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Immobilisation of Tyrosine Protein Kinase Src on Magnetic Microparticles for Myelin Basic Protein Phosphorylation

Authors: Bednářová Šárka | Morávek Ondřej | Kupčík Rudolf | Fojtíková Markéta | Bílková Zuzana | Slováková Marcela

Year: 2025

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Immobilisation of Tyrosine Protein Kinase Src on Magnetic Microparticles for Myelin Basic Protein Phosphorylation	Phosphorylation is a very important post-translational modification of proteins in terms of their proper function and structure. A detailed study of protein phosphorylation can help clarify the pathogenesis of many diseases such as cancer or neurodegenerative diseases [1]. To study substrate phosphorylations, which are possible by various approaches, we chose the use of protein kinases. Protein kinases were conjugated with magnetic particles, with advantages such as easy and fast handling and separation from the mixture, and no contamination of the final product. For the best possible enzyme activity, we optimised immobilization conditions with various beads, also with the aim of minimising nonspecific sorption by using BSA [2]. The available particles with a carboxyl-modified surface were searched (Thermo Fisher, Chemicell, and Invitrogen). Phosphorylation of the peptide substrate by immobilised Src kinase was followed by analysis with MALDI-LTQ Orbitrap XL MS. The highest level of peptide phosphorylation (93.2% after 48 hours) was achieved with Src kinase immobilised on Sera Mag Speed Beads. In terms of storage stability, the activity of the immobilised enzyme after 4 weeks of storage was reduced by only 10.85%. Finally, the immobilised Src kinase was used to phosphorylate the native bovine myelin basic protein (MBP), known as an intrinsic disordered protein, which undergoes dynamic conformational ensembles with only a weak tertiary interaction. Naturally, MBP does not carry tyrosine phosphorylation [3]. Phosphorylation on tyrosines was demonstrated for soluble and immobilised Src protein kinase by MALDI-LTQ Orbitrap XL mass spectrometry and Western blot with immunodetection and pIMAGO detection.	Immobilisation; Src kinase; Phosphorylation