Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.
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.
Kyriakakis
Phillip
P
Biology Department; University of Massachusetts Boston; Boston, Massachusetts, USA.
Tipping
Marla
M
Abed
Louka
L
Veraksa
Alexey
A
D000818:Animals; D002460:Cell Line; D004265:DNA Helicases; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification*; D004331:Drosophila melanogaster; D010450:Endopeptidases; D005822:Genetic Vectors; D009687:Nuclear Proteins; D010957:Plasmids
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D010450:Endopeptidases; C099488:TEV protease; C074323:KZ protein, Drosophila; D004265:DNA Helicases