title: Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes.
abstract: A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.
authors.affiliation: Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: Akatsuki
authors.initials: A
authors.lastname: Kimura
authors.forename: Naruhiko
authors.initials: N
authors.lastname: Adachi
authors.forename: Masami
authors.initials: M
authors.lastname: Horikoshi
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology; D006639:Histidine / Q000031:analogs & derivatives / Q000235:genetics; D006657:Histones / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D006657:Histones; D011506:Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D000123:Acetyltransferases; D005947:Glucose; D005690:Galactose