RESUMEN
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.
Asunto(s)
Vesículas Extracelulares , Edición Génica , Sistemas CRISPR-Cas/genética , Células HEK293 , Humanos , Proproteína Convertasa 9/genéticaRESUMEN
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
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Proteínas de Unión al Calcio , Insuficiencia Cardíaca , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Corazón , Ratones , ARN MensajeroRESUMEN
Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.
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Proteínas Morfogenéticas Óseas/inmunología , Factores de Diferenciación de Crecimiento/inmunología , Factores Inmunológicos/inmunología , Interferón-alfa/inmunología , Proteínas Asociadas a Pancreatitis/inmunología , Linfocitos T Reguladores/inmunología , Proteínas Morfogenéticas Óseas/genética , Factores de Diferenciación de Crecimiento/genética , Humanos , Factores Inmunológicos/genética , Interferón-alfa/genética , Proteínas Asociadas a Pancreatitis/genéticaRESUMEN
The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.
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Sistemas CRISPR-Cas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Edición Génica/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteína 9 Asociada a CRISPR/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Doxiciclina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Transgénicos , ARN Guía de Kinetoplastida/genética , Recombinación Genética/efectos de los fármacos , Reproducibilidad de los Resultados , Activación Transcripcional/efectos de los fármacos , Transfección/métodos , Transgenes/genéticaRESUMEN
The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic ß-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
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Ensayos Analíticos de Alto Rendimiento , Animales , Células CHO , Cricetulus , ADN/biosíntesis , ADN/genética , Células HEK293 , Humanos , Proteómica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismoRESUMEN
Aim: Extracellular vesicles (EVs) are desirable delivery vehicles for therapeutic cargoes. We aimed to load EVs with Cre recombinase protein and determine whether functional delivery to cells could be improved by using endosomal escape enhancing compounds. Materials & methods: Overexpressed CreFRB protein was actively loaded into EVs by rapalog-induced dimerization to CD81FKBP, or passively loaded by overexpression in the absence of rapalog. Functional delivery of CreFRB was analysed using a HEK293 Cre reporter cell line in the absence and presence of endosomal escape enhancing compounds. Results: The EVs loaded with CreFRB by both active and passive mechanisms were able to deliver functional CreFRB to recipient cells only in the presence of endosomal escape enhancing compounds chloroquine and UNC10217938A. Conclusion: The use of endosomal escape enhancing compounds in conjunction with EVs loaded with therapeutic cargoes may improve efficacy of future EV based therapeutics.
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Endosomas/metabolismo , Vesículas Extracelulares/química , Integrasas/química , Nanocápsulas/química , Transporte Biológico , Cloroquina/química , Cloroquina/metabolismo , Liberación de Fármacos , Elementos de Facilitación Genéticos , Vesículas Extracelulares/metabolismo , Expresión Génica , Células HEK293 , Humanos , Integrasas/genética , Integrasas/metabolismo , Tamaño de la Partícula , Multimerización de Proteína , Transducción de SeñalRESUMEN
BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
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Colesterol/sangre , Hipercolesterolemia/genética , Hígado/metabolismo , Proproteína Convertasa 9/genética , Animales , Modelos Animales de Enfermedad , Edición Génica , Genoma , Humanos , Hipercolesterolemia/metabolismo , Ratones , Ratones TransgénicosRESUMEN
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
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Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Edición Génica/normas , Genoma/genética , Mutación , Especificidad por Sustrato/genética , Animales , Proteínas Asociadas a CRISPR/genética , Femenino , Humanos , Mutación INDEL , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9/genética , Transgenes/genéticaRESUMEN
The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Reparación del ADN por Unión de Extremidades , Mutación INDEL , Programas Informáticos , Células A549 , Algoritmos , Secuencia de Bases , Línea Celular , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/metabolismo , Conjuntos de Datos como Asunto , Francisella/enzimología , Humanos , Motivos de Nucleótidos , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimología , Especificidad por Sustrato , ADN Polimerasa thetaRESUMEN
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
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Sistemas CRISPR-Cas , Edición Génica , Fenotipo , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/genética , Adenoviridae/genética , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Ratones Transgénicos , Transducción Genética , Transgenes , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/patología , Deficiencia de alfa 1-Antitripsina/terapiaAsunto(s)
Genómica/métodos , Edición de ARN , Interferencia de ARN , Animales , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
The synthesis of the first examples of tellurophenes exhibiting phosphorescence in the solid state and under ambient conditions (room temperature and in air) is reported. Each of these main-group-element-based emitters feature pinacolboronates (BPin) as ring-appended side groups. The nature of the luminescence observed was also investigated using computational methods.
RESUMEN
Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells.
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Descubrimiento de Drogas , Proteínas Recombinantes/biosíntesis , Baculoviridae , Células Cultivadas , Escherichia coli , Expresión Génica , Vectores Genéticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloración y Etiquetado , TransfecciónRESUMEN
Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further detailed in-vitro and preclinical studies were undertaken. Large amounts of the recombinant protein of high purity and free of unwanted side products, such as lipopolysaccharides (LPS), were required. Since GMCSF is of mammalian origin and it requires proper disulfide bond formation, we intended to use the baculovirus expression vector system (BEVS) for the expression of the recombinant fusion protein. However, despite previous reports on the expression of several other immunotoxins by this system, the A1 derived fusion proteins revealed an inhibitory effect on baculoviral particle formation and even caused cell death in insect cells. This observation was further pursued and confirmed by the use of other baculoviral specific promoters. The salient features of this finding are described below.
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Baculoviridae/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Vectores Genéticos/química , Inmunotoxinas/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Animales , Baculoviridae/fisiología , Western Blotting , Línea Celular , Disulfuros , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Toxina Shiga/química , Toxina Shiga/genética , Spodoptera , Transfección , Células Tumorales CultivadasRESUMEN
As has been widely reviewed elsewhere, the pharmaceutical industry is experiencing an 'innovation deficit' as evidenced by the decline in new chemical entity output. This decline, compounded by increased costs and regulatory requirements highlights the need to significantly revise strategic options across the drug-discovery spectrum. Within such revision(s), much of the focus has been on outsourcing to reduce, or at least contain, costs, but if the underlying predominance of 'closed collaborations' is not challenged to allow better use of combined knowledge and, thus, move towards a more genuine collaborative process then a 'numbers only' approach will not bring medium-to-long-term survival. There are many problems to confront in evolving new sustainable strategies, a real need to think differently exists and should to be cultivated. This article reviews current outsourcing and collaboration strategies to provide a perspective on how great knowledge sharing could help revise the drug-discovery process.
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Conducta Cooperativa , Servicios Externos/organización & administración , Descubrimiento de Drogas , Servicios Externos/economíaRESUMEN
We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.
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Colorantes Fluorescentes/química , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/metabolismo , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Bioensayo , Humanos , Concentración 50 Inhibidora , Péptidos/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.
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Bioensayo/métodos , Evaluación Preclínica de Medicamentos/métodos , Metaloproteinasa 12 de la Matriz/metabolismo , Fragmentos de Péptidos/análisis , Tripsina/metabolismo , Animales , Bovinos , Cromatografía Liquida , Reacciones Falso Negativas , Reacciones Falso Positivas , Estudios de Factibilidad , Fluorescencia , Humanos , Cinética , Luz , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fragmentos de Péptidos/química , Dispersión de Radiación , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca(2+) sensor, Case16, in the presence of a low Ca(2+) concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca(2+)-dependent response when only two out of four Ca(2+)-binding pockets of calmodulin (CaM) are occupied with Ca(2+) ions. In such a "half Ca(2+)-bound state", Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca(2+) sensor Case12 at saturating Ca(2+) concentration. Based on this structure, we postulate that cpGFP-based Ca(2+) sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca(2+) sensors in living cells.