Directed Evolution of a Probe Ligase with Activity in the Secretory Pathway and Application to Imaging Intercellular Protein–Protein Interactions

Previously,
we reported a new method for intracellular protein
labeling in living cells called PRIME (probe incorporation mediated
by enzymes). PRIME uses a mutant of Escherichia coli lipoic acid ligase (LplA) to catalyze covalent probe ligation onto
a 13-amino acid peptide recognition sequence. While our first demonstration
labeled proteins with a coumarin fluorophore, subsequent engineering
produced alkyl azide and trans-cyclooctene ligases
as well as an interaction-dependent form of the coumarin PRIME method
(ID-PRIME). One major limitation of the PRIME methodologies is that
LplA mutants have very low activity in the secretory pathway. Here,
we extend PRIME labeling to oxidizing compartments such as the endoplasmic
reticulum and the cell surface. We used yeast-display evolution and
four rounds of selection to isolate LplA mutants with improved picolyl
azide ligation activity. Then we compared the ligation activities
of the evolved mutants both in vitro and on the mammalian
cell surface. We characterized the picolyl azide ligation activity
of the most active LplA variant in vitro, in the
endoplasmic reticulum, and at the mammalian cell surface. Finally,
we used the optimized LplA variant to label neurexin and neuroligin
interactions at the mammalian cell surface in just 5 min. Compared
to another method for imaging these protein–protein interactions
(GFP recomplementation across synapses), our optimized ID-PRIME ligase
is faster, more sensitive, and does not trap interacting proteins
in a complex (nontrapping).