Date

Monday 30 Mar 2026

Time

15:15 - 15:45

Location

BW018

Supervisor

Sander van Kasteren

2nd reviewer

Kim Bonger

Jury

Alexander Kros

Bioorthogonal click chemistry enables efficient, reliable and selective reactions in biological systems between bioorthogonal reactive groups. Metabolic labelling of proteins with these reactive groups allows conjugation to fluorescent dyes, affinity tags or other probes. In this study we label interleukine-1β (IL-1β) with threonine (Thr) analogue β-ethynylserine (βES) which bears a terminal alkyne enabling click chemistry. Thr is encoded by four different mRNA codons (ACA, ACC, ACG and ACU) present at non-random frequencies in coding DNA of Escherichia coli (E. coli). This study uses a threonine-derived non-canonical amino acid tagging method (THRONCAT) to investigate βES incorporation efficiency at the four Thr codons to enable site-specific or maximal βES incorporation. Recombinant IL-1β as a model protein was produced in E. coli ArcticExpress (DE3)RP cells in full, minimal and Thr-free growth medium supplemented with 0-4 mM βES. BES-labelling of His6 tag purified IL-1β was assessed with in-gel fluorescence after conjugation to azide-Cy5, intact protein ESI LC-MS and LC-MS/MS analyses. Four Il1β mutants, generated with site-directed mutagenesis PCR, containing five of the same Thr codons were used as a measure of βES incorporation per codon. Intact protein LC-MS analysis revealed that approximately 100% of the ACA protein mutants (produced in Thr-free medium with 4 mM βES) were labelled with βES, up to five residues (maximal incorporation) per protein. BES incorporation efficiency is reduced at the other Thr codons, corroborated by in-gel fluorescence, preventing site-specifically incorporating βES with codon optimisation. An IL-1β activity assay revealed no clear difference in activity between unlabelled and βES-labelled IL-1β. This study demonstrates that βES incorporation efficiency is highest at the ACA codon enabling maximal βES-labelling of recombinant proteins by Thr codon optimisation to ACA, useful for fluorescence imaging and pull-down assays.