RESUMO
Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.
Assuntos
Reatores Biológicos , Proteínas Recombinantes , Transfecção , Transfecção/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Humanos , Animais , Linhagem Celular , Técnicas de Cultura de Células/métodos , Expressão GênicaRESUMO
BACKGROUND: Prospectively and systematically collected real-world data on vedolizumab are scarce. We aimed to assess the long-term clinical effectiveness of vedolizumab in inflammatory bowel disease (IBD). METHODS: This study was a prospective, observational, multicentre study. Overall, 286 patients with active IBD were included (Crohn's disease, n = 169; ulcerative colitis, n = 117). The primary outcomes were clinical response at week 12 and clinical remission at week 52, based on the Harvey Bradshaw Index and the partial Mayo Clinic score. Secondary outcomes included clinical remission at week 12, clinical response at week 52, corticosteroid-free clinical remission at week 52, changes in biochemical measures, and health-related quality of life (HRQoL). RESULTS: At baseline, 88% of the patients were exposed to anti-TNF and 41% of the patients with Crohn's disease had undergone ⩾1 surgical resection. At week 12, clinical response was 27% and remission 47% in Crohn's disease; corresponding figures in ulcerative colitis were 52% and 34%. Clinical response, remission and corticosteroid-free remission at week 52 were 22%, 41% and 40% in Crohn's disease and 49%, 47% and 46% in ulcerative colitis, respectively. A statistically significant decrease in median faecal-calprotectin and C-reactive protein was observed at 12 and 52 weeks in patients with Crohn's disease and ulcerative colitis. The HRQoL measures Short Health Scale and EuroQol 5-Dimensions improved in both Crohn's disease and ulcerative colitis patients (p < 0.001). Clinical disease activity at baseline was inversely associated with clinical remission at week 52. CONCLUSION: Vedolizumab proved effective for the treatment of refractory IBD in clinical practice.
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Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
Assuntos
Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Domínios FERM , Humanos , Modelos Moleculares , Receptores de LDL/química , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Adenosina Trifosfatases , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ftalimidas , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/fisiologia , Inibidores de Proteínas Quinases , Especificidade por SubstratoRESUMO
Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His6-tagged ß-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response.
RESUMO
Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against ß-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Imagem Molecular , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Humanos , Soluções , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.
Assuntos
Caspases/fisiologia , Linfócitos/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Sequência de Bases , Western Blotting , Caspases/genética , Caspases/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células Jurkat , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação de Sentido Incorreto/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/genética , Transcrição Gênica/fisiologiaRESUMO
OBJECTIVE: Many patients with nonerosive reflux disease (NERD) have insufficient relief on proton pump inhibitors (PPIs). Some patients have a hypersensitive esophagus and may respond to transient receptor potential vanilloid 1 (TRPV1) antagonists. Aim. To investigate the effect of the TRPV1 antagonist AZD1386 on experimental esophageal pain in NERD patients. MATERIAL AND METHODS: Enrolled patients had NERD and a partial PPI response (moderate-to-severe heartburn or regurgitation ≥3 days/week before enrolment despite ≥6 weeks' PPI therapy). Fourteen patients (21-69 years, 9 women) were block-randomized into this placebo-controlled, double-blinded, crossover study examining efficacy of a single dose (95 mg) of AZD1386. On treatment days, each participant's esophagus was stimulated with heat, distension, and electrical current at teaching hospitals in Denmark and Sweden. Heat and pressure pain served as somatic control stimuli. Per protocol results were analyzed. RESULTS: Of 14 randomized patients, 12 were treated with AZD1386. In the esophagus AZD1386 did not significantly change the moderate pain threshold for heat [-3%, 95% confidence interval (CI), -22;20%], distension (-11%, 95% CI, -28;10%), or electrical current (6%, 95% CI, -10;25%). Mean cutaneous heat tolerance increased by 4.9°C (95% CI, 3.7;6.2°C). AZD1386 increased the maximum body temperature by a mean of 0.59°C (95% CI, 0.40-0.79°C), normalizing within 4 h. CONCLUSIONS: AZD1386 had no analgesic effect on experimental esophageal pain in patients with NERD and a partial PPI response, whereas it increased cutaneous heat tolerance. TRPV1 does not play a major role in heat-, mechanically and electrically evoked esophageal pain in these patients. ClinicalTrials.gov identifier: D9127C00002.
Assuntos
Benzimidazóis/uso terapêutico , Refluxo Gastroesofágico/tratamento farmacológico , Limiar da Dor/efeitos dos fármacos , Dor/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Adulto , Idoso , Análise de Variância , Benzimidazóis/farmacocinética , Temperatura Corporal/efeitos dos fármacos , Estudos Cross-Over , Dilatação/efeitos adversos , Método Duplo-Cego , Estimulação Elétrica/efeitos adversos , Feminino , Azia/tratamento farmacológico , Temperatura Alta/efeitos adversos , Humanos , Refluxo Laringofaríngeo/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Dor/etiologia , Inibidores da Bomba de Prótons/uso terapêutico , Adulto JovemRESUMO
Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.
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Antígenos de Superfície/análise , Proteínas de Membrana/análise , Análise Serial de Proteínas/métodos , Extratos de Tecidos/análise , Anticorpos/genética , Anticorpos/imunologia , Antígenos de Superfície/imunologia , Feminino , Humanos , Limite de Detecção , Proteínas de Membrana/imunologia , Tonsila Palatina/citologia , Placenta/citologia , Gravidez , Proteínas Recombinantes , Reprodutibilidade dos Testes , Extratos de Tecidos/imunologiaRESUMO
Functional bowel disorders (FBDs) are common disorders that are characterized by various combinations of abdominal pain and/or discomfort, bloating and changes in bowel habits. At present, diagnosing FBDs often incurs considerable health-care costs, partly because unnecessary investigations are performed. Patients are currently diagnosed as having an FBD on the basis of a combination of typical symptoms, normal physical examination and the absence of alarm features indicative of an organic gastrointestinal disease. Basic laboratory investigations, such as a complete blood count, measurement of the erythrocyte sedimentation rate and serological tests for celiac disease, are useful in the initial evaluation. No further investigations are needed for most patients who have typical symptoms and no alarm symptoms. The most important alarm symptoms include signs of gastrointestinal bleeding, symptom onset above 50 years of age, a family history of colorectal cancer, documented weight loss and nocturnal symptoms. The presence of alarm symptoms obviously does not exclude an FBD, but further investigation is needed before confirmation of the diagnosis. For patients with predominant and severe diarrhea, a more thorough diagnostic work-up should normally be considered, including colonoscopy with colonic biopsies and a test for bile-acid malabsorption.