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In Vitro Phosphorylation Using Tyrosine Kinase Src Immobilised on Magnetic Beads and Detection of Activity

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

Year: 2025

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	In Vitro Phosphorylation Using Tyrosine Kinase Src Immobilised on Magnetic Beads and Detection of Activity	Any deviation in the degree or location of protein phosphorylation leads to a change in the structural conformation of proteins, which results in a decrease or loss of their function. As a result, it leads to the development of various diseases, as tumour growth or neurodegenerative diseases.1 Knowledge of the pathways related to phosphorylation is therefore important for designing new therapeutic strategies.2 In this context, protein kinases play an important role in cell signalling and are often monitored. Soluble kinases are commonly used to study the phosphorylation of recombinant proteins in vitro. To control the process of protein phosphorylation, stable reaction conditions must be ensured in catalysis,3 therefore tyrosine kinase Src was covalently attached to magnetic microparticles Sera-Mag speed beads. To monitor kinase activity and quality of peptide/protein (cSrc, myelin basic protein, recombinant alpha synuclein) phosphorylation we have chosen highly sensitive biochemical and analytical methods, such as Western blotting using phosphospecific antibodies, phospho-specific coloring and visualization method pIMAGOTM, and MALDI mass spectrometry. TiO2 based affinity chromatography method have been used for the specific enrichment of phosphopeptides before MS analysis. Immobilized kinase Src have shown the advantage of easy and efficient separation from the final product, especially advantageous in the case of large-scale production of phosphorylated recombinant proteins. Another significant advantage has shown the storage stability for 4 weeks without change in enzyme activity. Phosphorylation of myelin basic protein with soluble and immobilized Src kinase was confirmed by all the methods mentioned. Phosphorylation at Tyr39 of alpha-synuclein with soluble and immobilized kinase was confirmed by mass spectrometry and Western blot.	Phosphorylation; Src kinase; Immobilisation; MBP