Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium

Marcel E. Meima, Karin E. Weening, Pauline Schaap

    Research output: Contribution to journalArticle

    28 Citations (Scopus)

    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.
    Original languageEnglish
    Pages (from-to)283-288
    Number of pages6
    JournalProtein Expression and Purification
    Volume53
    Issue number2
    DOIs
    Publication statusPublished - Jun 2007

    Keywords

    • Actin-Related Protein 2-3 Complex
    • Animals
    • Bacterial Proteins
    • Base Sequence
    • Chromatography, Affinity
    • Cloning, Molecular
    • DNA Primers
    • Dictyostelium
    • Gene Expression
    • Genetic Vectors
    • Green Fluorescent Proteins
    • Luminescent Proteins
    • Peptide Mapping
    • Plasmids
    • Protozoan Proteins
    • Recombinant Fusion Proteins

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