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Recombinant DNA; myosin; cytoskeleton; expression vector; cellular slime mold; transformation
Abstract:
We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products.
