High-performance liquid chromatographic separation and renaturation of protein kinase subunits: Application to catalytic subunit of phosphorylase kinase

This chapter describes various methods used for the separation of phosphorylase kinase subunits under denaturing conditions and subsequent reactivation of the γ subunit and for renaturation of the bacterial expressed form of the subunit of phosphorylase kinase. For high-performance liquid chromatographic (HPLC) separation of phosphorylase kinase subunits, the subunits are eluted according to size, with the δ subunit eluting first between 43 and 50% acetonitrile and α subunit eluting last at about 58% acetonitrile. Phosphorylase kinase is injected directly to the column previously equilibrated in 0.1% TFA and the γ subunit is eluted at approximately 50% acetonitrile and 0.09% TFA, with a pH of 2.5. The γ subunit prepared in this way is pure, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For renaturation of HPLC-isolated γ subunit of phosphorylase kinase, the HPLC-purified γ subunit is diluted to 1–5 μg/ml in an ice-cold solution containing one part assay buffer and four parts buffer A plus 60–100 μg/ml calmodulin and 1.5 mg/ml phosphorylase b or bovine serum albumin. This results in a final pH of 8.2 and final concentrations of no more than 5% acetonitrile and 0.01% TFA. The reactivation mixture is kept on ice for several days to attain optimum catalytic activity.