High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.
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.
Jeon
Won Bae
WB
The Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Bobcock Drive, 53706-1549, USA.
Aceti
David J
DJ
Bingman
Craig A
CA
Vojtik
Frank C
FC
Olson
Andrew C
AC
Ellefson
Jason M
JM
McCombs
Janet E
JE
Sreenath
Hassan K
HK
Blommel
Paul G
PG
Seder
Kory D
KD
Burns
Brendan T
BT
Geetha
Holalkere V
HV
Harms
Amy C
AC
Sabat
Grzegorz
G
Sussman
Michael R
MR
Fox
Brian G
BG
Phillips
George N
GN
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
D029681:Arabidopsis Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012645:Selenomethionine