Bi-substrate analogue ligands for affinity chromatography of protein kinases.
Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where 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 pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins 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 mode of the affinity ligand.
Loog
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Institute of Chemical Physics, Tartu University, Estonia. mart.loog@medkem.uu.se
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Järv
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D000818:Animals; D047390:Casein Kinase II; D002846:Chromatography, Affinity / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D015394:Molecular Structure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D008024:Ligands; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases