HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae.
We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers. These plasmids allow single-step PCR-based tagging and expression under control of the endogenous promoters or the inducible GAL1 promoter. HB tagging of several budding yeast ORFs demonstrated efficient biotinylation of the HB tag in vivo by endogenous yeast biotin ligases. No adverse effects of the HB tag on protein function were observed. The HB tagging plasmids presented here are related to previously reported epitope-tagging plasmids, allowing PCR-based tagging with the same locus-specific primer sets that are used for other widely used epitope-tagging strategies. The Sequences for the described plasmids were submitted to GenBank under Accession Numbers DQ407918-pFA6a-HBH-kanMX6 DQ407927-pFA6a-RGS18H-kanMX6 DQ407919-pFA6a-HBH-hphMX4 DQ407928-pFA6a-RGS18H-hphMX4 DQ407920-pFA6a-HBH-TRP1 DQ407929-pFA6a-RGS18H-TRP1 DQ407921-pFA6a-HTB-kanMX6 DQ407930-pFA6a-kanMX6-PGAL1-HBH DQ407922-pFA6a-HTB-hphMX4 DQ407931-pFA6a-TRP1-PGAL1-HBH DQ407923-pFA6a-HTB-TRP1 DQ407924-pFA6a-BIO-kanMX6 DQ407925-pFA6a-BIO-hphMX4 DQ407926-pFA6a-BIO-TRP1.
Tagwerker
Christian
C
University of California Irvine, Department of Biological Chemistry, School of Medicine, Irvine, CA 92697-1700, USA.
Zhang
Hongwei
H
Wang
Xiaorong
X
Larsen
Liza S Z
LS
Lathrop
Richard H
RH
Hatfield
G Wesley
GW
Auer
Bernhard
B
Huang
Lan
L
Kaiser
Peter
P
D001483:Base Sequence; D001710:Biotin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002846:Chromatography, Affinity; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D000939:Epitopes; D006639:Histidine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004271:DNA, Fungal; D000939:Epitopes; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D001710:Biotin