Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag

Article abstract of DOI:10.1002/anie.201108130

Clicking into place: Cu-free click chemistry is combined with the aldehyde tag protein modification strategy to produce heterobifunctional protein fusions. This method relies on linkers that utilize orthogonal triazole and oxime chemistries (see scheme) to expand on conjugation with the fGly residue and enables site-specific protein conjugation to full-length human antibodies. Copyright

Full text of DOI:10.1002/anie.201108130

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Chemie
DOI: 10.1002/anie.201108130
Protein Chemistry
Synthesis of Heterobifunctional Protein Fusions Using Copper-Free
Click Chemistry and the Aldehyde Tag**
Jason E. Hudak, Robyn M. Barfield, Gregory W. de Hart, Patricia Grob, Eva Nogales,
Carolyn R. Bertozzi,* and David Rabuka*
Heterobifunctional protein fusions are gaining interest as
next-generation biopharmaceuticals.[1–5] Combining proteins
with disparate functions can enable multidrug therapy with
a single chemical entity,[6,7] add a targeting element to an
otherwise nonspecific therapeutic,[8,9] or improve the phar-
macokinetic profile of a rapidly cleared molecule.[10,11]
Indeed, heterobifunctional proteins, such as immunoglobul-
in G (IgG) Fc domain fusions, are among the top-selling
biotherapeutics on the market today.[12] These biomolecules
are primarily generated as genetic fusions. The DNA
sequences that encode the individual protein components
are fused in tandem to direct the expression of a single
polypeptide that comprises the two proteins joined together
at their N and C termini, respectively. However, this limited
topology is not ideal for every protein combination, as some
polypeptides require unmodified termini for optimal bioac-
tivity[13] or can suffer from expression difficulties as a result of
folding and processing issues.[3,14,15]
expressed a Fab fragment bearing an unnatural keto amino
acid to which a maleimide-funtionalized linker was conju-
gated by oxime formation.[21] This, in turn, enabled further
conjugation to a single cysteine residue that was engineered
into a protein toxin. Implicit in this work is the need for
a protein–protein coupling reaction with intrinsically fast
kinetics, of which thiol to maleimide addition is a paragon
example. In this direction, Bundy and Swartz have imple-
mented cell-free protein synthesis to install azide and alkyne
amino acids into green fluorescent protein for Cu-catalyzed
dimerization.[22] This approach, however, suffered from low
protein expression as well as Cu-induced protein damage.
The strain-promoted 1,3-dipolar cycloaddition of cyclo-
octynes and azides, also termed the Cu-free azide–alkyne
cycloaddition, is a bioorthogonal reaction that is well suited
for protein–protein conjugation.[23–26] The cyclooctyne reagent
can be tuned for fast kinetics and the reaction proceeds
selectively under a wide range of conditions.[27–31] However,
harnessing these qualities for heterobifunctional protein
conjugate synthesis first requires a practical route for the
site-specific introduction of the necessary reactive partners.
The genetically encoded aldehyde tag offers a simple
means of site-specific protein functionalization.[32–34] The tag
consists of a succinct five-residue sequence (CxPxR) that is
recognized by the formylglycine-generating enzyme (FGE).
FGE oxidizes the genetically-encoded cysteine residue to the
aldehyde-bearing residue formylglycine (fGly) during protein
expression in either E. coli or mammalian cells (Scheme
1A).[35] The aldehyde can then be modified by hydrazone or
oxime formation (Scheme 1B).[36] Thus, the aldehyde tag
serves as a means for site-specific introduction of azides or
cyclooctynes onto recombinant proteins through small-mol-
An alternative approach to generating protein–protein
fusions is through chemical conjugation. Native chemical
ligation of C-terminal thioesters with b-amino thiols is
a
powerful method for generating protein–protein
fusions,[16–18] but at least one coupling partner must be
linked at its terminus. In principle, greater topological
diversity can be achieved by introducing bioorthogonal
functional groups at specific amino acid side chains of the
two proteins.[19,20] As a recent example, Hutchins et al.
[*] J. E. Hudak, Prof. C. R. Bertozzi
Departments of Chemistry and Molecular and Cell Biology
Howard Hughes Medical Institute
University of California, Berkeley, CA 94720 (USA)
E-mail: crb@berkeley.edu
Dr. R. M. Barfield, Dr. G. W. de Hart, Dr. D. Rabuka
Redwood Bioscience Inc.
5703 Hollis St., Emeryville, CA 94608 (USA)
E-mail: drabuka@redwoodbioscience.com
Dr. P. Grob, Prof. E. Nogales
Department of Molecular and Cell Biology, Howard Hughes Medical
Institute, Lawrence Berkeley National Laboratory
University of California, Berkeley, CA 94720 (USA)
[**] We thank Prof. Zev Gartner, Ellen Sletten, Brian Belardi, and Brian
Carlson for materials, discussion, and manuscript critique. This
work was funded by grants from the NIH to D.R. (1RC1EB010344-
01) and C.R.B. (GM59907). J.E.H. was supported by an NSF
predoctoral fellowship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201108130.
Scheme 1. Aldehyde tag enables site-specific protein modification.
A) FGE recognizes the sequence CxPxR and converts Cys into fGly by
oxidation of the sulfhydryl group to an aldehyde. B) The aldehyde
reacts with an aminooxy reagent to form a stable oxime.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://angewandte.org/open.
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increase conjugation yields with fGly at any pH tested, and
may have been inhibitory in this set of reactions.[37] We
obtained maximal protein labeling after 24 h at 378C (Fig-
ure S2A in the Supporting Information). The reactions of
hIgG with a peptide probe, aminooxy-FLAG (AO-FLAG),[32]
were dependent on the reagent concentration and required
over ten equivalents (100 mm) of AO-FLAG for optimal
labeling (Figure S2B in the Supporting Information). These
results highlight the limitations of an exclusively oxime-based
conjugation approach with sterically encumbered reactants,
and also provided the impetus to explore Cu-free azide–
alkyne cycloadditions for protein–protein assembly.[20,38]
We generated three linkers of various lengths (1–3,
Figure 1B) that each contain an azide attached by a tetrae-
thyleneglycol (TEG) spacer to an aminooxy moiety. For the
cyclooctyne component, we chose the commercially available
dibenzoazacyclooctyne (DIBAC).[30,39] Linkers 1–3 were
treated with aldehyde-tagged MBP and subsequently an
excess amount of the dibenzoazacyclooctyne fluorophore
DIBAC-488. Robust labeling was observed by fluorescence
gel scanning, which was dependent on the presence of the
azide-functionalized linker (Figure 2). In contrast, direct
labeling of MBP-fGly with AO-AF488 produced weaker
labeling under similar conditions (Figure S3 in the Supporting
Information). Furthermore, removal of excess azide linker
before reaction with DIBAC-488 allowed the use of 15-fold
less of the fluorophore reagent without affecting the yields
(Figure 2). Linker 2, which contains an aminooxy acetyl
group, was the least efficient labeling reagent, as determined
by MALDI-TOF MS analysis (Figure 2, see also Figure S4 in
the Supporting Information). One concern was the possible
side reactivity of DIBAC reagents with free thiols, which has
been noted with other reactive cyclooctynes.[40,41] Our experi-
ments with MBP could not address this issue, as the protein
has no free cysteine residues. Thus, we performed a similar
reaction with aldehyde-tagged human serum albumin (HSA),
which contains a natural free cysteine residue. Treatment of
aldehyde-tagged HSA with DIBAC-488 alone gave no
significant labeling (Figure S5 in the Supporting Informa-
tion). Thus, the low to sub-millimolar concentrations of
Figure 1. Bifunctional “click” linkers. A) Aldehyde-tagged, fGly-contain-
ing proteins are first treated with an azido-aminooxy bifunctional
linker. Cu-free click chemistry can then be performed for covalent
attachment to any DIBAC-functionalized molecule. B) Heterobifunc-
tional linkers for introducing azides and cyclooctynes onto aldehyde-
tagged proteins.
ecule linkers. Once installed, these rapidly reacting functional
groups enable the assembly of protein–protein conjugates
(Figure 1A). Herein, we employed this approach in the
synthesis of heterobifunctional chemical protein fusions of
unprecedented complexity;
a full length human IgG
(155 kDa) coupled to either human growth hormone (hGH,
26 kD) or the maltose-binding protein (MBP, 42 kDa). This
site-selective coupling highlights the potential of Cu-free click
chemistry in state-of-the-art controlled protein assembly.
To expand on previous reports of fGly conjugation, we
initially identified the optimal conditions for oxime formation
on aldehyde-tagged recombinant proteins. MBP was chosen
as a model monomeric globular protein, whereas human IgG1
(hIgG) served as a more complex and clinically relevant
conjugation substrate. Additionally, both MBP and hIgG
demonstrate more than 90% Cys-to-fGly conversion when
expressed in bacterial[32] and mammalian[33] cell hosts, respec-
tively (see the Supporting Information for conversion ana-
lytical data). Conjugations with aminooxy Alexa Fluor 488
(AO-AF488) in various buffers were strongly pH dependent,
with yields reaching over 70% between pH 4–5 (Figure S1
and Table S1 in the Supporting Information). Aniline,
a reported catalyst of oxime formation, did not appear to
Figure 2. Reaction of fGly-containing MBP (30 mm) with bifunctional
linkers 1–3 and subsequently with DIBAC-488. Lanes 1–6: protein
treated with linkers (pH 4.5, 328C, 16 h) and then excess DIBAC-488
(16 h, 48C). Lane 7: fGly-MBP treated with DIBAC-488 alone, without
prior linker conjugation. Lanes 8–10: linker was removed then azide-
tagged MBP (15 mm) was treated with DIBAC-488 (2 equiv, 16 h, 48C).
Top row: fluorescent scan; bottom row: coomassie stain.
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DIBAC reagents that were used in our procedures do not
appear to produce unwanted side reactions.[31]
To demonstrate the power of the Cu-free click chemistry
approach, we generated conjugates of full-length hIgG with
hGH[32,34] or MBP (Figure 3A). These constructs are partic-
ularly relevant to ongoing efforts to increase the serum
halflife of protein therapeutics (hGH-hIgG)[10,42] or to achieve
dual binding specificities in a single molecule (MBP-
hIgG).[5,43] Our strategy for fusing the protein pairs included
the synthesis of bifunctional linker 4, which comprises
DIBAC tethered by a TEG spacer to an aminooxy group
(Figure 1). We envisioned that an aldehyde-tagged protein
could be treated with linker 1 and then combined with
a protein that is conjugated to linker 4 to form a chemically
and topologically defined protein homo or heterodimer.
As a next step, we explored protein–peptide conjugations
by using a DIBAC-FLAG conjugate as a model peptide (see
the Supporting Information). Aldehyde-tagged MBP was
treated with linkers 1–3, and the purified conjugate was
coupled with DIBAC-FLAG. The Cu-free click reactions
labeled MBP-fGly more efficiently than treatment with AO-
FLAG alone, as demonstrated by immunoblot (Figure S6 in
the Supporting Information). In more detailed comparisons,
DIBAC-FLAG reactions were faster at room temperature
than the corresponding AO-FLAG reactions at 378C and
required lower reagent concentrations (Table S2 and Fig-
ure S7 in the Supporting Information).
Figure 3. Protein–protein conjugation of hIgG with hGH and MBP. A) Aldehyde-tagged protein functionalized with azide 1 (hGH-Az) reacts
specifically with protein functionalized with 4 (hIgG-DIBAC). As hIgG is a homodimer, two molecules of hGH-Az can react with hIgG-DIBAC to
form a trimer. B) Western blot analysis of hIgG-hGH and hIgG-MBP chemical conjugations, nonreduced to highlight mono- and diconjugation.
rxn: after reaction at 48C for 16 h. pur: after purification. Top blots: ponceau stain. Middle blots: blot probed with a-hGH or a-MBP and
subsequently by a-mIgG HRP. Bottom blots: same blot probed with a-hIgG 647. * Denotes single conjugate; ** denotes diconjugate. C) Flow
cytometry analysis of SKOV3 cells treated with aldehyde-tagged a-HER2-hIgG. Chemically conjugated hGH-hIgG and MBP-hIgG labeled the cell
surface by a-HER2 binding, whereas azide-modified hGH-Az or MBP-Az alone did not. Blue=a-hlgG; red=a-hGH; green=a-MBP.D) Negative
stain TEM image of C-terminal-tagged MBP-hIgG conjugates. A gallery of 2D class-averages of negatively stained MBP-hIgG shows a flexible
additional density at the tip of one of the hIgG density lobes (arrows) that is consistent with a C-terminal attachment. The left panel displays
a simulated density map of unconjugated IgG; the averages on the right are overlaid with a 2D docking of IgG1 alone (red) or with additional
MBP crystal structures (light and dark blue; Protein Data Bank (PDB) 1IGY, 1JW4).
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We treated linkers 1 or 4 with hGH, MBP, and hIgG
separately, then subsequently treated the conjugates with
DIBAC-488, azide Alexa Fluor 647 (Az-647), or the comple-
mentary Cu-free click protein partner. The oxime-conjugated
proteins were efficiently labeled with dye and formed the
expected homo and heterodimers (Figures S8 and S9 in the
Supporting Information). Next, we established large-scale
conjugation conditions for the reaction of azide-modified
linker 1-hGH or linker 1-MBP with DIBAC-modified linker
4-hIgG (2:1 azide protein/DIBAC protein, 48C, phosphate-
buffered saline (PBS)). The resulting hIgG–protein chemical
fusions (Figure 3A) were purified and analyzed by immuno-
blot and transmission electron microscopy (TEM).
In conclusion, we have demonstrated that Cu-free click
chemistry in conjunction with the aldehyde tag can produce
protein–protein chemical conjugates of unprecedented size
and complexity. The synthetic route capitalizes on small-
molecule linkers that can increase reaction yields, lower the
necessary reagent concentrations, and decrease the reaction
time. The method should expand the topologies of available
protein fusions and allow the exploration of alternate points
of protein–protein attachment.
Possible applications in the antibody drug discovery space
include antibody-dependent enzyme prodrug therapies
(ADEPT) and antibody targeted immunotoxins.[46–48] Fur-
thermore, the approach can be extended to protein–synthetic
polymer conjugations and surface immobilization[49,50] along
with designing protein conjugates that extend serum half-
life,[51] or for vaccine development.[52]
The hIgG construct used in this study has the aldehyde tag
at the C termini of its two identical heavy chains. Thus, each
fully assembled hIgG unit presents two sites for conjugation.
As shown in Figure 3B, the reactions of DIBAC-functional-
ized hIgG with azide-functionalized hGH or MBP produced
two species with higher molecular weights in a nonreducing Experimental Section
General protein conjugation: A buffered solution (optimal pH 4.5) of
gel, which we attribute to the formation of mono and
diconjugated proteins. Further confirmation of the product
identities was obtained by immunoblot probing for hGH,
MBP, and hIgG. Under reducing conditions, we detected the
protein-conjugated hIgG heavy chain (Figure S10 in the
Supporting Information). Over 70% of hIgG was conjugated
(over two steps; oxime formation and cycloaddition) accord-
ing to densitometry analysis.
aldehyde-tagged protein (10–50 mm) was treated with aminooxy
reagent (0.2–1 mm, 10–20 equiv) and agitated at 358C for 16 h.
Proteins were purified from low molecular weight reagents by buffer
exchange or analyzed directly by SDS-PAGE. Subsequent Cu-free
azide-alkyne cycloaddition reactions were conducted at 378C for 1 h
or at 48C for 16 h in the case of protein–protein conjugations.
Received: November 18, 2011
Published online: March 12, 2012
The generality of this approach to antibody–protein
conjugation was assessed by generating similar fusions with
a human antibody against the HER2/neu receptor, a common
breast and ovarian cancer marker and target of the clinically
approved antibody drug Herceptin.[44] The anti-HER2/neu
antibody was tagged with the aldehyde tag at the C terminus
then conjugated to hGH and MBP by using the same protocol
described for hIgG. We confirmed that the antibody–protein
chemical conjugates retained antigen binding activity by using
cell-based assays. The HER2-overexpressing cell line SKOV3
was incubated with the antibody–protein conjugates and
analyzed by flow cytometry staining with anti-hGH, anti-
MBP, and anti-hIgG antibodies. As shown in Figure 3C and
Figure S11 in the Supporting Information, the chemically
conjugated antibody fully retained its ability to bind its target
on SKOV3 cells and delivered its associated hGH or MBP
domain to the cell surface. Importantly, the low-pH con-
ditions of the initial oxime-forming reaction did not appear to
impact antigen binding. No labelling was detected for azide-
modified hGH-Az/MBP-Az alone or on Jurkat T cells, which
do not express HER2.
As further proof of the structure of the conjugates, we
performed a TEM analysis of the MBP-hIgG conjugate by
using negative staining as well as single-particle alignment
and classification. The resulting averaged 2D densities show
characteristic three-lobed views of the IgG[45] and a clear
additional density that is comparable in size with one or two
molecules of MBP at the end of one of the lobes, which is
consistent with a C-terminal attachment. This was verified by
2D docking of IgG and MBP crystal structures to some of the
class averages, as illustrated in Figure 3D.
Keywords: antibodies · bioorganic chemistry · click chemistry ·
oximes · proteins
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