Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastorisReportar como inadecuado




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Journal of Structural and Functional Genomics

, Volume 15, Issue 4, pp 191–199

First Online: 15 November 2014Received: 19 September 2014Accepted: 08 November 2014

Abstract

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.

KeywordsEpitope tagging RNA polymerase Transcription Yeast AbbreviationsRNAPRNA polymerase

TAPTandem affinity purification

TCEPTris2-carboxyethylphosphine

SDSSodium dodecyl sulfate

PAGEPolyacrylamide gel electrophoresis

CBBCoomassie Brilliant Blue

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Autor: Toshiaki Higo - Noriyuki Suka - Haruhiko Ehara - Masatoshi Wakamori - Shin Sato - Hideaki Maeda - Shun-ichi Sekine - Takash

Fuente: https://link.springer.com/







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