Site-specific conjugation of RAFT polymers to proteins via expressed protein ligation

Article abstract of DOI:10.1039/c3cc38976f

Site-specific protein conjugates with RAFT polymers were synthesized using expressed protein ligation. Stable micelles were formed from both linear block copolymer and Y-shaped conjugates.

Full text of DOI:10.1039/c3cc38976f

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Site-specific conjugation of RAFT polymers to proteins
via expressed protein ligation†
Cite this: Chem. Commun., 2013,
49, 2566
Yan Xia,z Shengchang Tang and Bradley D. Olsen*
Received 15th December 2012,
Accepted 4th February 2013
DOI: 10.1039/c3cc38976f
www.rsc.org/chemcomm
Site-specific protein conjugates with RAFT polymers were synthe- narrowly dispersed polymers synthesized by controlled radical
sized using expressed protein ligation. Stable micelles were formed polymerization. Considering the large variety of polymers that
from both linear block copolymer and Y-shaped conjugates.
can be synthesized using controlled radical polymerization, the
extension of EPL to these polymers will enrich the diversity of
Protein–polymer conjugates are of increasing interest for pharma- well-defined protein–polymer conjugates.
ceutical and biotechnological applications, due to their improved
Herein, we report the first examples of direct EPL to a range
pharmacokinetics and decreased immunogenicity as compared to of polymers prepared using RAFT polymerization. Furthermore,
the unmodified proteins.1–3 Many of these applications require we demonstrate the ability to perform orthogonal conjugation
homogeneous, site-specific conjugates for uniform, consistent reactions at the same site, enabling the synthesis of different
performance and biological activity.4 The majority of reported bioconjugate architectures that form stable micelles in solution.
protein–polymer conjugation reactions rely on the attachment to
RAFT polymerization is a versatile living polymerization
lysine or cysteine residues on the protein surface.1–3,5 The technique for preparing functional polymers with pre-defined
presence of multiple lysines on the protein surface often leads MW and narrow PDI.19 However, most RAFT agents are composed
to heterogeneity in both the position and the number of conjuga- of trithiocarbonates or dithioesters, which we observed to react
tion sites when lysine is targeted as a reactive site. Cysteine is less with cysteine rapidly under NCL conditions to form cyclic dithio-
abundant in the proteome, but cysteine-based conjugation can be carbamate or thioamide, respectively (Fig. S1, ESI†). Therefore,
complicated when disulfide bonds are structurally important for for conjugation to the C-terminus of a protein, it is important
the protein secondary or tertiary structures. Numerous alternative to prepare RAFT agents with protected cysteine and remove
methods have been developed to allow site-specific conjugation to the RAFT agent end group after RAFT polymerization to avoid
proteins.6–8 Among them, intein-mediated ligation, or expressed interference and consumption of the cysteine end group before
protein ligation (EPL),9 takes advantage of self-splicing intein and NCL can be attempted.
chemo- and regio-selective native chemical ligation (NCL) between
We synthesized trithiocarbonate and dithioester RAFT agents
a N-terminal cysteine and a C-terminal thioester.10,11 EPL has with protected cysteine groups, and polymerizations using these
been used to conjugate small molecules,12–14 nucleic acids,15 RAFT agents proceeded smoothly to yield poly(dimethylacryl-
peptides,9 and dendrimers.16 The Chilkoti group recently reported amide) (PDMA), poly(N-isopropylacrylamide) (PNIPAM), and
conjugation of an atom transfer radical polymerization (ATRP) poly(polyethylene-glycol methacrylate) (PPEGMA) with molar
initiator onto protein using EPL and subsequent ATRP from the mass of 6–21 kDa and PDI o 1.1 (Table 1). The RAFT agent
protein initiator to form protein–polymer conjugate.17 Sumerlin and cysteine end groups were then sequentially removed and
and coworkers first demonstrated protein–polymer conjugates deprotected (see experimental detail in ESI†).
using reversible fragmentation chain transfer (RAFT) polymeriza-
A model protein, green fluorescent protein (GFP), was cloned
tion via grafting-from.18 However, to the best of our knowledge, EPL into the expression vector with the intein–maltose binding domain
has not been used to directly conjugate proteins to pre-formed, fused to the C-terminus of GFP to produce a gene encoding a
protein suitable for EPL conjugation. The C-terminal residue on the
Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, MA, USA. E-mail: bdolsen@mit.edu; Tel: +1 617-715-4548
† Electronic supplementary information (ESI) available: Experimental details,
NMR, GPC, LC-MS, CD, UV-Vis spectra and additional SDS-PAGE. See DOI:
10.1039/c3cc38976f
protein of interest is known to have a significant effect on the intein
cleavage, and glycine has been reported to maximize the cleavage
efficiency.13 Therefore, two glycine residues were encoded
between the C-terminus of GFP and the intein. A His6 tag was
also coded at the N-terminus of the GFP to allow purification of
the protein–polymer conjugate by metal affinity chromatography.
‡ Current address: Department of Chemistry, Stanford University, Stanford, CA,
USA.
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Table 1 Characteristics and conjugation of RAFT polymers to GFP
the subsequent conjugation reactions were performed using
10 fold excess of polymer at room temperature for 12 h without
additional reducing agent. Using these conditions, all RAFT
polymers were successfully conjugated to GFP at moderate
conversions (Fig. 1). The lower conversion of PPEGMA is likely
due to the increased steric hindrance at the cysteine chain end
as compared to other linear polymers.
a
Polymer
Mn (kDa)
PDIa
Conjugation yieldb (%)
PDMA
PDMA
PPEGMA
PDMA-b-PNIPAM
PDMAc
6.7
21.0
9.2
14.8
6.4
1.02
1.06
1.04
1.08
1.04
62
52
29
57
60
a
Determined by GPC in DMF (0.02 M LiBr) using refractive index (RI)
Since NCL retains the cysteine residue at the linkage, we
next sought to take advantage of this cysteine for subsequent
conjugation to the same site on protein to form a Y-shaped
protein–polymer conjugates. This strategy provides a comple-
mentary strategy to recently reported dibromomaleimide cou-
pling20,21 to conjugate proteins to two different functionalities,
in the present case placing proteins at the interface between
two polymer blocks (Scheme 1). The site of protein attachment
may have an impact on the stability or activity of proteins.
To enable two subsequent site-specific conjugations at the
same site, a GFP mutant was prepared with no accessible surface
cysteines, which was confirmed by the absence of conjugation in
the presence of excess maleimide-terminated PEG (Mn = 5 kDa)
and TCEP. To avoid interference with the thiol–maleimide
coupling at the newly formed cysteine, the RAFT agent at the
polymer chain end was removed via desulfurization by radical-
induced reduction.22 Complete removal of RAFT agent was
evidenced by the disappearance of the absorption peak of RAFT
agent at 310 nm. Cysteine-functionalized PDMA was then
conjugated to GFP under our standard NCL conditions with a
60% yield. The reaction mixture was purified to remove excess
PDMA, and no attempt was made to isolate the GFP–PDMA from
unreacted GFP. The product from the first ligation reaction was
then subjected to 10 eq. PNIPAM (Mn = 7 kDa) with a maleimide
end group. The band corresponding to GFP–PDMA has
completely moved to higher MW, as shown by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
indicating successful second conjugation (Fig. 2). This resulted
in a Y-shaped conjugate, GFP–Y–PDMA–PNIPAM, and the final
conjugate was purified by anion exchange chromatography. We
also synthesized a PDMA-b-PNIPAM with similar MWs in each
block (7 kDa each) as compared to the PDMA and PNIPAM used
in Y-shaped conjugates. This linear BCP conjugate was simply
b
and MALLS detectors. Calculated using densitometry from SDS-PAGE
analysis. PDMA with a hydride end group.
c
The GFP–intein fusion protein was expressed in Escherichia coli,
and the cell lysate was loaded onto chitin resin to bind the
fusion protein. After incubation with 50 mM sodium mercapto-
ethanesulfonate (MESNa) for >12 h at 4 1C, GFP was cleaved
off the chitin resin with a sulfoethyl thioester group at the
C-terminus (Fig. S2, ESI†).
We first used a PDMA (entry 1 in Table 1) polymer to
investigate the conditions and efficiency of polymer conjugation
to GFP–thioester. Conjugation conversions reached 50–60%
after 6 h at all temperatures investigated, namely 4 1C, room
temperature, and 37 1C, and stayed constant afterwards,
although the reaction proceeded faster as temperature was
increased (Fig. S3(a)–(c), ESI†). Increasing the equivalence of
polymer from 10 fold to 20 fold or adding additional reducing
agent, such as tris(2-carboxyethyl) phosphine (TCEP) did not
increase the conversion (Fig. S3(d) and (e), ESI†). Therefore, all
Fig. 1 SDS-PAGE of (1) GFP–thioester after chitin column purification; crude
conjugates of GFP with (2) PDMA (entry 1 in Table 1), (3) PDMA (entry 2 in
Table 1), (4) PPEGMA (entry 3 in Table 1), (5) PDMA-b-PNIPAM (entry 4 in Table 1),
(6) PDMA (entry 5 in Table 1).
Scheme 1 Synthesis of linear (top) and Y-shaped (bottom) GFP–block copolymer conjugates and their proposed micellar assembly with different protein location in
respective micelles.
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Fig. 4 TEM of the micelles resulted from (a) linear and (b) Y-shaped BCP
conjugates at 50 1C. The scale bar corresponds to 50 nm.
Fig. 2 SDS-PAGE of the conjugation reactions and purification of the conju-
gates. (1) GFP–thioester after intein cleavage; (2) crude conjugate of GFP with
PDMA-b-PNIPAM (entry 4 in Table 1); (3) purified conjugate of GFP with PDMA-b-
PNIPAM by ammonium sulfate precipitation; (4) crude conjugate of GFP with
PDMA (entry 5 in Table 1); (5) crude Y-shaped conjugate of GFP–PDMA and
PNIPAM; (6) purified Y-shaped conjugate of GFP–Y–PDMA–PNIPAM by anion
exchange chromatography.
Y-shaped GFP–PDMA–PNIPAM BCP conjugates formed stable,
thermo-responsive micelles. This demonstrates that the general
EPL/RAFT bioconjugation strategy can be applied to prepare
various protein–polymer conjugate materials for encapsulated
enzymes and protein therapeutics.
We would like to thank DTRA for funding under project
BA12PHM159, the Biophysical Instrumentation Facility for the
Study of Complex Macromolecular Systems and the Center for
Materials Science and Engineering (NSF-0070319 and NIH
GM68762) for the use of instruments. We thank Prof. Hadley
Sikes for the use of FPLC, Carla Thomas for assistance with
TEM, and Christopher Lam for providing the GFP gene.
purified by ammonium sulfate precipitation to remove uncon-
jugated GFP (Fig. 2). Circular dichroism (CD) and UV-vis
spectra of both types of GFP–polymer conjugates showed
minimal change from the spectra recorded with the unconju-
gated GFP (Fig. S9 and S10, ESI†), indicating the native protein
conformation is reserved by the conjugation procedures.
Thermo-responsive micellization of these protein–polymer
conjugates was observed regardless of the molecular architecture,
as studied using dynamic light scattering (DLS, Fig. 3). At 25 1C,
both conjugates existed in the monomer form with Rh of 5.5 and
3.5 nm for the linear and Y-shaped BCP conjugates, respectively;
the lower Rh for the Y-shaped conjugate is consistent with the
more compact molecular configuration of the branched polymer.
Upon heating to 50 1C, above the lower critical solution tempera-
ture (LCST) of PNIPAM, micelles were rapidly formed in both
cases: the linear conjugate had a Rh of 15.3 nm and the Y-shaped
conjugate had a Rh of 12.7 nm. Micellization was completely
reversible. Upon cooling back to 25 1C, the conjugate monomer
form was restored within minutes. Transmission electron micro-
scopy (TEM) of dehydrated micelles adsorbed to formvar-coated
grids and negatively stained revealed the formation of spherical
micelles for both bioconjugate architectures (Fig. 4).
Notes and references
1 A. S. Hoffman and P. S. Stayton, Prog. Polym. Sci., 2007, 32, 922.
2 M. A. Gauthier and H.-A. Klok, Chem. Commun., 2008, 2591.
3 R. M. Broyer, G. N. Grover and H. D. Maynard, Chem. Commun.,
2011, 47, 2212.
4 G. G. Kochendoerfer, Curr. Opin. Chem. Biol., 2005, 9, 555.
5 M. W. Jones, G. Mantovani, S. M. Ryan, X. Wang, D. J. Brayden and
D. M. Haddleton, Chem. Commun., 2009, 5272.
6 J. M. Antos and M. B. Francis, Curr. Opin. Chem. Biol., 2006, 10, 253.
7 A. J. de Graaf, M. Kooijman, W. E. Hennink and E. Mastrobattista,
Bioconjugate Chem., 2009, 20, 1281.
8 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009,
48, 6974.
9 T. W. Muir, D. Sondhi and P. A. Cole, Proc. Natl. Acad. Sci. U. S. A.,
1998, 95, 6705.
10 P. Dawson, T. Muir, I. Clark-Lewis and S. Kent, Science, 1994,
266, 776.
11 T. M. Hackeng, J. H. Griffin and P. E. Dawson, Proc. Natl. Acad. Sci.
U. S. A., 1999, 96, 10068.
12 T. J. Tolbert and C.-H. Wong, J. Am. Chem. Soc., 2000, 122, 5421.
13 R. Y. P. Lue, G. Y. J. Chen, Y. Hu, Q. Zhu and S. Q. Yao, J. Am. Chem.
Soc., 2004, 126, 1055.
14 R. J. Wood, D. D. Pascoe, Z. K. Brown, E. M. Medlicott, M. Kriek,
C. Neylon and P. L. Roach, Bioconjugate Chem., 2004, 15, 366.
15 M. Lovrinovic, L. Fruk, H. Schroder and C. M. Niemeyer, Chem.
Commun., 2007, 353.
We demonstrated the successful, site-specific conjugation of
RAFT homopolymers and block copolymers to a model protein,
GFP, via EPL. Sequential EPL and thiol–maleimide ligation
allowed the synthesis of Y-shaped conjugate with two different
polymers attached at the same site on protein. Both linear and
16 I. van Baal, H. Malda, S. A. Synowsky, J. L. J. van Dongen,
T. M. Hackeng, M. Merkx and E. W. Meijer, Angew. Chem., Int. Ed.,
2005, 44, 5052.
17 W. Gao, W. Liu, T. Christensen, M. R. Zalutsky and A. Chilkoti, Proc.
Natl. Acad. Sci. U. S. A., 2010, 107, 16432.
18 P. De, M. Li, S. R. Gondi and B. S. Sumerlin, J. Am. Chem. Soc., 2008,
130, 11288.
19 G. Moad, E. Rizzardo and S. H. Thang, Acc. Chem. Res., 2008,
41, 1133.
20 M. E. B. Smith, F. F. Schumacher, C. P. Ryan, L. M. Tedaldi,
D. Papaioannou, G. Waksman, S. Caddick and J. R. Baker, J. Am.
Chem. Soc., 2010, 132, 1960.
21 M. W. Jones, R. A. Strickland, F. F. Schumacher, S. Caddick,
J. R. Baker, M. I. Gibson and D. M. Haddleton, J. Am. Chem. Soc.,
2011, 134, 1847.
22 Y. K. Chong, G. Moad, E. Rizzardo and S. H. Thang, Macromolecules,
2007, 40, 4446.
Fig. 3 DLS of (a) linear and (b) Y-shaped BCP conjugates showing reversible
temperature dependent micellization.
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2568 Chem. Commun., 2013, 49, 2566--2568
This journal is The Royal Society of Chemistry 2013