RESUMEN
The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.
Asunto(s)
Escherichia coli , Fragmentos Fab de Inmunoglobulinas , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/química , Escherichia coli/genética , Pichia , Adalimumab/uso terapéutico , Isótopos de Carbono , Anticuerpos Monoclonales , TrastuzumabRESUMEN
The photo-control of protein activity can often be achieved via the photo-control of protein structure. Intramolecular cross-linkers that change length upon photoisomerization provide a means to photo-control protein structure by linking to pairs of Cys residues in a protein sequence. In this protocol, we describe general methods for introducing intramolecular cross-linkers, both UV light switchable and red-light switchable, under either denaturing or native conditions.
Asunto(s)
Compuestos Azo/química , Reactivos de Enlaces Cruzados/química , Cisteína/química , Proteínas/química , Secuencia de Aminoácidos , Compuestos Azo/síntesis química , Técnicas de Química Sintética/métodos , Cromatografía Líquida de Alta Presión/métodos , Reactivos de Enlaces Cruzados/síntesis química , Cisteína/síntesis química , Luz , Modelos Moleculares , Procesos Fotoquímicos , Conformación Proteica , Pliegue de Proteína , Proteínas/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrofotometría Ultravioleta/métodos , Rayos UltravioletaRESUMEN
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development, and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis noninvasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, AsLOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photoactivated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate human eIF4E-dependent translation initiation in a mechanistically defined manner.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Optogenética/métodos , Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/genética , Regulación hacia Abajo/genética , Humanos , Unión Proteica/genéticaRESUMEN
Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.