Bi-substrate analogue ligands for affinity chromatography of protein kinases

Novel affinity ligands, consisting of ATP-resembling part coupled with spec ificity determining peptide fragment, were proposed for purification of pro tein kinases, Following this approach affinity sorbents based on two closel y similar ligands AdoC-Aoc-Arg(4)-Lys and AdoC-Aoc-Arg(4)-NH(CH2)(6)NH2, wh ere AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pi g heart homogenate was selectively isolated using MgATP as eluting agent. P rotein kinase with acidic specificity determinant (CK2) as well as other pr oteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mo de of the affinity ligand, (C) 2000 Federation of European Biochemical Soci eties. Published by Elsevier Science B.V. All rights reserved.