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Optimisation of tyrosine protein kinase immobilisation for in vitro substrate phosphorylation

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

Year: 2023

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Optimisation of tyrosine protein kinase immobilisation for in vitro substrate phosphorylation	Aberrant phosphorylation of proteins has been associated with the development of numerous cancers and neurodegenerative diseases, including Alzheimer's and Parkinson's diseases [1]. Studying phosphorylation in vitro can help to clarify the causes of the development of these diseases. Nevertheless, employing soluble protein kinases for protein phosphorylation research can result in undesired sample contamination, which can impact subsequent analyses or applications [2]. Using kinases immobilised on a support with controlled termination of the phosphorylation process provides significant advantage. Immobilised enzymes are more resistant to reaction influences such as changes in pH, temperature, solvent or a medium containing contaminants [3]. An additional benefit is the rapid removal immobilized enzymes from the mixture. However, immobilisation itself can affect enzyme activity, which makes optimisation necessary before the use of enzymes in experiments. Detection of protein phosphorylation requires the use of sensitive methods such as mass spectrometry (MS). The radioactive labelling method utilizing a phosphorus isotope is prevalent. Similarly, certain chelating agents such as Zn2+, Mn2+, Ti4+ and Ca2+ selectively bind to phosphorylated proteins. Furthermore, specific immunochemical methods based on the use of antibodies against phosphorylated proteins are also employed. In this study, Src kinase was immobilised through covalent binding to Sera-Mag microparticles with carboxyl functional groups. Bovine serum albumin (BSA) was utilised during immobilisation to increase enzyme activity. Src kinase activity was monitored through analysis with MALDI-LTQ Orbitrap XL MS. After 48 hours, the phosphorylation rate of the peptide substrate was 93.2% for the kinase immobilised in BSA containing medium, whereas for the kinase immobilised in BSA-free medium, it was only 38%. During the 4-week storage period, BSA molecules obstructed the free surface of the particles [4], lead	tyrosine protein kinase; immobilisation; phosphorylation; synuklein