Production of milligram quantities of affinity tagged-proteins using automated multistep chromatographic purification.
A new chromatography system, AKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at > 90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine- and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.
Bhikhabhai
Rama
R
Department of Research and Development, GE Healthcare Company, Amersham Biosciences AB, SE-751 84 Uppsala, Sweden. rama.bhikhabhai@ge.com
Sjöberg
Anna
A
Hedkvist
Lotta
L
Galin
Markus
M
Liljedahl
Pia
P
Frigård
Tuomo
T
Pettersson
Niklas
N
Nilsson
Mats
M
Sigrell-Simon
Jill A
JA
Markeland-Johansson
Christine
C
D000345:Affinity Labels; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D010770:Phosphotransferases / Q000138:chemical synthesis / Q000302:isolation & purification; D011506:Proteins / Q000138:chemical synthesis / Q000302:isolation & purification*
D000345:Affinity Labels; D011506:Proteins; D010770:Phosphotransferases