Tandem affinity purification in Drosophila: the advantages of the GS-TAP system

Fly (Austin). Jul-Aug 2008;2(4):229-35. doi: 10.4161/fly.6669.

Abstract

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • DNA Helicases
  • Drosophila Proteins / genetics
  • Drosophila Proteins / isolation & purification*
  • Drosophila melanogaster
  • Endopeptidases
  • Genetic Vectors*
  • Nuclear Proteins
  • Plasmids

Substances

  • Drosophila Proteins
  • Nuclear Proteins
  • Endopeptidases
  • TEV protease
  • KZ protein, Drosophila
  • DNA Helicases