Doctoral dissertation (article) (G5)

The peptide-break technology : a novel approach for protein post-translational modification assays in drug discovery




List of AuthorsTong-Ochoa Natalia

PublisherUniversity of Turku

PlaceTurku

Publication year2019

ISBN978-951-29-7552-5

eISBN978-951-29-7553-2

URLhttp://urn.fi/URN:ISBN:978-951-29-7553-2

Self-archived copy’s web addresshttp://urn.fi/URN:ISBN:978-951-29-7553-2


Abstract

The work
presented in this thesis describes the development
of a universal platform for the biochemical
detection of protein post-translational modifications (PTM) using the quenching
resonance energy transfer (QRET) technique in an antibody-free system. The QRET
technique employed is based on the difference in the luminescence signal
produced by the target bound and unbound to a Ln3+-ligand in the
presence of soluble quenchers.

 In the project,
PTM monitoring was first demonstrated in a simple QRET tyrosine EGFR assay
using antibodies to recognize the modified substrate peptide, a commonality in
FRET assays, yet in a single-label approach. On the basis of this, a universal
PTM detection method called “the peptide-break technology” was further
developed as an antibody-free system. The approach relied on the peptide
dimerization concept of leucine zipper (LZ) coiled-coils.
The dimer was composed of an enzyme-substrate peptide and a detection Eu3+-peptide.
In the assay, the PTM addition to the substrate peptide disrupted the dimer,
leading to a low luminescence signal, whereas without PTM addition the dimer
formation proceeds and provides Eu3+-chelate protection leading to a
high luminescence. The peptide-break technology was successfully developed for
a variety of enzymes for phosphorylation, dephosphorylation, deacetylation, and
citrullination, using nanomolar concentrations without the need for antibodies
or enzyme reporters.

 Later, the
peptide-break technology was simplified and optimized to increase the freedom
for substrate peptide selection and design. The peptide dimerization was
mediated by the interaction between oppositely charged peptides. Finally, the
technology was further studied in a thermal shift assay, allowing the use of
high substrate peptide concentrations that are needed in assays with low
affinity or activity enzymes. The reported data suggest that this technology is
potentially applicable to other challenging PTM types, possibly providing new
assay solutions within academia as well as for the pharmaceutical industry.


Last updated on 2021-24-06 at 11:21