3 research outputs found
Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition
Biologics,
such as antibody–drug conjugates, are becoming
mainstream therapeutics. Consequently, methods to functionalize biologics
without disrupting their native properties are essential for identifying,
characterizing, and translating candidate biologics from the bench
to clinical practice. Here, we present a method for site-specific,
carboxy-terminal modification of single-chain antibody fragments (scFvs).
ScFvs displayed on the surface of yeast were isolated and functionalized
by combining intein-mediated expressed protein ligation (EPL) with
inverse electron-demand Diels–Alder (IEDDA) cycloaddition using
a styrene–tetrazine pair. The high thiol concentration required
to trigger EPL can hinder the subsequent chemoselective ligation reactions;
therefore, the EPL reaction was used to append styrene to the scFv,
limiting tetrazine exposure to damaging thiols. Subsequently, the
styrene-functionalized scFv was reacted with tetrazine-conjugated
compounds in an IEDDA cycloaddition to generate functionalized scFvs
that retain their native binding activity. Rapid functionalization
of yeast surface-derived scFv in a site-directed manner could find
utility in many downstream laboratory and preclinical applications
Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display
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
An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins
Expressing
antibodies as fusions to the non-self-cleaving Mxe GyrA
intein enables site-specific, carboxy-terminal chemical modification
of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein
fusion protein expression platforms typically yield insoluble inclusion
bodies that require refolding to obtain active antibody-intein fusion
proteins. Previously, we demonstrated that it was possible to employ
yeast surface display to express properly folded single-chain antibody
(scFv)-intein fusions, therefore permitting the direct small-scale
chemical functionalization of scFvs. Here, directed evolution of the
Mxe GyrA intein was performed to improve both the display and secretion
levels of scFv-intein fusion proteins from yeast. The engineered intein
was shown to increase the yeast display levels of eight different
scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein
(GFP)-intein fusion proteins can be secreted from yeast, and while
fusion of the scFvs to the wild-type intein resulted in low expression
levels, the engineered intein increased scFv-intein production levels
by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins
retained their respective binding and fluorescent activities, and
upon intein release, EPL resulted in carboxy-terminal azide functionalization
of the target proteins. The azide-functionalized scFvs and GFP were
subsequently employed in a copper-free, strain-promoted click reaction
to site-specifically immobilize the proteins on surfaces, and it was
demonstrated that the functionalized, immobilized scFvs retained their
antigen binding specificity. Taken together, the evolved yeast intein
platform provides a robust alternative to bacterial intein expression
systems