A rapid purification protocol for the mitogen-activated p70 S6 kinase.
Reentry into the cell cycle from quiescence is stimulated by mitogens and supported by increased levels of protein synthesis. Initiation of polypeptide synthesis is the step most often subject to regulation. This is controlled by phosphorylation of a number of initiation factors and the single 40S ribosomal protein S6. The kinase responsible for S6 phosphorylation is p70(S6k). In the past, the apparent low cellular abundance of p70(S6k) resulted in the development of laborious large-scale purification procedures. Here a rapid and low-cost protocol which yields p70(S6k) purified to near homogeneity and with high specific activity is described. With respect to previous strategies, this novel scheme takes advantage of recently improved ion-exchange media in the first and second purification steps, thus allowing the effective removal of bulk proteins and the major Ser/Thr phosphatases. The third step of purification consists of a single affinity chromatography column in which the ligand is a 20-residue peptide containing the structural motif required for recognition and binding by p70(S6k). This novel protocol allows the rapid purification of large amounts of p70(S6k) and will facilitate the screening of libraries of natural or synthetic compounds aimed at the identification of p70(S6k) inhibitory molecules.
Ferrari
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Tumor Biology Center, Institute for Experimental Cancer Research, Freiburg, D-79011, Germany. stefferrari@hotmail.com
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D008099:Liver / Q000201:enzymology; D011817:Rabbits; D019893:Ribosomal Protein S6 Kinases / Q000302:isolation & purification*
D019893:Ribosomal Protein S6 Kinases