Improved method for rapid purification of protein kinase from streptomycetes |
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Institution: | 1. Département d''orthopédie dento-faciale, Faculté dentaire de Nantes, CHU de Nantes, 1 place Alexis Ricordeau, Nantes 44093, France;2. Nantes Université, CHU Nantes, Service de chirurgie maxillo-faciale et stomatologie, Nantes F-44000, France;3. Nantes Université, Oniris, UnivAngers, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, Nantes F-44000, France;4. Nantes Université, UnivAngers, CHU Nantes, INSERM, CNRS, CRCI2NA, Nantes F-44000, France |
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Abstract: | Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, ~ 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylaminde gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues. |
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