RESUMO
Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.
Assuntos
Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Nicotiana/genética , Plantas Tóxicas , Solanum tuberosum/genética , Hidrolases de Éster Carboxílico/imunologia , Linhagem Celular , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Glicosilação , Hibridomas , Região Variável de Imunoglobulina/imunologia , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Plantas Geneticamente Modificadas , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/metabolismo , Protoplastos/metabolismo , Solanum tuberosum/metabolismo , Nicotiana/metabolismo , Nicotiana/ultraestrutura , Transformação GenéticaRESUMO
Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.