RRC ID 6685
Author Knetsch ML, Tsiavaliaris G, Zimmermann S, Rühl U, Manstein DJ.
Title Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum.
Journal J. Muscle Res. Cell. Motil.
Abstract 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.
Volume 23(7-8)
Pages 605-11
Published 2002
PMID 12952059
MeSH Amino Acid Sequence Ampicillin Resistance / genetics Animals Bacterial Proteins / genetics Base Sequence Cytoskeletal Proteins / genetics* DNA Primers Dictyostelium / genetics* Genes, Reporter Genetic Vectors Green Fluorescent Proteins Luminescent Proteins / genetics Molecular Sequence Data Myosins / genetics Protozoan Proteins / genetics Recombinant Fusion Proteins / chemistry Restriction Mapping / methods Transfection
IF 2.052
Times Cited 23
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