Bacterial expression and purification of interleukin-2 tyrosine kinase: single step separation of the chaperonin impurity.
Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here, we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl2/ATP/KCl incubation procedure to remove the co-purifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets.
Joseph
Raji E
RE
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 4208 MBB, Ames, IA 50011, USA.
Andreotti
Amy H
AH
D000818:Animals; D018833:Chaperonins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D018833:Chaperonins