title: Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium.
abstract: We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS-PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified.
authors.affiliation: School of Life Sciences, University of Dundee, MSI/WTB/JBC complex, Dow Street, Dundee DD15EH, UK.
authors.forename: Marcel E
authors.initials: ME
authors.lastname: Meima
authors.forename: Karin E
authors.initials: KE
authors.lastname: Weening
authors.forename: Pauline
authors.initials: P
authors.lastname: Schaap
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins