High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.

J Struct Funct Genomics 2005 ;6(2-3):143-7

The Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Bobcock Drive, 53706-1549, USA.

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.

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http://dx.doi.org/10.1007/s10969-005-1908-7DOI Listing
June 2006
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