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Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG
journal contribution
posted on 2020-08-01, 00:03 authored by Thomas Kjeldsen, Wouter F. J. Hogendorf, Christian W. Tornøe, Jonathan Anderson, Frantisek Hubalek, Carsten E. Stidsen, Jan L. Sorensen, Thomas Hoeg-JensenCovalent
cross-linking of biomolecules can be useful in pursuit
of tissue targeting or dual targeting of two receptors on cell surfaces
for avidity effects. Long linkers (>10 kDa) can be advantageous
for
such purposes, and poly(ethylene glycol) (PEG) linkers are most commonly
used due to the high aqueous solubility of PEG and its relative inertness
toward biological targets. However, PEG is non-biodegradable, and
available PEG linkers longer than 5 kDa are heterogeneous (polydisperse),
which means that conjugates based on such materials will be mixtures.
We describe here recombinant linkers of distinct lengths, which can
be expressed in yeast, which are polar, and which carry orthogonal
reactivity at each end of the linker, thus allowing chemoselective
cross-linking of proteins. A conjugate between insulin and either
of the two trypsin inhibitor peptides/proteins exemplifies the technology,
using a GQAP-based linker of molecular weight of 17 848, having
one amine at the N-terminal, and one Cys, at the C-terminal. Notably,
yeast-based expression systems typically give products with mixed
disulfides when expressing proteins that are equipped with one unpaired
Cys, namely, mixed disulfides with glutathione, free Cys amino acid,
and/or a protein homodimer. To obtain a homogeneous linker, we worked
out conditions for transforming the linker with mixed disulfides into
a linker with a homogeneous disulfide, using excess 4-mercaptophenylacetic
acid. Subsequently, the N-terminal amine of the linker was transformed
into an azide, and the C-terminal Cys disulfide was reduced to a free
thiol and reacted with halo-acetyl insulin. The N-terminal azide was
finally conjugated to either of the two types of alkyne-containing
trypsin inhibitor peptides/proteins. This reaction sequence allowed
the cross-linked proteins to carry internal disulfides, as no reduction
step was needed after protein conjugations. The insulin–trypsin
inhibitor conjugates were shown to be stabilized toward enzymatic
digestions and to have partially retained binding to the insulin receptor.
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insulin receptorprotein homodimerPEG Covalent cross-linkingprotein conjugationsN-terminal azideC-terminal Cys disulfide5 kDaavidity effectschemoselective cross-linkingyeast-based expression systemscross-linked proteinsreduction stepDually Reactive Long Recombinant Li...halo-acetyl insulinorthogonal reactivityGQAP-based linker4- mercaptophenylacetic acidN-terminal aminecell surfacesPEG linkersreaction sequence
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