title: A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4.
abstract: With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. Here, we show that TAP, using Protein A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein A and CBP. FLAG-His purification worked sufficiently well so that two cofactors of the Drosophila nuclear receptor protein dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. We show that the two proteins increase the ability of dHNF4 to bind DNA in vitro and to function in vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms.
authors.affiliation: Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, University of Toronto, Charles H. Best Institute, Toronto, Ontario, Canada.
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.forename: Heidi M
authors.initials: HM
authors.lastname: Sampson
authors.forename: Henry M
authors.initials: HM
authors.lastname: Krause
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G; D008297:Male; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D029721:Drosophila Proteins; D050883:HSC70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D051557:Hepatocyte Nuclear Factor 4; C089272:Hsp83 protein, Drosophila; D007074:Immunoglobulin G; D009842:Oligopeptides; D010455:Peptides; D013205:Staphylococcal Protein A; D006639:Histidine; D004247:DNA; C085706:FLAG peptide