Construction of a small-molecule-integrated semisynthetic split intein for in vivo protein ligation

Article abstract of DOI:10.1039/b712843f

A new split intein-based protein ligation tool that is synthetically accessible and can be used for protein semisynthesis on the cell surface and potentially inside cells has been constructed. The Royal Society of Chemistry.

Full text of DOI:10.1039/b712843f

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Construction of a small-molecule-integrated semisynthetic split intein
for in vivo protein ligation{
Tomomi Ando,a Shinya Tsukiji,a Tsutomu Tanakaa and Teruyuki Nagamune*abc
Received (in Cambridge, UK) 22nd August 2007, Accepted 17th September 2007
First published as an Advance Article on the web 10th October 2007
DOI: 10.1039/b712843f
limitation.5 We focused on the rational design of a new split intein
A new split intein-based protein ligation tool that is syntheti-
that overcomes these drawbacks. The Ssp DnaB intein was chosen
as our starting point, because it was recently found that the DnaB
intein split at residue 11, generating IN and IC, is capable of trans-
splicing in bacteria6 and also in vitro.7 The IN fragment contains
only 11 amino acids, so is suitable for preparation and further
modification by conventional solid-phase peptide synthesis (SPPS).
However, the in vitro study reported a reactant concentration,
required for trans-splicing of the pair of these IN and IC fragments,
of over 10 mM,7 which will restrict its broad use for cellular
applications. Consistent with this, we observed no trans-splicing of
the IN–IC pair under submicromolar concentrations (vide infra).
Therefore, inspired by previous two-8 and three-hybrid strategies,9
we aimed to confer high affinity to the complementary IN–IC pair
by integrating an auxiliary receptor–ligand interaction as outlined
in Scheme 1. Covalently fusing a small-molecule ligand and its
cognate receptor protein to the C-terminus of IN and the
N-terminus of IC, respectively, should generate a semisynthetic
peptide–protein trans-splicing system in which the short synthetic
cally accessible and can be used for protein semisynthesis on the
cell surface and potentially inside cells has been constructed.
The ability to incorporate site-specifically a range of chemical
probes such as fluorescent molecules, photocrosslinkers, unnatural
amino acids, and post-translational modifications into cellular
proteins will provide invaluable tools for the study of living
systems. Consequently, the development of strategies that enable
the specific modification of proteins in cellular contexts is an
increasingly active area of chemical biology. Although several
chemical labeling methods have been described to date, most of
them allow only limited types of modification at the side chain of
specific residue(s) within remote fusion protein-/peptide-tags.1 A
protein semisynthesis approach2 is a powerful alternative strategy.
The semisynthesis of proteins is based on the chemoselective
ligation of a chemically modified peptide to a recombinant protein,
thus enabling the precise incorporation of an unlimited variety of
chemical probes into the target protein.2 Despite such significant
potential, the utility of this approach is currently limited to in vitro
conditions due to the lack of protein ligation techniques which are
applicable in vivo.3,4 Here we report a new split intein-based
protein ligation method that can be used on the cell surface and
potentially inside cells.
IN fragment can heterodimerize with the recombinant IC efficiently
to reconstitute the splicing-active complex.
To test our design strategy, a series of model IN peptides and IC
proteins were prepared, in which the chemical probe, the
N-terminal extein, is a biotin tag, and the target protein, the
C-terminal extein, is the monomeric red fluorescent protein
(mRFP) (Fig. 1(A)). We used E. coli dihydrofolate reductase
(eDHFR) and trimethoprim (TMP) as the receptor–ligand pair,
because TMP has nanomolar affinity for eDHFR and can be
easily derivatized without substantial loss of its binding affinity.10
We synthesized a 49-carboxy-substituted TMP (Scheme S1, ESI{)
and attached it to the C-terminus of IN via a linker consisting a
tetramer of PEG4-based amino acid.11 The peptides were readily
Inteins are attractive scaffolds for the creation of in vivo protein
ligation tools, because they can split into two halves to mediate
protein splicing in trans.5 The trans-splicing process allows N- and
C-terminal extein polypeptides, either synthetic or recombinant in
origin, to be joined together by a native peptide bond with
concomitant removal of the intein complex.5 In pioneering work,
Giriat and Muir demonstrated the feasibility of in vivo protein
semisynthesis by using the naturally split Synechocystis sp. (Ssp)
DnaE intein.4 However, the general applicability of the Ssp DnaE
intein as well as other existing split inteins would be severely
limited by their moderate-to-large sizes (ca. 36–180 amino acids),
which make chemical synthesis of intein fragments extremely
difficult or impractical. In addition, for many trans-splicing inteins,
the intrinsic low affinity between the complementary fragments is a
aDepartment of Chemistry and Biotechnology, School of Engineering,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656,
Japan
bDepartment of Bioengineering, School of Engineering, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
E-mail: nagamune@bioeng.t.u-tokyo.ac.jp; Fax: +81-3-5841-8657;
Tel: +81-3-5841-7328
cCenter for NanoBio Integration, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo, 113-8656, Japan
{ Electronic supplementary information (ESI) available: Full experimental
procedures. See DOI: 10.1039/b712843f
Scheme 1 Schematic representation of the small-molecule-integrated
semisynthetic split intein.
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Fig. 2 Effects of linker length of TMP-integrated IN peptide on trans-
splicing efficiency. The reactions were performed between pairs of 0.5 mM
of IN peptide and IC-3 at 25 uC for 20 h and analyzed by Western blotting
using SAv-HRP.
We also investigated the effect of linker length of TMP-
integrated IN peptide on trans-splicing efficiency and found the
tetramer of PEG4-amino acid to be optimal among peptides we
tested (Fig. 2). It was clearly indicated that a linker of appropriate
length is essential for the reconstitution of splicing-active intein
complex in this system. Importantly, at a reaction concentration of
5 mM, the formation of P-6 could be detected using IN-1 and IC-3,
whereas the pair of IN-2 and IC-3 still showed a three-fold higher
trans-splicing efficiency by virtue of the auxiliary interaction
(Fig. S3, ESI{).
Fig. 1 In vitro trans-splicing assays. (A) The IN peptides and IC proteins
used in this study. The LRESG and SIEQD sequences in IN and IC
fragments are native exteins of Ssp DnaB intein. TMP is shown in pink.
(B) Protein products of the trans-splicing between IN-2 and IC-3. (C)
Western blotting analysis of P-6 formation using SAv-HRP. The reactions
were performed between pairs of 0.5 mM of IN peptide and IC protein at
25 uC for 20 h.
synthesized by standard SPPS and purified by reversed-phase
HPLC. Each of the protein constructs were bacterially expressed
and purified from the soluble fraction by affinity chromatography.
Initial trans-splicing assays were performed by incubating each
of the IN and the IC at an equimolar concentration of 0.5 mM and
analyzed by Western blotting using streptavidin–horseradish
peroxidase conjugate (SAv-HRP). While the incubation of IN-1
and IC-3 for 20 h resulted in no splicing (Fig. 1(C), lane 4), the
formation of splicing product P-6 was clearly observed in the
reaction of IN-2 and IC-3 (Fig. 1(C), lane 3). The product band
showed cross-reactivity to the anti-His-tag antibody as expected
(data not shown). The formation of splicing product P-6, excised
intein fragment P-7, and C-terminal cleavage product P-8 was
further confirmed by MALDI-TOF-MS analysis of the reaction
mixture.12 In contrast, no splicing was observed between IN-2 and
active site-mutated (S155A) IC-4 (Fig. 1(C), lane 6), which is
consistent with product formation occurring via protein trans-
splicing. The total yield of P-6 was estimated to be ca. 50%.13 This
value lies in the range reported for other split inteins.7,9 In
addition, kinetic experiments revealed that the trans-splicing
reaction follows first-order kinetics with a kobs of 1.1 6 1024 s21
(Fig. S1, ESI{).14 It should be noted that a significant amount of
splicing product P-6 could be detected even within 1 h (Fig. S1,
ESI{).
To demonstrate the in vivo applicability of this ligation tool, we
next performed the semisynthesis of a protein on the surface of
mammalian cells. We chose cell membrane-bound mRFP as the
model protein (Fig. 3(A)). Chinese hamster ovary (CHO) cells
Fig. 3 On-cell trans-splicing. (A) The IC constructs used in this study and
the splicing product. IgS is the Igk signal sequence and TMD is the
transmembrane domain of the platelet-derived growth factor receptor. (B)
Biotinylation of cell surface-displayed mRFP. CHO cells expressing IC-9
were treated with 0.5 mM of either IN-1 (top) or IN-2 (bottom) at 25 uC for
8 h. The biotinylation was detected with SAv-488. Confocal images show
Alexa Fluor 488 fluorescence (left) and overlays of mRFP fluorescence
and DIC images (right). (C) Western blotting analysis of the biotinylation
of mRFP-TMD (lane 1) using SAv-HRP. Negative controls are shown
with IN-1 (lane 2) or with cells expressing IC-10 (lanes 3 and 4).
To further confirm that the interaction between TMP ligand
and eDHFR plays a critical role in this peptide–protein trans-
splicing system, the reactions were carried out using IN-2 and IC-3
in the presence of a 20-fold molar excess of TMP, and using IN-2
and IC-5 lacking the eDHFR portion. In neither case was the
product formed (Fig. S2, ESI{ and Fig. 1(C), lane 5). These results
provide clear evidence that the specific eDHFR–TMP interaction
facilitates the trans-splicing reaction.
4996 | Chem. Commun., 2007, 4995–4997
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3 As a few examples, Yao and coworkers demonstrated that native
chemical ligation and expressed protein ligation can be performed in
living cells: D. S. Y. Yeo, R. Srinivasan, M. Uttamchandani,
G. Y. J. Chen, Q. Zhu and S. Q. Yao, Chem. Commun., 2003,
2870–2871; R. Y. P. Lue, G. Y. J. Chen, Y. Hu, Q. Zhu and S. Q. Yao,
J. Am. Chem. Soc., 2004, 126, 1055–1062.
4 I. Giriat and T. W. Muir, J. Am. Chem. Soc., 2003, 125, 7180–7181.
5 See the following review and references cited therein: V. Muralidharan
and T. W. Muir, Nat. Methods, 2006, 3, 429–438.
6 W. Sun, J. Yang and X.-Q. Liu, J. Biol. Chem., 2004, 279, 35281–35286.
7 C. Ludwig, M. Pfeiff, U. Linne and H. D. Mootz, Angew. Chem., Int.
Ed., 2006, 45, 5218–5221.
8 T. Ozawa, S. Nogami, M. Sato, Y. Ohya and Y. Umezawa, Anal.
Chem., 2000, 72, 5151–5157; T. Ozawa, A. Kaihara, M. Sato,
K. Tachihara and Y. Umezawa, Anal. Chem., 2001, 73, 2516–2521;
T. Ozawa, M. Takeuchi, A. Kaihara, M. Sato and Y. Umezawa, Anal.
Chem., 2001, 73, 5866–5874.
9 H. D. Mootz and T. W. Muir, J. Am. Chem. Soc., 2002, 124,
9044–9045; H. D. Mootz, E. S. Blum, A. B. Tyszkiewicz and T. W. Muir,
J. Am. Chem. Soc., 2003, 125, 10561–10569; H. D. Mootz, E. S. Blum
and T. W. Muir, Angew. Chem., Int. Ed., 2004, 43, 5189–5192.
10 L. W. Miller, Y. Cai, M. P. Sheetz and V. W. Cornish, Nat. Methods,
2005, 2, 255–257; N. T. Calloway, M. Choob, A. Sanz, M. P. Sheetz,
L. W. Miller and V. W. Cornish, ChemBioChem, 2007, 8, 767–774.
11 We designed the linker length by predicting the tertiary structure of the
fusion complex of IN and eDHFR-IC using the crystal structures of Ssp
DnaB intein (PDB ID: 1MI8) and eDHFR-methotrexate complex
(1DRE).
12 MALDI-TOF-MS analysis was performed in negative mode with
sinapic acid as a matrix: P-6, calc. 28806, obs. 28789; P-7, calc. 35279,
obs. 35286; P-8, calc. 28037, obs. 28020.
13 Because we could not distinguish the bands of P-6 and P-8 by SDS-
PAGE/Western blotting due to the similar molecular weight, we
estimated the yield of P-6 by determining the peak ratio of P-6 and P-8
by MALDI-TOF-MS analysis (ca. 55 : 45). However, the potential
differences in ionization energy cannot be fully ruled out.
14 This rate constant is comparable to that for the split Ssp DnaE intein
(0.7 6 1024 s21: D. D. Martin, M.-Q. Xu and T. C. Evans, Jr.,
Biochemistry, 2001, 40, 1393–1402), the rapamaycin-inducible split
VMA intein (1.9 6 1024 s21),9 and the split Ssp DnaB intein (IN–IC
pair) (0.4 6 1024 s21)7.
15 We confirmed that the construct IC-9 is capable of trans-splicing with
IN-2 in a crude cell lysate (Fig. S4, ESI{). It was demonstrated that over
10 mM of IN-1 was required to produce the splicing product P-11 in an
amount comparable to that obtained using 0.5 mM of IN-2 in the cell
lysate (Fig. S4, ESI{), indicating again the advantage of the present
ligation system.
16 J. S. Wadia and S. F. Dowdy, Curr. Opin. Biotechnol., 2002, 13, 52–56;
A. Joliot and A. Prochiantz, Nat. Cell Biol., 2004, 6, 189–196;
M. C. Morris, J. Depollier, J. Mery, F. Heitz and G. Divita, Nat.
Biotechnol., 2001, 19, 1173–1176; T. Takeuchi, M. Kosuge,
A. Tadokoro, Y. Sugiura, M. Nishi, M. Kawata, N. Sakai, S. Matile
and S. Futaki, ACS Chem. Biol., 2006, 1, 299–303; M. Okuyama,
H. Laman, S. R. Kingsbury, C. Visintin, E. Leo, K. L. Eward,
K. Stoeber, C. Boshoff, G. H. Williams and D. L. Selwood, Nat.
Methods, 2007, 4, 153–159.
were transiently transfected with plasmid encoding IC-9, and cell
surface expression of the intein-mRFP fusion was confirmed by
immunofluorescence staining using FITC-labeled anti-HA and
anti-Myc antibodies (data not shown).15 Cells were incubated with
IN-2 for 8 h and washed to remove excess peptide. Cells were then
stained with Alexa Fluor 488-labeled streptavidin (SAv-488) and
observed by confocal laser scanning microscopy. The images
showed that only surfaces of transfected cells were specifically
labeled by biotin (Fig. 3(B)). Cells treated with SAv-488 alone
showed no fluorescence staining (data not shown). The formation
of biotinylated P-11 was unambiguously confirmed by Western
blotting using SAv-HRP (Fig. 3(C), lane 1). No labeling was
observed after incubation with IN-1 or on cells expressing IC-10
that lacks the intein domain (Fig. 3(C), lanes 2–4). These results
are in accordance with the in vitro experiments described above. To
our knowledge, this work represents the first demonstration of a
semisynthesis of cell surface protein on living cells.
In conclusion, we have designed and constructed a new split
intein tool for in vivo protein ligation. The small-molecule-ligand-
integrating strategy allowed the creation of semisynthetic split
intein in which the IN fragment is short, hence synthetically
accessible, but is capable of trans-splicing efficiently through the
intein fragment complementation assisted by the auxiliary ligand–
receptor interaction. Given the high orthogonality of eDHFR–
TMP interaction in mammalian cells,10 this system should be
applicable inside living cells by combining it with intracellular
peptide delivery methods.4,16 Because inteins are promiscuous with
respect to the extein sequences, the present protein ligation tool
would find general use for semisynthesis of proteins containing a
variety of chemical probes in cellular contexts.
We are grateful to Toshihiro Ninomiya, Dr Shuji Takeda, and
Satoshi Goda for their early contribution to this work. This work
was supported by the Center for NanoBio Integration.
Notes and references
1 Selected reviews: I. Chen and A. Y. Ting, Curr. Opin. Biotechnol., 2005,
16, 35–40; T. Gronemeyer, G. Godin and K. Johnsson, Curr. Opin.
Biotechnol., 2005, 16, 453–458; L. W. Miller and V. W. Cornish, Curr.
Opin. Chem. Biol., 2005, 9, 56–61; K. M. Marks and G. P. Nolan, Nat.
Methods, 2006, 3, 591–596.
2 C. J. A. Wallace, Curr. Opin. Biotechnol., 1995, 6, 403–410; T. W. Muir,
Annu. Rev. Biochem., 2003, 72, 249–289; D. Schwarzer and P. A. Cole,
Curr. Opin. Chem. Biol., 2005, 9, 561–569; B. L. Nilsson, M. B.
Soellner and R. T. Raines, Annu. Rev. Biophys. Biomol. Struct., 2005, 34,
91–118.
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