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) followe...

Full description

Saved in:
Bibliographic Details
Published in:Journal of structural and functional genomics 2014-12, Vol.15 (4), p.191-199
Main Authors: Higo, Toshiaki, Suka, Noriyuki, Ehara, Haruhiko, Wakamori, Masatoshi, Sato, Shin, Maeda, Hideaki, Sekine, Shun-ichi, Umehara, Takashi, Yokoyama, Shigeyuki
Format: Article
Language:English
Subjects:
Biochemistry
Bioinformatics
Biology
Biomedical and Life Sciences
Chromatography, Affinity - methods
Crystallization
Forestry Management
Fungal Proteins - biosynthesis
Fungal Proteins - genetics
Fungal Proteins - isolation & purification
Histidine - biosynthesis
Histidine - genetics
Histidine - isolation & purification
Human Genetics
Life Sciences
Methods
Microbial Genetics and Genomics
Multiprotein Complexes - biosynthesis
Multiprotein Complexes - genetics
Multiprotein Complexes - isolation & purification
Pichia - chemistry
Pichia - genetics
Pichia - metabolism
Pichia pastoris
Plant Genetics and Genomics
Proteins
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
RNA polymerase
Yeast
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1345-711X
1570-0267
DOI:10.1007/s10969-014-9190-1