title: High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.
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.
authors.affiliation: The Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Bobcock Drive, 53706-1549, USA.
authors.forename: Won Bae
authors.initials: WB
authors.lastname: Jeon
authors.forename: David J
authors.initials: DJ
authors.lastname: Aceti
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.forename: Frank C
authors.initials: FC
authors.lastname: Vojtik
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Olson
authors.forename: Jason M
authors.initials: JM
authors.lastname: Ellefson
authors.forename: Janet E
authors.initials: JE
authors.lastname: McCombs
authors.forename: Hassan K
authors.initials: HK
authors.lastname: Sreenath
authors.forename: Paul G
authors.initials: PG
authors.lastname: Blommel
authors.forename: Kory D
authors.initials: KD
authors.lastname: Seder
authors.forename: Brendan T
authors.initials: BT
authors.lastname: Burns
authors.forename: Holalkere V
authors.initials: HV
authors.lastname: Geetha
authors.forename: Amy C
authors.initials: AC
authors.lastname: Harms
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Sabat
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000302:isolation & purification*; D002247:Carbon Isotopes / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli; D009587:Nitrogen Isotopes / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029681:Arabidopsis Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012645:Selenomethionine