Immobilized ERK2 and GSK-3beta magnetic microparticles for targeted protein phosphorylation

Introduction: Micro and nanoparticles with immobilized enzymes as recoverable, stable and specific catalysts are applied in a variety of technologies, in biomedical applications and widely in research. Magnetic microparticles... [ view full abstract ]

Introduction: Micro and nanoparticles with immobilized enzymes as recoverable, stable and specific catalysts are applied in a variety of technologies, in biomedical applications and widely in research. Magnetic microparticles bring the advantage of the non-contaminating and very specific and sensitive reaction on their substrates, peptides and proteins. This work is based on the use of proline-directed protein kinases: extracellular signal-regulated kinase (ERK2) and glycogen synthase kinase 3β (GSK-3β) immobilized to magnetic microparticles for targeted recombinant tau 1-441 phosphorylation.
Methods: In order to obtain highly efficient carrier for the targeted phosphorylation of peptides/proteins, two kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, and metal cations Ni2+ or Co3+ functionalities. Relevant methods of covalent immobilization, non-oriented and oriented, were chosen and reaction conditions were optimized. Phosphorylation of low molecular peptides and operational and storage stabilities of kinase-superparamagnetic particles were performed. Soluble and immobilized ERK2 and GSK-3β were applied for recombinant tau 1-441 phosphorylation. Tryptic phosphopeptides enrichment was performed by ion-metal affinity chromatography using TiO2 magnetic nanoparticles. The level and the position of phosphorylation sites were identified by using MALDI-LTQ-Orbitrap MS. Western blot was carried out to confirm phosphorylation of tau 1–441 (ENZO and rPeptide) by soluble or immobilized ERK2 and GSK-3β kinases using specific anti-tau and anti-phospho-tau antibodies.
Results: The effect of immobilization was confirmed due to the enzymes ability to phosphorylate low molecular synthetic peptides. The ERK2 and GSK-3βkinases immobilized on SeraMag beads using the carbodiimide chemistry proved the ability to phosphorylate peptides in 10 cycles without significant loss in activity. Using carriers with immobilized ERK2 or GSK-3β we performed the efficient phosphorylation of model recombinant protein tau 1-441. Presence and position of phosphorylation along the peptide chains were detected by MALDI MS and confirmed also by Western blot with anti-tau phospho-specific antibodies.
Discussion: Present results document very well the ability of ERK2 and GSK-3β superparamagnetic particles to phosphorylate the target peptides and recombinant tau 1-441 with desired efficiency and specificity. The process of phosphorylation can be better controlled; subsequent purification of phosphorylated tau can be omitted.
Acknowledgement: EU project NADINE No. 246513