Bi-substrate analogue ligands for affinity chromatography of protein kinases
| Autor: | Jaak Järv, Pia Ek, Asko Uri, Mart Loog |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Swine
Protein subunit Biophysics Protein Serine-Threonine Kinases Ligands Affinity chromatography Biochemistry Chromatography Affinity Protein kinase Substrate Specificity Bi-substrate inhibitor Structural Biology Chemical specificity Genetics Animals Binding site Bovine serum albumin Casein Kinase II Protein kinase A Molecular Biology Molecular Structure biology Kinase Chemistry Cell Biology Cyclic AMP-Dependent Protein Kinases biology.protein Pyruvate kinase |
| Zdroj: | FEBS Letters. 480:244-248 |
| ISSN: | 0014-5793 |
| DOI: | 10.1016/s0014-5793(00)01948-7 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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