RESUMO
We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N-acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level.
Assuntos
Acetiltransferases/genética , Membrana Celular/metabolismo , Peptídeos/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Acetiltransferases/metabolismo , Animais , Biotina/química , Biotina/metabolismo , Células CHO , Cricetulus , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Engenharia Genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/metabolismo , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
The presence of anti-myelin antibodies (Abs) in patients with early multiple sclerosis (MS) and in MS animal models has led to renewed interest in the role of B cells, plasma cells and their products in the pathogenesis of the disease, and in their therapeutic potential. Here, we present a novel strategy based on filamentous phage display of the myelin oligodendrocyte glycoprotein immunodominant epitope (MOG 36-44) fused to the main coat protein. Filamentous phages are well characterized, both structurally and genetically. We found that the fibrous shape of the phage (1000 nm long and 6 nm wide) enables penetration into the central nervous system (CNS) when administered nasally. Thus, intranasal treatment of experimental autoimmune encephalomyelitis (EAE) in mice, with phage MOG, showed improved neuronal function, reduced levels of proinflammatory cytokines, particularly monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-gamma) and IL-6, but no change in IL-10 or IL-12 levels. Moreover, the treatment induced depletion of the autoantibodies against MOG and prevented demyelination resulting in improved clinical scores and the reduced inflammation in the CNS and periphery in EAE mice compared to untreated sick animals.
