"clickable" elastins: Elastin-like polypeptides functionalized with azide or alkyne groups

Article abstract of DOI:10.1039/b903903a

Elastin-like polypeptides (ELPs) functionalized with azide or alkyne groups were produced biosynthetically and coupled via the Cu-catalyzed azide-alkyne cycloaddition to a variety of (bio)molecules.

Full text of DOI:10.1039/b903903a

Showcasing research from Jan van Hest’s laboratory,
based at Radboud University Nijmegen, in The
Netherlands
As featured in:
Chemical Communications
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Number 27 | 21 July 2009 | Pages 3969–4132
Title: Clickable elastins: Elastin-like polypeptides functionalized
with azide or alkyne groups
Temperature-responsive elastin-like polypeptides (ELPs)
functionalized with azide or alkyne groups were produced
biosynthetically and coupled via the Cu-catalyzed azide–alkyne
cycloaddition to a variety of (bio)molecules, namely fluorescent
probes, a polymer, and the enzyme CalB.
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‘‘Clickable’’ elastins: elastin-like polypeptides functionalized with azide
or alkyne groupsw
Rosalie L. M. Teeuwen,ab Sander S. van Berkel,a Tim H. H. van Dulmen,a Sanne Schoffelen,a
Silvie A. Meeuwissen,a Han Zuilhof,b Frits A. de Wolfc and Jan C. M. van Hest*a
Received (in Cambridge, UK) 24th February 2009, Accepted 7th April 2009
First published as an Advance Article on the web 21st April 2009
DOI: 10.1039/b903903a
Elastin-like polypeptides (ELPs) functionalized with azide or
alkyne groups were produced biosynthetically and coupled via
the Cu-catalyzed azide–alkyne cycloaddition to a variety of
(bio)molecules.
been constructed for the formation of hydrogels for tissue
engineering.18–21 Block copolymers of different types of elastin
have been prepared to form micelles with stimulus-responsive
behavior.22 Although recombinant DNA technology has
facilitated the production of these fusion proteins, it is still
laborious to design each fusion protein starting from the
genetic level.
Bioconjugation offers the possibility to introduce biological
functionality into hybrid materials by connecting biomolecules
and synthetic molecules with each other. This methodology
has become increasingly important for the development of new
materials for applications in the fields of diagnostics, medicine
and tissue engineering.1–3
Conjugation of ELPs to (bio)molecules is a more versatile
approach, not only giving rise to ELP–protein conjugates but
also to a variety of biohybrid materials. Block copolymers of
elastin and PEI or PAA have been constructed for the
modification of surfaces applied in tissue engineering.23,24
ELPs modified with drugs25–29 or fluorescent probes30,31 have
been used in tumor tissue targeting. In all these examples ELPs
were reacted at the C-terminus, on lysine or cysteine residues
using the more traditional N-hydroxysuccinimide ester or
maleimide–thiol coupling chemistry. These conjugation methods
use functional groups that are abundantly present in proteins,
which results in multiple point attachment.
The structural protein elastin4,5 has in this respect drawn
significant attention from the materials science community,
because of its striking stimulus-responsive character. Upon
heating, the water-soluble and unorganized protein undergoes
a phase transition, forming a hydrophobic b-spiral structure,
which results in the formation of aggregates.6–8 This lower
critical solution temperature (LCST) behavior is completely
reversible and can be mimicked by elastin-like polypeptides
(ELPs), which are composed of the amino acid repeat
sequence Val-Pro-Gly-Xaa-Gly (Val = valine, Pro = proline,
Gly = glycine), in which the fourth amino acid residue Xaa
can be any natural amino acid except proline.9,10 ELPs can
be readily produced in high yields by molecular biology
techniques using E. coli as the expression system.11,12 The
LCST can be varied over an extensive temperature range by
changing the hydrophobicity of the fourth residue, the length
of the polypeptide, ionic strength of the medium and the ELP
concentration.9,13–15
Site-specific coupling to proteins can be improved by using
orthogonal functionalities. A well-known example is the
Cu-catalyzed azide–alkyne cycloaddition (CuAAC).32 This
method has been thoroughly investigated and applied in
bioconjugation.33–35 The non-natural reactive groups (azide
or alkyne) can be introduced into a protein using single-site
replacement strategies36 or auxotrophic expression37–40 by
replacing methionine 1 with azidohomoalanine 2 (AHA) or
homopropargylglycine 3 (HPG), (Fig. 1).
Here we present the biosynthesis of ‘‘clickable’’ elastins and
their use as versatile modular building blocks by conjugation
of the ELPs via the CuAAC reaction. This is demonstrated by
the conjugation to three different moieties, namely fluorescent
probes, a polymer, and a protein, resulting in three different
hybrid materials, which display the characteristics of each of
the ‘‘clicking’’ partners.
The unique properties of elastin open up the use of this
material for a variety of applications. The LCST character of
ELPs has been applied to position proteins fused to ELPs in a
controlled fashion for diagnostics,16,17 and to aggregate
and purify fusion proteins after fermentative production
from a cell lysate.13 Block copolymers of elastin and silk,
and elastin polymers containing cell adhesion domains have
The ELPs were produced using protein engineering. After
cloning ELP genes with recursive directional ligation,15 the
proteins were expressed in the auxotrophic E. coli strain
a Radboud University Nijmegen, Institute for Molecules and Materials,
Department of Organic Chemistry, P.O. Box 9010,
6500 GL Nijmegen, The Netherlands.
E-mail: j.vanhest@science.ru.nl
b Wageningen UR, Laboratory for Organic Chemistry,
P.O. Box 8026, 6700 EG Wageningen, The Netherlands
c Wageningen UR, Agrotechnology & Food Innovations B.V.,
P.O. Box 17, 6700 AA Wageningen, The Netherlands
w Electronic supplementary information (ESI) available: ELP
cloning, protein production, characterization procedures, including
MALDI-TOF and turbidity measurements, syntheses of fluorescent
probes, and click conditions. See DOI: 10.1039/b903903a
Fig. 1 Structure of methionine 1 and its analogs azidohomoalanine 2,
homopropargylglycine 3, and CuAAC ligand 4.
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4022 | Chem. Commun., 2009, 4022–4024

Scheme 1 Synthesis of alkyne and azido fluorophores. Reagents and
conditions: (i) ethylene carbonate, K2CO3, toluene, Ar atmosphere,
115 1C, 24 h (98%); (ii) TBDMSCl, DMF, imidazole, rt 2 h (95%);
(iii) (a) Mg, EtBr2, Et2O, rt, 2 h, (b) TBDMS–xanthone (at 0 1C),
THF, rt, 1.5 h, (c) MeOH, TFA, rt, 30 min; (5 (89%), 7 (33%))
(iv) MsCl, CH2Cl2, DMAP, 0 1C to rt, 4 h (25%); (v) NaN3, DMF,
60 1C, 36 h (27%); (vi) DiAD, PPh3, CH2Cl2, 0 1C to rt 48 h (72%);
(vii) K2CO3, MeOH, rt, 18 h (99%).
B834(DE3)pLysS according to literature procedures,41 and
purified using inverse transition cycling.13 Proteins were
analyzed by SDS-PAGE, MALDI-TOF, and their transition
temperature was determined (ESIw). Incorporation of the
methionine analogs (i.e. 2 and 3) producing AHA–ELP and
HPG–ELP was confirmed by tryptic digest (ESIw).
As a first example to demonstrate the coupling capacity
of the clickable ELPs, fluorescent probes were employed,42–45
in particular the strongly emitting TokyoGreen based
fluorophores.46–48 Since derivatives of TokyoGreen suitable
for CuAAC chemistry were not readily available, we first
developed azide-containing and alkyne-containing green
fluorescent probes 6 and 8 (Scheme 1, ESIw).
Fig. 3 (a) SDS-PAGE gel of HPG–ELP labeled with coumarin-
functionalized PEG2000. (b) SDS-PAGE gel of HPG–ELP labeled
with azido-functionalized CalB. Reaction mixture (lane1), CalB–ELP
conjugate after removal of unreacted AHA–CalB using LCST (lane 2).
(c) Comparison of the hydrolytic activity of CalB–ELP conjugate,
AHA–CalB, and HPG–ELP. (d) Normalized turbidity profile of
HPG–ELP (0.1 mg mLÀ1) and CalB–ELP (0.07 mg mLÀ1) in PBS
measured at 350 nm.
expected, fluorescence was absent in the negative controls
using fluorophore 5.
As an example of a polymer–protein hybrid material
coumarin-functionalized PEG200051 was reacted with HPG–
ELP. After dialysis the reaction was analyzed with
SDS-PAGE (Fig. 3a). The Coomassie stained gel showed
two bands of comparable intensity, indicating a yield of
approximately 50% for the formation of the polymer–protein
hybrid. The upper band showed fluorescence upon irradiation
with UV light, which confirmed the reaction of HPG–ELP
with the coumarin-functionalized PEG2000. The reason that
functionalization did not go to completion can be partly
explained by the fact that a fraction of the ELP produced still
contained methionine instead of HPG. Furthermore, coupling
of macromolecules remains an inherently difficult process.
AHA–ELP and HPG–ELP were both successfully labeled
with ClickGreen derivatives 6 and 8, respectively, in the
presence of Cu(I)Br and tristriazole ligand 449,50 (Fig. 2). As
As
a third example an azido-functionalized enzyme,
Candida antarctica lipase B (CalB),52 was ‘‘clicked’’ to
HPG–ELP (Fig. 3b, lane 1). This reaction proceeded with
a
yield comparable to the polymer–protein hybrid.
Unreacted AHA–CalB (34 kDa) was removed using the LCST
characteristics of the CalB–ELP conjugate (Fig. 3b, lane 2),
while FPLC was used to further purify CalB–ELP from
unreacted HPG–ELP (ESIw).
Fig. 2 SDS-PAGE gel of labeling AHA–ELP (A) and HPG–ELP (H)
with fluorescent probes 6 and 8, respectively. Final concentrations:
Cu(I)Br (4.8 mM), 4 (9.6 mM), 5, 6 and 8 (90 mM).
Subsequently, the hydrolytic activity of the CalB part of the
CalB–ELP conjugate was determined by measuring the
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 4022–4024 | 4023

hydrolysis of p-nitrophenol butyrate at 405 nm (Fig. 3c). Its
activity was compared to HPG–ELP and AHA–CalB, which
had been subjected to ‘‘click’’ conditions. The activity of the
conjugate was 50% of AHA–CalB. The LCST of the ELP part
of CalB–ELP was also determined (Fig. 3d). The LCST shifted
to 46 1C, and the inverse transition was slightly broader. These
observations are in accordance with literature reports on the
characteristics of ELP fusion proteins prepared by genetic
engineering.13,53
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The authors thank Process on a Chip (PoaC/NWO-ACTS)
and the Dutch National Research School Combination-
Catalysis (NRSC-C) for financial support. Dr A. Martens
is acknowledged for the design of the aIII-adapter oligo’s
and for providing the vector pMTL23-d-BsaI-aIII, and
Dr T. Dirks is acknowledged for supplying us with the
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4024 | Chem. Commun., 2009, 4022–4024