Page 45 - Tyrosine-Based Bioconjugations - Jorick Bruins

2.2. Results and discussion
Laminarinase A. Given the inaccessibility of native tyrosine residues by mTyr,11, 12 it was envisioned that an exposed tyrosine required installation in a protein of interest by means of a short spacer. Thus, tetra-glycyltyrosine (G4Y) was genetically fused to the C-terminus of a model protein laminarinase A (LamA), a hyperthermostable endo-β-1,3-glucanase from Pyrococcus furiosus, which contained an N-terminal His-tag for purification.16, 27 Modifications were achieved via site-directed mutagenesis and expression in Escherichia coli, resulting in a C- terminal G4Y fusion (LamA–G4Y, here referred to as Y-tag).
Figure 1. SPOCQ labelling of G4Y-tagged laminarinase A by reaction of BCN-lissamine 1 with in situ generated 1,2-quinone.
Purified LamA–G4Y was subjected to oxidation by catalytic mTyr (7.5 mol %) to generate the intermediate 1,2-quinone, anticipated to undergo in situ SPOCQ with BCN-modified lissamine 1, present in 4-fold excess (Figure 1). Gratifyingly, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated the conversion of LamA–G4Y into the labelled
Protein labelling via SPOCQ
43
2