Facile
Chemical Functionalization of Proteins through Intein-Linked Yeast
Display
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Abstract
Intein-mediated expressed protein
ligation (EPL) permits the site-specific
chemical customization of proteins. While traditional techniques have
used purified, soluble proteins, we have extended these methods to
release and modify intein fusion proteins expressed on the yeast surface,
thereby eliminating the need for soluble protein expression and purification.
To this end, we sought to simultaneously release yeast surface-displayed
proteins and selectively conjugate with chemical functionalities compatible
with EPL and click chemistry. Single-chain antibodies (scFv) and green
fluorescent protein (GFP) were displayed on the yeast surface as fusions
to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released
from the yeast surface with either a sulfur nucleophile (MESNA) or
a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine
azide permitted the simultaneous release and azido functionalization
of displayed proteins, but nonspecific reactions with other yeast
proteins were detected, and cleavage efficiency was limited. In contrast,
MESNA released significantly more protein from the yeast surface while
also generating a unique thioester at the carboxy-terminus of the
released protein. These protein thioesters were subsequently reacted
with a cysteine alkyne in an EPL reaction and then employed in an
azide<i>–</i>alkyne cycloaddition to immobilize the
scFv and GFP on an azide-decorated surface with >90% site-specificity.
Importantly, the immobilized proteins retained their activity. Since
yeast surface display is also a protein engineering platform, these
approaches provide a particularly powerful tool for the rapid assessment
of engineered proteins