Site-specific protein modifications through pyrroline-carboxy-lysine residues

Article abstract of DOI:10.1073/pnas.1105197108

Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol) s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.

Full text of DOI:10.1073/pnas.1105197108

Site-specific protein modifications through
pyrroline-carboxy-lysine residues
Weijia Ou1, Tetsuo Uno1, Hsien-Po Chiu, Jan Grünewald, Susan E. Cellitti, Tiffany Crossgrove, Xueshi Hao,
Qian Fan, Lisa L. Quinn, Paula Patterson, Linda Okach, David H. Jones, Scott A. Lesley, Ansgar Brock, and
Bernhard H. Geierstanger2
Genomics Institute of the Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121-1125
Edited* by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved May 11, 2011 (received for review April 4, 2011)
Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine
that is generated by the pyrrolysine biosynthetic enzymes when
the growth media is supplemented with D-ornithine. Pcl is readily
incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase
pair into proteins expressed in Escherichia coli and in mammalian
cells. Here, we describe a broadly applicable conjugation chemistry
that is specific for Pcl and orthogonal to all other reactive groups
on proteins. The reaction of Pcl with 2-amino-benzaldehyde or
2-amino-acetophenone reagents proceeds to near completion at
neutral pH with high efficiency. We illustrate the versatility of
the chemistry by conjugating Pcl proteins with poly(ethylene gly-
col)s, peptides, oligosaccharides, oligonucleotides, fluorescence,
and biotin labels and other small molecules. Because Pcl is geneti-
cally encoded by TAG codons, this conjugation chemistry enables
enhancements of the pharmacology and functionality of proteins
through site-specific conjugation.
acids will face similar hurdles to achieve site-specificity without
deleterious effects to the protein of interest.
The most elegant way to generate homogenously, site-specifi-
cally modified proteins is the in vivo incorporation of unnatural
amino acids (7–9). Over 70 unnatural amino acids featuring a
wide array of functionalities can be incorporated at TAG codons
using specific tRNA/tRNA synthetase pairs engineered through
an in vivo selection process to be orthogonal to the cellular
machinery of the host cells. A set of reactive unnatural amino
acids carrying keto (10), azido (11), and alkyne (12) moieties are
most frequently used for bioconjugation through oxime forma-
tion and “click-chemistry.” Although oxime formation requires
acidic pH and some click-chemistry reactions require a copper
catalyst for efficient conjugation, unnatural amino acid technol-
ogy provides unprecedented opportunities for protein labeling
and conjugation through chemical functionalities that are not
available among the common 20 amino acids.
protein engineering ∣ protein medicinal chemistry ∣ desmethylpyrrolysine ∣
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, was
discovered in several methyltransferases in methanogenic
archaea (Fig. 1) (Selenocysteine being the 21st amino acid)
(13–17). In Methanosarcinae, the TAG codon, a canonical termi-
nation signal, is primarily repurposed to signal Pyl incorporation
(18). Two of the three Pyl biosynthetic genes, pylC and pylD, along
with the pyrrolysl-tRNA/tRNA synthetase pair can be transferred
to Escherichia coli (19) as well as mammalian cells (20), and
facilitate the incorporation of pyrroline-carboxy-lysine (Pcl)
rather than Pyl when D-ornithine (D-Orn) is added to the growth
media (Fig. 1) (20, 21). Here we show that Pcl can be incorpo-
rated into a broad range of target proteins at TAG codons in both
E. coli and mammalian cells, and we report a specific conjugation
chemistry that enables coupling of synthetic compounds to Pcl in
recombinant proteins. This combination provides a powerful
platform for site-specific modification of proteins.
amber suppression
ust as small molecule drug candidates are optimized through
J
medicinal chemistry, protein biotherapeutics need to be opti-
mized in terms of their pharmacological and physiochemical
properties, selectivity, and immunogenicity (1–3). Such protein
medicinal chemistry efforts may, for example, involve choosing
an appropriate expression system to achieve a particular glycosy-
lation state of the protein (2). Changes in the primary protein
sequence through recombinant DNA technology can enhance
solubility and stability or alter immunogenicity and selectivity
of the protein drug. Only a few of the canonical 20 amino acids
are commonly used to modify the properties of proteins through
chemical derivatization. Conjugation of poly(ethylene glycol)s
(PEGs) for serum half-life extension, chemical haptenation to
increase the immunogenicity of antigens, and other conjugations
to effect the physicochemical and pharmacological properties of
proteins drugs and vaccines are primarily performed through
lysines, cysteines, and the N terminus (4). Amine reactive re-
agents are broadly available, but conjugation results in heteroge-
nous mixtures with often substantially reduced activity relative to
the unmodified protein. Homogenous products can be produced
by optimizing reaction conditions such that only the N terminus is
derivatized, but accessibility and effects on activity may limit this
site-specific modification approach. Alternatively, a solvent ex-
posed cysteine can be introduced through mutagenesis, but native
cysteines may need to be mutated to achieve derivatization at a
single desired site. Often, this type of mutagenesis is not a viable
option because additional cysteine residues can interfere with
protein folding, causing aggregation or dimerization, and in pro-
teins expressed in mammalian cells, often form cysteine or glu-
tathione adducts that need to be disrupted through reduction
prior to conjugation with thiol-specific reagents (5). Site-specific
bioorthogonal chemical or enzymatic derivatization can also
be accomplished through a number of peptide tags (4, 6) but
requires adding a stretch of nonendogenous sequence to the pro-
tein of interest. Any chemistry utilizing the standard 20 amino
Results
Pcl Protein Production in E. coli and Mammalian Cells. Pyl-containing
proteins can only be produced in small amounts from E. coli.
Pcl incorporation at a single site was, however, supported at
high yields in E. coli and mammalian cells when 1–5 mM
D-Orn was added to the growth media (20). So far, more than
350 Pcl mutants of over 30 different proteins have been produced,
Author contributions: W.O., T.U., S.E.C., S.A.L., A.B., and B.H.G. designed research; W.O.,
T.U., H.-P.C., J.G., S.E.C., T.C., X.H., Q.F., L.L.Q., P.P., L.O., D.H.J., A.B., and B.H.G. performed
research; W.O., T.U., H.-P.C., J.G., S.E.C., X.H., and A.B. contributed new reagents/analytic
tools; W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., S.A.L., A.B., and B.H.G. analyzed data;
and W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., A.B., and B.H.G. wrote the paper.
Conflict of interest statement:
A patent application regarding the biosynthesis of
pyrroline-carboxy-lysine and site-specific protein modifications via chemical derivatization
of pyrroline-carboxy-lysine and pyrrolysine residues has been filed.
*This Direct Submission article had a prearranged editor.
1W.O. and T.U. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: bgeierst@gnf.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1105197108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1105197108
PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10437–10442

O
strated by the improved yield of four hFAS-TE mutants with
additional nucleotide changes at the þ1 codon (Fig. 2B). Incor-
poration efficiency cannot be predicted at this time but because
of the ease of expression, many sites can quickly be scanned for
each new application. Our observations for Pcl are consistent
with reports (18, 24, 25) that Pyl, unlike selenocysteine, incor-
poration does not require a conserved mRNA element (22, 23).
Expression of Pcl proteins has so far been tested in shake flask
culture, small bioreactors and wavebags™ and feasibility of large
scale production needs to be evaluated in the future.
NH
H2N
O
D-Orn
into media
Pyl
OH
N
Pyl Biosynthetic Genes
Pcl
NH
H
N
O
O
2
O
NH
2
H2N
O
PylC
PylD
H
N
H N
2
2
O
+ L-Lys
N
N
NH
2
OH
N
H
H
O
OH
NH
NH
2
HO
2
HO
O
O
+ PylT tRNA
PylS RS
Pcl
Nascent
protein
Pcl
Pcl-Specific Bioorthogonal Conjugation Chemistry. It has been sug-
EF-TU
mRNA
gested previously that Pyl is chemically reactive (26). Previously,
AUC
UAG
1
2-amino-benzaldehyde (2-ABA) was used to derivatize Δ -pyrro-
line-5-carboxylate, a molecule analogous to the pyrroline ring of
Pyl, in order to study its role in the biosynthesis of proline (27).
Here, we show that Pcl (and Pyl) incorporated into proteins can
with high efficiency and specificity be derivatized with 2-ABA or
2-amino-acetophenone (2-AAP) moieties (Fig. 3A, SI Appendix).
The reactions proceeded to near completion at pH 5 and pH 7.5
at molar ratios of 6∶1 of 2-ABA or 2-AAP using 17 μM Pcl
protein (Fig. 3 B and C). Conjugation of 2-ABA to the Pcl residue
was confirmed by mass spectrometric (MS) analysis (SI Appendix:
Fig. S1). Additional conjugation events were only observed at very
high 2-ABA concentration (>80 mM), both for the Pcl protein
(Fig. 3D) and also for a control protein that did not contain
Pcl (Fig. 3E). These data suggest that other reactive groups of the
protein can only be modified at extreme reactant concentrations.
The 2-ABA/2-AAP reaction with Pcl appears to be highly chemo-
selective as none of the 12 lysines, six cysteines (in three disulfide
bonds), or eight histidines of hRBP4 used as test case in Fig. 3
were derivatized.
Site-specific conjugation of Pcl proteins also proceeded with
high efficiency with derivatized 2-AAP or 2-ABA reagents
(Table 1). For example, near complete conjugation to hRBP4
Phe122Pcl protein (4.3 μM or 0.9 μM) occurred for a short
PEG (2-AAP-PEG8 at 9.1 mM) at pH 7.5 (Fig. 3F) and pH 5.0
(Fig. 3G) while wild-type protein showed no indication of any
reaction (Fig. 3 H and I). Pyl and Pcl had similar reactivity as
was demonstrated by the derivatization of a mixture of Pyl- and
Pcl-containing mEGF protein with TU3627-014 (Fig. 3 A, J, and
K). The reactions of 2-ABA and 2-AAP reagents proceeded with
high efficiency at 4 °C, room temperature and 37 °C, at low pro-
tein (>1 μM) and reagent (typically <1 mM) concentrations and
reached near completion after 16 h at neutral pH (Table 1).
AUC
Ribosome
E. coli or mammalian cell
Fig. 1. Biosynthetic generation and incorporation of Pcl into proteins
produced in E. coli or mammalian cells using the Pyl machinery from Metha-
nosarcinae maize and D-Orn added to the media (20).
including enzymes, membrane receptors, growth factors, Fc-
fusion proteins, antibodies, and antibody fragments (Fig. 2, SI
Appendix). Expression yields in E. coli were typically 25–40%
but some reached 80% of wild-type protein using a two-plasmid
expression system (Fig. 2 A and B). Transient cotransfection of
HEK293 cells with the Pyl machinery regularly produced Pcl
protein at 10–20% of the corresponding wild-type proteins with
the best yield reaching ∼65% (Fig. 2 C and D). As with any site-
directed mutagenesis, yields for a mutant protein may be altered
by effects on folding, stability, and solubility. Lower than wild-
type yields are expected due to competition between Pcl-charged
tRNA and release factor at the TAG codon. In fact, truncated
protein products consistent with translation termination were
generally observed but were easily separated during purification,
for example with the use of a purification tag at the C terminus
of the protein of interest. There are likely some contextual effects
on the incorporation efficiency in the DNA or mRNA as illu-
mTNF-α
WT T2450 WT Q21
FKBP
WT
I90
mEGF
hFAS-TE
B
A
hFAS-TE
250
200
150
100
50
WT Y10
-
-
+
-
-
+
-
-
+
-
-
+
D-Orn
0
Structure and Stability of Pcl Conjugates. Nuclear magnetic reso-
nance (NMR) spectroscopy studies of the Pcl amino acid reacted
with 2-ABA or 2-AAP suggested the structure of the Pcl protein
adducts shown in Fig. 4A. The reaction of 2-ABA with Pcl, but
not with the alternative imine isomer, rapidly generated a minor
and a major product and proceeded to completion (SI Appendix:
Fig. S2). A detailed analysis of the purified adduct by 1H-13C
heteronuclear multiple bond correlation (HMBC) NMR (Fig. 4B,
SI Appendix: Fig. S3) suggested that the formation of a pyrrolo-
quinazoline ring was stabilized through cyclization of the 2-
ABA/2-AAP carbonyl moiety with the ϵ-amide of Pcl (for NMR
data of the 2-AAP adduct, see SI Appendix: Fig. S4 and S5).
The formation of the Pcl protein adduct was reversible (SI
Appendix: Fig. S6); the stability depended on the chemical struc-
ture of the reactant (SI Appendix: Fig. S7). For 2-AAP and its
derivatives, 50% of the conjugate reverted to unmodified Pcl
protein in 4 to 10 h (at 37 °C, pH 7.4). For derivatized forms
of 2-ABA, decay to 50% conjugate occurred over 20 to 30 h. MS
analysis and repeated reactions demonstrated regeneration of
the Pcl side chain by reversion of the conjugation by simple hydro-
lysis rather than modification to a new structure. The Pcl-2-
ABA (or 2-AAP) linkage was further stabilized by a simple and
mEPO
mIgG1 Fc
D
C
4
3
2
1
0
D-Orn:
-
-
+
+
+
+
+
+
+
+
+
+
+
Fig. 2. Incorporation of Pcl into proteins produced in E. coli or mammalian
cells (20). (A) Examples of single-site Pcl incorporation into hFAS-TE, mTNF-α,
FKBP, and mEGF using E. coli as expression host. Wild-type (WT) and Pcl
proteins from equal volume cultures grown with (þ) and without (−) D-Orn
were compared. (B) hFAS-TE Pcl protein yields as a function of incorporation
site. For some sites, yields were increased by changing the first nucleotide
after the TAG codon resulting in a silent or conservative mutation. Pcl is
abbreviated as Z. Examples of single-site Pcl incorporation into mouse EPO
(C) and mIgG1 Fc domain constructs (D) using transient transfection and
HEK293 cells. Protein yields varied with incorporation site relative to WT
protein. Pcl incorporation was verified by mass spectrometry. For additional
information, see SI Appendix.
10438
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www.pnas.org/cgi/doi/10.1073/pnas.1105197108
Ou et al.

100 B
100
23270.0
23283.6
in E. coli and mammalian cells, make Pcl a versatile and broadly
applicable protein modification platform. To illustrate this versa-
tility, we synthesized and successfully conjugated over 50 differ-
ent Pcl reactive reagents ranging from low molecular weight
molecules for fluorescent labeling to PEG polymers (Table 1,
Fig. 5, SI Appendix: Figs. S8–S14).
C
A
O
NH
2
O
NH
2
23166.8
23167.2
2-ABA
2-AAP
23290.8
23400
23364.0
23400
mass
24200
mass
0
0
NH
2
O
23000
23800
24200
23000
23800
100
100
H
N
23120.8
23476.0
23682.8
O
Site-specific PEGylation is a preferred method to extend the
serum half-life of biotherapeutic proteins as it preserves activity
and generates a homogeneous product (3, 28). We synthesized
2-AAP and 2-ABA activated PEGs of 0.5, 2.4, 5, 10, 20, 30, and
40 kDa molecular mass and coupled them to Pcl proteins (Fig. 5,
SI Appendix). Coupling of 2-ABA-activated PEGs (Fig. 5A) was
more efficient and resulted in more stable adducts than coupling
with 2-AAP-PEGs. In fact, 2-AAP-PEG/Pcl adducts required
NaBH3CN reduction for effective purification (Fig. 5B). The
reduced conjugates appeared as single species on SDS-PAGE
(Fig. 5). Coupling efficiencies varied with length of the PEG from
>95% for short PEGs (Fig. 3 F and G) to typically 50% for 30
and 40 kDa PEGs (Fig. 5 A and B). In one instance, coupling
of a 40 kDa PEG to a hIgG4 Pcl mutant proceeded nearly to
completion (Fig. 5C), suggesting that PEGylation efficiency is
not limited by the conjugation chemistry but rather by accessibility
of the Pcl side chain for reactions with large and flexible PEGs.
This example further illustrated the high specificity of the PEG
attachment to the Pcl residue in the IgG4 heavy chain as only
the heavy chain was shifted on a reducing gel (Fig. 5D), highlight-
ing the homogeneity of the PEGylated protein product.
Another method to modify the pharmacology of protein drugs
is glycoslyation. This posttranslational modification occurs natu-
rally in proteins expressed in eukaryotic cells but usually not in
E. coli. Glycosylated proteins typically feature a mixture of com-
plex oligosaccharide structures attached at specific serines and
threonines (O-linked glycosylation) or asparagines (N-linked gly-
cosylation). To explore the feasibility of chemically conjugating
defined sugars to proteins expressed in E. coli, we activated a
disaccharide and linked it to a 2-ABA moiety (SI Appendix:
Fig. S8). Subsequent coupling of the sugar to a Pcl protein
proceeded to completion as monitored by MS.
The conjugation of a phospolipid, to be potentially used as
membrane anchor, was also successfully tested (SI Appendix:
Fig. S9). In addition, a biotin analog (SI Appendix: Fig. S10) and
a fluorescent dye (SI Appendix: Fig. S11) were conjugated nearly
to completion using 0.1 to 1 mM 2-ABA reagent and 10 μM
protein, illustrating the potential of Pcl conjugation for standard
protein labeling and detection strategies.
Chemical haptenation of protein antigens, for example with
nitro-phenol reagents, has long been used for the efficient gen-
eration of antibodies (29, 30). Similarly, the immunogenicity of
vaccine antigens can be enhanced by adjuvants such as aluminum
oxide particles and by adding immune stimulators such as toll-like
receptor (TLR) agonists to these formulations (31). As colocali-
zation of such reagents with the antigen may boost efficacy (32),
we tested whether several immune stimulatory entities can be
conjugated to proteins. Indeed, site-specific coupling of 2-ABA
nitro-phenyl haptens to model antigens proceeded to >93%
(SI Appendix: Fig. S12) while the conjugation of two CpG oligo-
phosphothioate TLR9 agonists (33) (SI Appendix: Fig. S13) and
a T-cell epitope peptide (34) (SI Appendix: Fig. S14) reached
approximately 80% completion.
O
O
D
E
23327.2
23532.8
7
O
23270.0
2-AAP-PEG8
NH
2
O
23889.6
23168.8
O
O
23737.2
23800
O
TU3627-014
23724.2
mass
mass
24200
0
0
23000
23400
23800
23724.2
24200
23000
23400
100
100
100
7295.2
G
F
J
7309.6
7688.4
7666.0
23167.6
23167.8
mass
mass
mass
7200 7300 7400 7500 7600 7700
100
0
0
0
23000
23400
23800
24200
23000
23400
23800
24200
100
100
7500.4
23091.0
23091.8
I
H
K
7515.2
7520.0
7543.6
7265.2
7273.6
mass
7200 7300 7400 7500 7600 7700
mass
mass
0
0
0
23000
23400
23800
24200
23000
23400
23800
24200
Fig. 3. Specific reactions of Pcl- and Pyl-containing proteins with 2-ABA and
2-AAP reagents (A). Shown are intact LC-ESI mass spectra of reaction mix-
tures. Unmodified proteins are highlighted with stars. 2-ABA (B) and
2-AAP (C) at 6∶1 reactant to protein formed nearly complete adducts
[17 μM hRBP4 Phe122Pcl; molecular mass unreacted: 23167.2 Da and
23166.8 Da observed (obs.), 23167.7 Da calculated (cal).; reacted with
0.1 mM 2-ABA (B): 23270.0 Da obs., 23270.8 Da cal., reacted with 0.1 mM
2-AAP (C): 23283.6 Da obs., 23284.8 Da cal., 12 h, 22 °C, pH 5.0)]. At very high
concentrations, 2-ABA (or 2-ABA dimers, tetramers and hexamer) formed
additional adducts with the Pcl protein (D) (17 μM hRBP4 Phe122Pcl,
80 mM 2-ABA, 12 h, 22 °C, pH 7.5) but also with a control protein (E) that
did not contain any Pcl (100 mM 2-ABA, 15;400∶1 reactant to protein;
6.5 μM hRBP4 Phe62OMePhe unreacted: 23120.8 Da obs., 23,122 Da cal.,
100 mM 2-ABA, 12 h, 22 °C, pH 7.4). 2-AAP-PEG8 (at 9.1 mM) reacted with
hRBP4 Phe122Pcl to ∼94% at pH 7.5 (PBS; 2;100∶1 reactant to protein;
4.3 μM protein; unreacted: 23167.6 Da obs., 23167.7 cal.; reacted:
23724.2 Da obs., 23724.3 Da cal.) (F) and to ∼96% at pH 5.0 (200 mM sodium
acetate; 10;500∶1 reactant to protein; 0.86 μM protein; unreacted:
23167.8 Da obs., reacted: 23724.2 Da obs.) (G). Wild-type hRBP4 (23091.2
and 23092.0 Da obs., 23,092 Da cal.) did not react with 9.1 mM 2-AAP-
PEG8 at identical conditions (H, pH 7.5 and I, pH 5.0). Expression of mEGF
Tyr10TAG in E. coli in the presence of D-Orn and biosynthetic genes pylB, pylC
and pylD resulted in a mixture of Pcl and Pyl protein (J). When reacted
with 1 mM 2-AAP-derivative TU3627-014 (0.01 mM protein) for 16 h at
22 °C in 10× PBS, pH 7.4, both Pcl and Pyl protein reacted to near completion
and to similar extents (K) (mEGF Tyr10Pcl unreacted: 7295.2 Da obs.,
7,296 cal.; reacted: 7500.4 Da obs., 7,501 Da cal.; mEGF Tyr10Pyl unreacted:
7309.6 Da obs., 7,310 Da cal; reacted: 7515.2 Da obs., 7,515 Da cal.). See SI
Appendix for additional information.
quantitative reduction step with sodium cyanoborohydride
(Fig. 4A and Fig. 5 B and C), a reaction that is commonly used
during the N-terminal PEGylation of proteins (28). The structure
of this hydrolytically stable form was determined by NMR for
2-ABA and Pcl (Fig. 4C, SI Appendix: Fig. S3). For the respective
Pcl protein adduct, no degradation was observed over a period
of 96 h at 22 °C and pH 7.4 (SI Appendix: Fig. S7). The ability
to fine tune the stability of the coupling by the choice of reactive
group and linker provides the opportunity to design releasable
conjugates, for example for antibody drug conjugates. Alterna-
tively, reduction results in a hydrolytically stable linkage that can-
not be destroyed by boiling in SDS gel buffer (Fig. 5 A and B).
Although we have not fully evaluated the utility of all these
modifications, the examples never-the-less established the chem-
istry to produce site-specific PEG, oligosaccharide, small mole-
cule, oligonucleotide, and peptide-protein conjugates through
the Pcl amino acid. In general, the reactions of all reagents with
MW <2;000 Da proceeded to >95% completion after 16 h
(Table 1) in a pH and temperature range where most proteins are
stable as long as the solubility of the reagents was not limiting. As
with all biopharmaceuticals, the immunogenicity of each protein
Versatility of the Pcl Conjugation Chemistry and Applications. The
stability, specificity, and efficiency of the coupling chemistry com-
bined with the ease of Pcl incorporation into proteins expressed
Ou et al.
PNAS
∣
June 28, 2011
∣
vol. 108
∣
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10439

Table 1. Functionalization of Pcl proteins
Reagent
Structure(s)
Coupling [%]
Fig.
3, 5
O
NH2
O
NH2
PEGs (linear and branched)
50 to >95
H
N
H
N
O
O
O
O
O
O
n
n
O
O
OH OH
H2N
O
Disaccharide
>98
>94
SI Appendix: Fig. S8
SI Appendix: Fig. S9
OH
O
OH
H
O
HO
N
HO
OH
O
OH
O
O
DOPE phospholipid
H2N
O
O
O
H
N
O
O P O
OH
O
O
O
O
Biotin
>98
>88
SI Appendix: Fig. S10
SI Appendix: Fig. S11
O
O
HN
H2N
O
O
NH
N
H
N
O
O
H
O
S
O
Fluorescein
O
O
NH2
NH2
OH
H
H
COOH
N
O
N
O
O
O
O
O
O
O NH2
Nitro-phenyl haptens
>93
SI Appendix: Fig. S12
NO2
O2N
NO2
H
N
H
N
O
O
N
H
O
O
O
NH2
CpG- phosphothioate
∼80
>83
SI Appendix: Fig. S13
SI Appendix: Fig. S14
H
N
O
O
tcgtcgttttcggcgcgcgccg
O
O
NH2
PADRE peptide (T-cell epitope)
G(D-A)K(cyclohexyl-A) VAAWTLKA(D-A)G
O
Pcl reagents tested and coupling efficiencies at pH 7.4 and 22 °C. For details and results, see Fig. 3, Fig. 5, and SI Appendix: Figs. S8–S14 as indicated
drug containing unmodified or modified Pcl residues will need
to be assessed thoroughly during clinical development. Several
applications of the technology are being pursued currently.
Preliminary results from low oxygen fermentations in E. coli sug-
gest that Pyl incorporation is intrinsically more efficient than Pcl
(20). However, under standard expression conditions, the activity
of PylB, the enzyme responsible for the methylation of the pyr-
rolysine precursor, or a required cofactor appears to limit the
production of Pyl proteins in mammalian and E. coli cells (20).
The pyrrolysyl-tRNA/tRNA synthetase pair has naturally
evolved in Methanosarcinae to support amber suppression and
to be orthogonal to other endogenous pairs in those species as
well as in mammalian cells (35), Saccharomyces cerevisiae (35)
and E. coli (19). This history of evolutionary pressure along
with the observation that even the unmodified pyrrolysyl-tRNA
synthetase PylS is relatively tolerant of nonpyrrolysine substrates
[but generally utilizes them with lower efficiency (35–42)], makes
it an attractive system for the incorporation of unnatural amino
acids either using wild-type (35, 38–42) or mutant pyrrolysyl-
tRNA synthetases (35, 39, 43–46). Pcl mutagenesis differs from
the unnatural amino acid technology in that a standard metabo-
lite, D-Orn, is processed by the biosynthesis enzymes of a natural
amino acid into a close analog of Pyl. The close structural
similarity of Pcl to Pyl likely explains the high efficiency of Pcl
incorporation using the unmodified pyrrolysyl-tRNA synthetase.
In nature, the occurrence of Pyl is limited to a specific position in
the active site of methylamine methyltransferases in certain or-
ganisms (47). Despite this fact, Pcl can be incorporated with high
efficiency and good yields at many different positions in a large
number of proteins (Fig. 2).
Discussion
Strategies for protein modification are indispensible for convert-
ing a protein into a research compound or into a pharmaceutical
agent. Site-specific protein modification methods are most
powerful as they allow the protein designer to choose sites were
modifications do not affect the protein’s natural function or do so
in a controlled manner. Incorporation of unnatural amino acids is
currently the most versatile way to accomplish this task (7–9). The
ability to introduce new chemical functionalities through syn-
thetic unnatural amino acids provides tools nearly on par with
what traditional medicinal chemistry can do for low molecular
weight compounds. Unnatural amino acids can themselves con-
stitute the desired modification (e.g., mimics of posttranslational
modifications, spectroscopic probes) or alternatively can be func-
tionalized after protein incorporation through chemical derivati-
zation (8, 9). Here, we show that the discovery of a bioorthogonal
conjugation chemistry for Pcl, a biosynthetically generated Pyl
analog, provides a competitive method for protein medicinal
chemistry and labeling through site-specific protein conjugation.
Pyl was discovered about ten years ago as the 22nd genetically
encoded proteinogenic amino acid (13–16). Unexpectedly, we
discovered that addition of D-Orn to growth media in the pre-
sence of the Pyl biosynthesis genes results in the generation
and incorporation of a demethylated Pyl analog, Pcl (20, 21)
(Fig. 1) rather than Pyl as had been suggested previously (24).
D-Orn appears to feed into the Pyl biosynthetic pathway down-
stream of PylB, as Pcl incorporation requires only PylC and PylD.
In addition, Pcl proteins can site-specifically be derivatized
with 2-ABA and 2-AAP reagents including PEGs, sugars,
peptides, and oligonucleotides (Table 1, Fig. 3, 5). Small mole-
cules especially can be coupled with high efficiency, making
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A
B
Fig. 4. Site-specific modification of Pcl proteins
through 2-ABA (R1 ¼ H) and 2-AAP (R1 ¼ CH3)
derivatives. Examples of derivatives are shown in
Table 1. (A) Structures of the protein adduct and
the hydrolytically stable form after reduction as in-
ferred from NMR studies with reacted Pcl amino
acid. Summary of the key through bond connectiv-
ities detected by 1H-13C-HMBC NMR spectroscopy
for the 2-ABA/Pcl amino acid adduct before
(B) and after reduction (C). The first number of
the crosspeak label identifies the 1H resonance
while the second number identifies the 13C reso-
nance of the respective atom or group. For num-
bering and structure, see inserts. Additional
information, discussion, and NMR data for the
2-AAP adduct are provided as SI Appendix. Pcl
was synthesized as a diastereomeric mixture (20).
The stereochemistry of Pcl in the protein is drawn
as observed for Pyl in proteins (26).
C
SI Appendix: Table S1. FAS-TE constructs with a purine at the þ1 codon posi-
tion after TAG (CTG2223ATT, TCC2307AGC, CGG2351AGG, and CAG2373AAT)
were constructed to reduce termination efficiency (49). hFAS-TE was
expressed in HK100 cells (20); mTNF-α, FKBP, and mEGF were expressed in
BL21(DE3) cells (Invitrogen) grown in terrific broth media (Sigma) at 37 °C
supplemented with 5 mM D-Orn. Proteins were purified using Ni-NTA
(Qiagen) chromatography and yields are listed in SI Appendix: Table S2.
Pcl incorporation into proteins in HEK293F cells was accomplished by transi-
ent transfection (20). TAG codons were introduced into pRS expression vec-
tors for human retinol binding protein (hRBP4), mouse erythropoietin
(mEPO), two mIgG1 Fc constructs (Fc1, Fc2), and a human IgG4 antibody
(hIgG4) (SI Appendix: Table S3). HEK293F cells were grown in suspension
in 293 Freestyle expression media. 5 mM D-Orn was added after transfection.
Proteins were purified using Ni-NTA or protein A-affinity chromatography.
Pcl incorporation was verified by LC-MS. See SI Appendix for additional
details.
site-specific conjugation through Pcl a viable option for the
development of antibody drug conjugates and imaging reagents
as well as many research reagents. Key advantages of the Pcl pro-
tein modification platform are universal and robust protein
expression and site-specific incorporation of the reactive amino
acid in E. coli and mammalian cells with the same cellular
machinery that evolved naturally. In contrast to most unnatural
amino acids that need to be chemically synthesized, Pcl is gener-
ated biosynthetically in the host cell from a readily available
media supplement, D-Orn. Most importantly, these features
are combined with an efficient, Pcl-specific conjugation chemistry
that works for many applications near neutral pH.
Methods
Reagents. 2-AAP, 2-ABA, and NaBH3CN were purchased from Sigma while
D-Orn was purchased for Chem-Impex. Pcl ((2S)-2-amino-6-(3,4-dihydro-2H-
pyrrole-2-carboxamido)hexanoic acid) and its isomer (S)-2-amino-6-(3,4-dihy-
dro-2H-pyrrole-5-carboxamido)hexanoic acid were synthesized as described
(20). The syntheses of all Pcl reactive reagents are described in SI Appendix.
Site-Specific Derivatization of Pcl and Pyl Proteins (Fig. 3). Ten microliter hRBP4
Phe122Pcl protein solutions were mixed with 89 μL 200 mM sodium acetate
buffer (pH 5.0) and 1 μL 10 mM 2-ABA (Fig. 3B) or 2-AAP (Fig. 3C) solution,
with 80 μL 10× PBS and 10 μL 0.8 M 2-ABA solution (Fig. 3D), with 10 μL 10×
PBS and 2 μL 100 mM 2-AAP-PEG8 (TU3205-044) solution (Fig. 3F), or with
90 μL 200 mM sodium acetate buffer and 10 μL 100 mM 2-AAP-PEG8 (Fig. 3G).
A 10 μL sample of a hRBP4 O-methyl-phenylalanine (OMePhe) mutant (50)
was mixed with 80 μL 10× PBS and 10 μL 1 M 2-ABA (Fig. 3E). The reactions
proceeded 14 h at 22 °C followed by 72 h at 4 °C before MS. Reactions with
wild-type hRBP4 protein at concentrations of 20 μM (Fig. 3H) and 4 μM (Fig. 3I)
Production of Pcl Proteins (Fig. 2). E. coli cells were cotransformed with pAra-
pylSTCD and an expression plasmid for the protein of interest as described
(20). The thioesterase domain of human fatty acid synthase (hFAS-TE) was
expressed using
a pMH4 plasmid. Mouse tumor necrosis factor alpha
(mTNF-α), human FK506-binding protein 1A (FKBP) and mouse epidermal
growth factor (mEGF) were cloned into pET vectors using PIPE (48). TAG
codons for single-site Pcl incorporation were introduced as shown in
hFAS-TE Thr2450Pcl
hFAS-TE Thr2450Pcl
IgG4 Pcl
IgG4 Pcl
B
C
D
A
2-ABA-PEG
Reduction
Boiled
+
+
-
+
+
-
+
+
+
-
-
2-AAP-PEG
Reduction
Boiled
+
+
-
+
+
-
+
+
+
-
-
-
-
2-ABA-PEG:
2-ABA-PEG:
-
-
+
+
-
-
+
+
+
+
HC-
PEG
PEG-
hFAS-TE
IgG4-
PEG
175
IgG4
HC
LC
80
58
46
30
hFAS-TE
25
17
Fig. 5. Site-specific PEGylation of Pcl proteins. SDS-PAGE of reaction mixtures of hFAS-TE Pcl protein with 30 kDa-linear 2-ABA-PEG (A) and 2-AAP-PEG
(B). hFAS-TE Thr2450Pcl (0.2 mM) was incubated with 1 mM PEG for 60 h at 4 °C in PBS (pH 7.4). Mixtures were either reduced with 50 mM NaBH3CN for
1 h at 37 °C or left untreated. All samples were passed over NAP5 size exclusion columns; some samples were boiled at 95 °C for 5 min prior to loading onto
the gel. (C) SDS-PAGE (nonreducing gel) of the reaction mixture of a hIgG4 Pcl mutant (0.01 mM) with a 40 kDa-linear 2-ABA-PEG (0.1 mM) detected near
quantitative conjugation after 24 h at 22 °C in 200 mM Na acetate buffer (pH 5.0). (D) SDS-PAGE (reducing gel) of the material after reduction (20 mM
NaBH3CN, 4 h, 22 °C) showed PEGylation of the IgG4 heavy chain only. See SI Appendix for additional information.
Ou et al.
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were performed in parallel. A mEGF Tyr10Pcl/Pyl protein mixture (Fig. 3J) was
reacted similarly with 1 mM of TU3627-014 (Fig. 3K).
sample until the adduct resonance at 5.6 ppm (SI Appendix: Fig. S2) comple-
tely disappeared. The sample was repeatedly lyophilized from dry D2O and
reconstituted in 0.5 mL D2O. The 1H-13C HMBC spectra of the reduced 2-ABA/
Pcl adduct (Fig. 4C) was recorded on a 600 MHz Bruker Avance instrument
with a delay of 50 ms. Signal assignments and through-bond correlations
for nonreduced and reduced 2-ABA/Pcl adducts are summarized in SI
Appendix: Fig. S3, Tables S7, S8, S9. Comparison with the 2-AAP adducts
of Pcl (SI Appendix: Fig. S4, Table S10) and of 3,4-dihydro-2H-pyrrole-2-
carboxylic acid (SI Appendix: Fig. S5, Tables S11, S12) support the adduct struc-
tures shown in Fig. 4A. A detailed discussion is provided as SI Appendix.
Protein and Peptide LC-MS and Verification of 2-ABA/Pcl Adduct Formation.
Intact protein MS was obtained on an automated QTOF II LC-MS system
(Waters). Nano-reverse-phase liquid chromatography (LC) MSMS of proteo-
lytic digests was performed using a LTQ Orbitrap hybrid mass spectrometer
(ThermoElectron). MS data were processed as described (20). LC-MS analysis
of the tryptic digest of the 2-ABA-derivatized hRBP4 Phe122Pcl protein
verified conjugation to the Pcl residue (SI Appendix: Fig. S1, Table S4).
NMR Characterization of Pcl Adducts (Fig. 4). Pcl and 2-ABA solutions in D2O
were mixed and reacted to near completion as monitored by NMR
(SI Appendix: Fig. S2, Tables S5, S6). The 2-ABA/Pcl adduct was purified by
HPLC, redisolved in d6-DMSO, and a 1H-13C HMBC spectrum (Fig. 4B) opti-
mized for the detection of three-bond-correlations using a 50 ms delay
(J ¼ 10 Hz) was acquired at 300 K on a 400 MHz Bruker Avance instrument
(Bruker Biospin). For the characterization of the reduced 2-ABA/Pcl adduct
(Fig. 4C), aliquots of a NaBH3CN solution in D2O were added to a second
Functionalization of Pcl Proteins (Table 1). Reactions were typically carried out
in 10× PBS at pH 7.4 and 22 °C using 10 μM Pcl protein and 100 μM 2-ABA/
2-AAP reagent. Preparation of reagents, reaction details, and detection of
the protein conjugates are given in SI Appendix: Figs. S8–S14.
ACKNOWLEDGMENTS. We thank Dr. Sarah Rue and Dr. Marc Nasoff for the
clone of the IgG4 antibody modified in this study.
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