Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins.
In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity.
Dian
Cyril
C
Department of Biochemistry and Biophysics, Stockholm University, Sweden.
Eshaghi
Said
S
Urbig
Thomas
T
McSweeney
Sean
S
Heijbel
Anna
A
Salbert
Gilles
G
Birse
Darcy
D
D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase