RESUMO
Recombinant proteins, in particular antibodies, have become fundamental in biomedical research where they are used in numerous therapeutic and diagnostic applications. For this reason there is an increasing demand for quick and economical production systems for recombinant proteins in mammalian cells.
Assuntos
Vetores Genéticos/metabolismo , Anticorpos de Cadeia Única/biossíntese , Animais , Formação de Anticorpos , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genéticaRESUMO
Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of beta-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.
