Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris.
We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.
Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.
Toshiaki
T
Higo
Noriyuki
N
Suka
Haruhiko
H
Ehara
Masatoshi
M
Wakamori
Shin
S
Sato
Hideaki
H
Maeda
Shun-ichi
S
Sekine
Takashi
T
Umehara
Shigeyuki
S
Yokoyama
D002846:Chromatography, Affinity / Q000379:methods*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D005656:Fungal Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine