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/fisiología , Activinas/aislamiento & purificación , Animales , Diferenciación Celular , Clonación Molecular , Humanos , Células Híbridas , Subunidades beta de Inhibinas/aislamiento & purificación , Ratones , Conejos , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Cloning and identification of cDNA related to erythroid terminal differentiation factor (MEDRF). METHODS: cDNA related to erythroid terminal differentiation from the Friend virus anemia (FVA) infected splenic erythroblasts of BALB/c mice were performed by using subtractive hybridization combined with PCR technique. The splenic proerythroblasts isolated were cultured in the presence of erythropoietin for 36 hrs. Subtractive cDNA clones of differentially expressed in the 36 hrs erythroblasts (sub cDNA-36) but absent in the uncultured proerythroblasts were observed. The sub cDNA-36 was then used for construction of subtractive cDNA library using the Bluescript-SK(+) phage vector system, and differentially screened by 32P-labelled PCR generated probes. The positive clones were analyzed by nucleotide sequencing. RESULTS: The results indicated that a 472 bp cDNA fragment which contained a 309 bp reading frame from 51 to 359 coding 102 amino acids was identified and it has been accepted by GenBank as a new cDNA sequence that without comparable homology of existing sequences (accession number: AA114369). Northern blot analysis demonstrated that it was differentially expressed in the 36 hrs cultured intermediate-late stages of erythroblasts. CONCLUSIONS: The newly found cDNA, which expresses specifically in intermediate-late stages of erythroblasts, not in stages of proerythroblasts, may be a new gene related to murine erythroid terminal differentiation.