Bi‐substrate analogue ligands for affinity chromatography of protein kinases

FEBS Letters

Uri, Asko²  Järv, Jaak²  Ek, Pia¹  Loog, Mart² 

[1] Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75 123 Uppsala, Sweden;Institute of Chemical Physics, Tartu University, 2 Jakobi St., 51014 Tartu, Estonia

关键词: Protein kinase;    Affinity chromatography;    Bi-substrate inhibitor;    AdoC;    adenosine-5′-carboxylic acid;    Aoc;    amino-octanoic acid;    DMF;    dimethylformamide;    IPTG;    isopropyl β-D-thiogalactopyranoside;    PMSF;    phenyl methyl sulfonyl fluoride;    SDS;    sodium dodecylsulfate;    TFA;    trifluoroacetic acid;   

DOI  :  10.1016/S0014-5793(00)01948-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown