Site-specific biotinylation of human myeloid differentiation protein 88 in Drosophila melanogaster Schneider 2 cell cytoplasm.
In this work we describe the production of site-specific biotinylated human myeloid differentiation factor 88 (MyD88). A vector containing a coding sequence for a peptide derived from the carboxyl terminus of the Klebsiella pneumoniae oxalacetate decarboxylase alpha subunit was used to allow expression and biotinylation of MyD88 in Drosophila melanogaster Schneider 2 cell cytoplasm. As estimated by a comparison of Schneider 2 lysate with standard protein, the maximum expression level was 1.3 mug 107 cells-1. About 4 mg of biotinylated protein was purified by affinity chromatography on monomeric avidin from a 1-L culture. Exogenous biotin added to the culture medium increased the biotinylation efficiency of the expressed protein. Biotinylated MyD88 produced in Drosophila cells was able to precipitate recombinant MyD88 expressed in human embryonic kidney cells. The stable expression of MyD88 in Drosophila Schneider 2 cells offers a convenient and attractive method for large-scale production, which may be required to clarify the role of MyD88 in the inflammatory response. Moreover, site-specific biotinylation of MyD88 provides a useful tag for interaction assays where high sensitivity is required.
Basile
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Tecnogen S.C.p.A. Molecular Biology Area, Piana di Monte Verna, Caserta, Italy. basile@tecnogen.it
Peticca
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D000818:Animals; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D004331:Drosophila melanogaster; D006801:Humans; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D011994:Recombinant Proteins; D001710:Biotin