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

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

Jeon WB, Aceti DJ, Bingman CA, Vojtik FC, Olson AC, Ellefson JM, McCombs JE, Sreenath HK, Blommel PG, Seder KD, Burns BT, Geetha HV, Harms AC, Sabat G, Sussman MR, Fox BG, Phillips GN Jr.

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

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.

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