We constructed and tested a series of cloning vectors designed to faci
litate protein production and purification in Dictyostelium discoideum
(Dd). These vectors carry the origin of replication of the Dd high-co
py-number plasmid Ddp2, expression cassettes consisting of the strong,
constitutive actin (act15) or the inducible discoidin (disI gamma) pr
omoters, 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 epito
pe tags correspond to an 11-amino-acid sequence from human c-myc, reco
gnised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence
recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commerci
ally available. The YL1/2 epitope offers a second affinity tag for the
purification of proteins under native conditions. The functional comp
etence of the vectors was tested by determining their ability to promo
te the expression of various Dd myosin constructs. High synthesis leve
ls were obtained for each vector, up to 1 mg of homogenous, functional
protein per g of cells was obtained after purification of the recombi
nant products.