Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum.
We generated and tested a set of cloning vectors designed to facilitate the production, purification and visualization of proteins in Dictyostelium discoideum. The vectors are derived from the Dictyostelium-E. coli shuttle vector pDXA-3H (6.1 kb), which carries the origin of replication of the Dd high-copy-number plasmid, Ddp2, a high-copy-number E. coli plasmid origin of replication, an act6 promoter driven G418 resistance cassette, the bacterial ampicillin resistance gene and an expression cassette. The new cloning vectors carry expression cassettes consisting of the strong constitutive actin-15 promoter, a translation start followed by a multiple cloning site, sequences for the addition of purification or visualization tags, and Dictyostelium polyadenylation and termination signals. Vectors designed to facilitate protein visualization in living Dictyostelium cells contain either coding sequences for the cyan (CFP) or yellow (YFP) variants of green fluorescent protein (GFP). Versions of the vectors for the production of N- and C-terminal fusions with the fluorescent proteins were generated. To facilitate protein purification, vectors for the production of glutathione-S-transferase (GST) fusion proteins and Strep- or FLAG-affinity-tagged proteins were generated. Additionally, a vector for the production of His8-tagged proteins was generated, which has the G418-resistance cassette replaced by a hygromycin resistance cassette.
Knetsch
Menno L W
ML
Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany.
Tsiavaliaris
Georgios
G
Zimmermann
Sabine
S
Rühl
Ursula
U
Manstein
Dietmar J
DJ
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003598:Cytoskeletal Proteins / Q000235:genetics*; D017931:DNA Primers; D004023:Dictyostelium / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009218:Myosins / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015183:Restriction Mapping / Q000379:methods; D014162:Transfection
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D009218:Myosins