RESUMEN
Hemophilia A and hemophilia B are X-linked inherited bleeding disorders caused by a deficiency of coagulation factor VIII and IX, respectively. Standard of care is prophylactic factor replacement therapy; however, the development of neutralizing antibodies against these factors represents serious complications underlining the need for alternative treatment approaches. Human coagulation factor X has a central role within the blood coagulation system making it an attractive target for the development of alternative treatment strategies for patients with hemophilia. This study focuses on a modified variant of the human coagulation factor X with enhanced hemostatic bypass activity due to insertion of a factor IX derived activation sequence. This molecule design leads to the direct activation of the modified factor X protein by factor XIa allowing it to bypass the need for coagulation factor VIIIa/factor IXa. The modified variant was able to correct in-vitro activated partial prothrombin time of human and murine factor VIII/factor IX deficient plasma. Furthermore, reduced blood loss in factor VIII knock-out mice was observed after intravenous application of the modified factor X variant. In conclusion, these data suggest that the factor X variant described here could potentially serve as a bypassing agent independent of the inhibitor status of hemophilia patients. However, more research is needed to further investigate the potential of this molecule.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor X/farmacología , Hemostáticos/farmacología , Animales , Factor X/uso terapéutico , Femenino , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Hemorragia/sangre , Hemorragia/tratamiento farmacológico , Hemostáticos/uso terapéutico , Humanos , Masculino , Ratones , Tiempo de Tromboplastina Parcial , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas Recombinantes de Fusión/química , Anticuerpos Monoclonales/genética , Células Cultivadas , Cromatografía de Afinidad , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Recombinantes de Fusión/genética , Robótica , Proteína Estafilocócica A/química , TransfecciónRESUMEN
BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.
Asunto(s)
Haptoglobinas/genética , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Ingeniería de Proteínas/métodos , Serina Endopeptidasas/genética , Animales , Vasos Coronarios/efectos de los fármacos , Cobayas , Haptoglobinas/farmacología , Hemo/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Óxido Nítrico/metabolismo , Fenotipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Serina Endopeptidasas/metabolismo , PorcinosRESUMEN
OBJECTIVE: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. METHODS: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. RESULTS: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. INTERPRETATION: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970-982.
Asunto(s)
Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Factor XII/uso terapéutico , Factor XIIa/antagonistas & inhibidores , Proteínas de Insectos/uso terapéutico , Trombosis Intracraneal/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Albúmina Sérica/uso terapéutico , Adulto , Anciano , Animales , Lesiones Traumáticas del Encéfalo/fisiopatología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Factor XII/genética , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neuroimagen , Agregación Plaquetaria/fisiología , Albúmina Sérica Humana , Adulto JovenRESUMEN
Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the alphabeta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both alpha and beta subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.
Asunto(s)
Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Óxido Nítrico/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Vasodilatadores/farmacología , Vasos Sanguíneos , Línea Celular , GMP Cíclico/metabolismo , Humanos , Oxidación-Reducción , Guanilil Ciclasa Soluble , UbiquitinaciónRESUMEN
Monoclonal antibodies (mAbs) are the fastest-growing segment in the drug market with eight of the top 20 selling drugs being mAbs and combined sales of close to 60 billion US$/year. The development of new therapeutic mAbs requires the purification of a large number of candidate molecules during initial screenings, subsequent affinity maturation campaigns, and finally the engineering of variants to improve half-life, functionality, or biophysical properties of potential lead molecules. A successful strategy to purify this ever-increasing number of mAbs in a timely manner has been the miniaturization and automation of the purification process using automatic liquid handlers (ALHs) such as Tecan's Evo or PerkinElmer's Janus platforms. These systems can be equipped with miniaturized columns, which are available in a wide variety of sizes and affinity matrices to cater to the need of the respective application. Various publications have described the setup of ALHs including the respective purification procedure. However, despite being very precise regarding the overall approach, most publications do not focus on the technical optimization and potential pitfalls, which can be crucial to obtain a robust process. To fill this gap, the present publication is aiming to point at some technical difficulties and suggesting potential ways to overcome these problems in order to facilitate the setup of new ALH systems for the purification of antibodies.
Asunto(s)
Anticuerpos Monoclonales , Automatización de Laboratorios , Cromatografía de Afinidad , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , HumanosRESUMEN
Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Encefalitis/fisiopatología , Endocannabinoides , Proteínas Inmediatas-Precoces/biosíntesis , Microglía/metabolismo , Fármacos Neuroprotectores/metabolismo , Fosfoproteínas Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Adulto , Animales , Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Supervivencia Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual , Encefalitis/patología , Encefalitis/prevención & control , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Alcamidas Poliinsaturadas , Proteína Fosfatasa 1 , Ratas , Ratas Wistar , Receptores de Cannabinoides/metabolismo , Distribución TisularRESUMEN
A novel mechanism for extending the circulatory half-life of coagulation factor VIII (FVIII) has been established and evaluated preclinically. The FVIII binding domain of von Willebrand factor (D'D3) fused to human albumin (rD'D3-FP) dose dependently improved pharmacokinetics parameters of coadministered FVIII in all animal species tested, from mouse to cynomolgus monkey, after IV injection. At higher doses, the half-life of recombinant FVIII (rVIII-SingleChain) was calculated to be increased 2.6-fold to fivefold compared with rVIII-SingleChain administered alone in rats, rabbits, and cynomolgus monkeys, and it was increased 3.1-fold to 9.1-fold in mice. Sustained pharmacodynamics effects were observed (ie, activated partial thromboplastin time and thrombin generation measured ex vivo). No increased risk of thrombosis was observed with coadministration of rVIII-SingleChain and rD'D3-FP compared with rVIII-SingleChain alone. At concentrations beyond the anticipated therapeutic range, rD'D3-FP reduced the hemostatic efficacy of coadministered rVIII-SingleChain. This finding might be due to scavenging of activated FVIII by the excessive amount of rD'D3-FP which, in turn, might result in a reduced probability of the formation of the tenase complex. This observation underlines the importance of a fine-tuned balance between FVIII and its binding partner, von Willebrand factor, for hemostasis in general.
Asunto(s)
Hemofilia A , Hemostáticos , Albúminas , Animales , Factor VIII , Semivida , Esperanza de Vida , Macaca fascicularis , Ratones , Conejos , RatasRESUMEN
ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.
Asunto(s)
Benzoatos/farmacología , Vasos Sanguíneos/fisiología , Endotelio Vascular/fisiología , Guanilato Ciclasa/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Benzoatos/síntesis química , Presión Sanguínea/efectos de los fármacos , Técnicas de Cultivo de Célula , GMP Cíclico/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Guanilato Ciclasa/efectos de los fármacos , Hemo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Arteria Pulmonar , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Guanilil Ciclasa Soluble , Porcinos , VasodilataciónRESUMEN
Cyclic guanosine monophosphate (cGMP), generated via the guanylate cyclase (GC)-catalyzed conversion from GTP, is unequivocally recognized as crucial second messenger, intimately involved in the regulation of a broad range of physiological processes such as long term potentiation, blood pressure regulation, or platelet aggregation (for review: Hobbs 2000). Since its first identification in rat urine by Ashman and co-workers (1963), various approaches have been conceived and established to quantify cGMP in biological samples, or to detect cGMP as the reaction product of enzymatic assays, allowing the determination of kinetic parameters. These approaches have evolved from laborious handling of small numbers of samples with average sensitivity to highly developed biochemical detection assays allowing the processing of very large numbers of samples. The present article focuses upon the history of biochemical cGMP detection from the pioneering work of the early years to the actual state-of-the-art approaches for the detection of this important biological messenger.
Asunto(s)
Bioensayo/métodos , GMP Cíclico/análisis , Guanilato Ciclasa/metabolismo , Animales , Bioensayo/historia , Bioensayo/tendencias , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Oxidative stress, a risk factor for several cardiovascular disorders, interferes with the NO/sGC/cGMP signalling pathway through scavenging of NO and formation of the strong intermediate oxidant, peroxynitrite. Under these conditions, endothelial and vascular dysfunction develops, culminating in different cardio-renal and pulmonary-vascular diseases. Substituting NO with organic nitrates that release NO (NO donors) has been an important principle in cardiovascular therapy for more than a century. However, the development of nitrate tolerance limits their continuous clinical application and, under oxidative stress and increased formation of peroxynitrite foils the desired therapeutic effect. To overcome these obstacles of nitrate therapy, direct NO- and haem-independent sGC activators have been developed, such as BAY 58-2667 (cinaciguat) and HMR1766 (ataciguat), showing unique biochemical and pharmacological properties. Both compounds are capable of selectively activating the oxidized/haem-free enzyme via binding to the enzyme's haem pocket, causing pronounced vasodilatation. The potential importance of these new drugs resides in the fact that they selectively target a modified state of sGC that is prevalent under disease conditions as shown in several animal models and human disease. Activators of sGC may be beneficial in the treatment of a range of diseases including systemic and pulmonary hypertension (PH), heart failure, atherosclerosis, peripheral arterial occlusive disease (PAOD), thrombosis and renal fibrosis. The sGC activator HMR1766 is currently in clinical development as an oral therapy for patients with PAOD. The sGC activator BAY 58-2667 has demonstrated efficacy in a proof-of-concept study in patients with acute decompensated heart failure (ADHF), reducing pre- and afterload and increasing cardiac output from baseline. A phase IIb clinical study for the indication of ADHF is currently underway.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Activadores de Enzimas/farmacología , Guanilato Ciclasa/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Animales , Benzoatos/farmacología , Enfermedades Cardiovasculares/fisiopatología , Ensayos Clínicos como Asunto , Sistemas de Liberación de Medicamentos , Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Humanos , Óxido Nítrico/metabolismo , Estrés Oxidativo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Riesgo , Guanilil Ciclasa Soluble , Sulfonamidas/farmacología , ortoaminobenzoatos/farmacologíaRESUMEN
Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).
Asunto(s)
Activadores de Enzimas/farmacología , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/química , Activadores de Enzimas/uso terapéutico , Humanos , Estructura MolecularRESUMEN
The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3â Å resolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.
Asunto(s)
Proteínas Bacterianas/química , Subtipo H5N1 del Virus de la Influenza A/enzimología , Neuraminidasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Desulfurococcaceae/metabolismo , Humanos , Gripe Humana/virología , Modelos Moleculares , Neuraminidasa/metabolismo , Conformación Proteica , Dominios ProteicosRESUMEN
The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.
Asunto(s)
GMP Cíclico/química , Guanilato Ciclasa/química , Modelos Moleculares , Animales , GMP Cíclico/metabolismo , Activación Enzimática , Guanilato Ciclasa/metabolismo , Hemo , Estructura Terciaria de Proteína , Ratas , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Thermoanaerobacter/enzimologíaRESUMEN
Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Robótica , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Automatización , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Células HEK293 , Humanos , Miniaturización , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMEN
Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.
Asunto(s)
Alanina/genética , Guanilato Ciclasa/genética , Hemo/metabolismo , Serina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Benzoatos/farmacología , Sitios de Unión/genética , Células CHO , Cricetinae , Cricetulus , GMP Cíclico/metabolismo , Dietilaminas/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genotipo , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Ácido Nítrico/metabolismo , Ácido Nítrico/farmacología , Óxidos de Nitrógeno , Oxadiazoles/farmacología , Protoporfirinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Quinoxalinas/farmacología , Homología de Secuencia de Aminoácido , Solubilidad , TransfecciónRESUMEN
We developed a DNA microarray for identification of Bacillus anthracis and other phylogenetic groupings within the "Bacillus cereus group". Nucleotide sequences of 16S-23S ribosomal DNA internal transcribed spacers containing genes for tRNA(Ile) from 52 B. anthracis strains were found to be identical to sequences from seven strains published previously and different from all other bacteria. When 42 oligonucleotide probes targeting polymorphic sites were immobilized on glass slides and hybridized to fluorescently labeled PCR amplification products, one or more mismatches could be discriminated in all but one cases. Hence, hybridization events were highly specific and identification of B. anthracis was straightforward.
Asunto(s)
Bacillus anthracis/clasificación , Bacillus anthracis/aislamiento & purificación , ADN Espaciador Ribosómico/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Bacillus anthracis/genética , Disparidad de Par Base , ADN Bacteriano/química , ADN Bacteriano/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Filogenia , ARN Bacteriano/genética , ARN de Transferencia de Isoleucina/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.
Asunto(s)
Biomarcadores de Tumor/análisis , Técnicas Biosensibles/instrumentación , Tecnología de Fibra Óptica/instrumentación , Telomerasa/análisis , Biomarcadores de Tumor/metabolismo , Técnicas Biosensibles/métodos , Extractos Celulares/análisis , Cartilla de ADN , Activación Enzimática , Diseño de Equipo , Tecnología de Fibra Óptica/métodos , Fluorescencia , Células HL-60/enzimología , Humanos , Sensibilidad y Especificidad , Telomerasa/metabolismoRESUMEN
The characterization of the interaction between a ligand and its receptor is crucial for a broad variety of applications in academia as well as in the pharmaceutical industry. Although various sophisticated high-throughput technologies have been established to investigate the binding of ligands to their receptors, classical filtration-based receptor binding assays still have some advantages when smaller number of samples need to be tested. Here we describe a technically easy, cheap, and reliable receptor binding assay that was successfully applied to determine the binding constant of the NO-independent activator of soluble guanylate cyclase, cinaciguat, and the impact of other small molecules on its interaction with the enzyme.
Asunto(s)
Benzoatos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Benzoatos/metabolismo , Guanilato Ciclasa/metabolismo , Ligandos , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa SolubleRESUMEN
In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.