| Literature DB >> 16211511 |
Won Bae Jeon1, David J Aceti, Craig A Bingman, Frank C Vojtik, Andrew C Olson, Jason M Ellefson, Janet E McCombs, Hassan K Sreenath, Paul G Blommel, Kory D Seder, Brendan T Burns, Holalkere V Geetha, Amy C Harms, Grzegorz Sabat, Michael R Sussman, Brian G Fox, George N Phillips.
Abstract
The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.Entities:
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Year: 2005 PMID： 16211511 DOI： 10.1007/s10969-005-1908-7
Source DB: PubMed Journal: J Struct Funct Genomics ISSN： 1345-711X