Return to search

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

Identiferoai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/627877
Date05 1900
CreatorsDiaz Galicia, Miriam Escarlet
ContributorsArold, Stefan T., Biological and Environmental Sciences and Engineering (BESE) Division, Mahfouz, Magdy M., Jaremko, Lukasz
Source SetsKing Abdullah University of Science and Technology
LanguageEnglish
Detected LanguageEnglish
TypeThesis
Rights2019-05-15, At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis became available to the public after the expiration of the embargo on 2019-05-15.

Page generated in 0.0023 seconds