by using talon metal affinity resin, a glass Econo-Column and a Sephadex G-50 column. Proteolysis is performed at 23Â°C for 40 min. The protein solution is then loaded again on a Talon resin column but this time the flow-through, containing CTPS without His tag, is collected. Finally the protein is loaded onto a 0.2-ml SourceQ15 medium column.
enzyme is purified by gel filtration on a HiLoad 26/60 Superdex 200 column. Active fractions are pooled and applied to a Mono Q HR 5/5 column. Final purifications step is performed on a HiLoad 26/60 Superdex 75 column. The isolated EcCTPS is found >90% pure as judged by SDS-PAGE and ESI-MS.
His6-tagged human CTP synthetase 1 is purified by loading onto a Ni2+-NTA column
His6-tagged wild type and mutant human CTP synthetase 1 enzymes are expressed and purified from yeast cell extracts with Ni2+-NTA resin; His6-tagged wild type human CTP synthetase 1 is expressed and purified from Escherichia coli by Ni2+-NTA chromatography and with PorosHQ-ion-exchange chromatography
Ni2+-IDA resin column chromatography
protein is purified by using HisTrap HP and Superdex 75 columns
recombinant wild-type and mutant enzyme L109A
the soluble histidine-tagged CTPS is purified using metal ion affinity chromatography and the histidine tag is subsequently removed using thrombin-catalyzed cleavage