RESUMEN
Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.
Asunto(s)
Osteosarcoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/metabolismo , Humanos , Osteosarcoma/genética , Receptores Acoplados a Proteínas G/genética , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-2/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Células Tumorales CultivadasRESUMEN
A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.
Asunto(s)
Anticuerpos/inmunología , Inmunohistoquímica/métodos , Receptores Acoplados a Proteínas G/inmunología , Anticuerpos/química , Anticuerpos/genética , Baculoviridae/genética , Baculoviridae/fisiología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fluorescencia , Humanos , Indicadores y Reactivos , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Factores de Tiempo , Volumetría , Transducción GenéticaRESUMEN
Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production.