Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent

Article abstract of DOI:10.1021/acs.bioconjchem.8b00354

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short ela

Full text of DOI:10.1021/acs.bioconjchem.8b00354

Subscriber access provided by - Access paid by the | UCSB Libraries
Article
Conjugation of Amphiphilic Proteins to
Hydrophobic Ligands in Organic Solvent
Antonietta Maria Lillo, Ciana Lopez, Trideep Rajale, Hung-Ju Yen, Harsha Magurudeniya,
M. Lisa Phipps, Eva Rose Murdock Balog, Timothy C. Sanchez, Srinivas Iyer, Hsing-
Lin Wang, Ryszard Michalczyk, Reginaldo C. Rocha, and Jennifer S Martinez
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00354 • Publication Date (Web): 06 Jul 2018
Downloaded from http://pubs.acs.org on July 8, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 15
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in
Organic Solvent
Antonietta M. Lillo,§ Ciana L. Lopez,† Trideep Rajale,† Hung Ju Yen,|| Harsha Magurudeniya,† M. Lisa
Phipps,† Eva Rose Balog,^ Timothy Sanchez,§ Srinivas Iyer,§ Hsing-Lin Wang,‡ Ryszard Michalczyk,§
Reginaldo C. Rocha,† Jennifer S. Martinez†#*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†
#
§Bioscience Division, Center for Integrated Nanotechnologies, and Institute for Materials Science, Los Alamos National
Laboratory, Los Alamos, New Mexico, United States 87545; ||Institute of Chemistry, Academia Sinica, Nankang, Taipei, Tai-
^
wan 11529; Department of Chemistry and Physics, University of New England, Biddeford, Maine, United States 04005;
‡Department of Materials Science and Engineering, Southern University of Science and Technology, Nanshan District, Shen-
zhen, China 518055.
Supporting Information Placeholder
ABSTRACT: Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment
within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-
like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs (p-phenylene vinylene oli-
gomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aque-
ous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate
and/or interact non-covalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low con-
jugation efficiency and contamination with strong non-covalent “conjugates”. After exploring the solubility of sEL in
various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates
virtually free of contaminating non-covalent complexes. When conjugating carboxylated ligands to the amphiphile
amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient.
When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is
avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur
in “one pot” and our purification procedure is a simple workup utilizing a combination of water and organic solvent
extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of
protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug
delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of
nanostructures (peptides) and engineering of biocompatible optoelectronics (biological polymers), to cite a few.
INTRODUCTION
Conjugations to biological materials are typically car-
ried out in biologically ambient conditions (that is,
aqueous solvent, ≤ 37 ºC, pH 6–8) so as not to disrupt
protein architecture and/or function. While some conju-
gations are straightforward, problems arise when am-
phiphilic proteins or polymers are conjugated to hydro-
phobic ligands.8-10 Hydrophobic ligands by definition
reorder water and ultimately avoid exposure to the aque-
ous phase by aggregating or by seeking hydrophobic re-
gions within the amphiphile.11-14 As a result, the ligand
availability for covalent conjugation is reduced and the
conjugation is inefficient. Considering that the interac-
tion between an amphiphile and a hydrophobic ligand
can be very strong and that covalent conjugation might
Protein post-translational modification has an im-
portant role in nature. Acylation, methylation, phos-
phorylation, glycosylation and sulfation, among other
chemical reactions, are continuously used by biological
systems to augment or alter protein function. What na-
ture has discovered and naturally selected through evo-
lution, scientists try to reproduce in the laboratory using
available chemical tools to yield conjugates with new
critical functions.1, 2 For example, recombinant antibod-
ies are conjugated to drugs for targeted delivery to tu-
mor cells3 or pathogens4, and natural polymers are
chemically modified to sense and interact with their en-
vironment.5-7
ACS Paragon Plus Environment
1

Bioconjugate Chemistry
Page 2 of 15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Scheme illustrating amine or carboxyl-mediated functionalization of an amphiphilic protein (short elastin, sEL) with poorly
water-soluble ligands in organic solvent. (A) Reaction conditions for obtaining the WsEL-RuCOOH conjugate. (B) Reaction conditions for
obtaining the DsEL-TAAy or DsEL-OPPVNH2 conjugates. (C) Procedure for the purification of covalently conjugated WsEL. (D) Procedure for
the purification of covalently conjugated DsEL. *As an alternative method, the water-solubilized protein from the previous step can be extracted
with dichloromethane. (E) Ligand structures.
be inefficient, it is not surprising that non-covalent in-
teractions are often favored over covalent conjugation.
Indeed, this dominating non-covalent interaction be-
tween hydrophobic ligands and amphiphiles is often ex-
ploited for encapsulation of hydrophobic drugs within
micelles.14-17 Our group has also experienced difficulty
in covalently conjugating amphiphilic polymers to hy-
drophobic ligands, and recently we have exploited the
very strong non-covalent interaction between elastin-
like polymer (ELP) and p -phenylene vinylene oligomer
(OPPV) for generating a composite elastin-like polymer
which exhibits both pH and temperature-dependent flu-
orescence emission.5 However, for most devices or ther-
apeutics, molecularly-defined and covalent conjugates
are the desired target.18-24 Research aimed to increase
the efficiency of covalent conjugation, and prevent/dis-
rupt non-covalent interactions is therefore very relevant.
One of the most commonly employed methods to mod-
ify proteins is the reaction of solvent-accessible protein
amines with carboxyl-containing ligands activated by 1-
straightforward for hydrophilic proteins, problems arise
when using amphiphilic proteins and poorly water-sol-
uble ligands, due to solubility mismatch of the reagents.
An efficient reaction typically requires ten-fold or
higher excess of ligand over protein, but when a hydro-
phobic ligand is used such ratios are difficult to achieve,
even with the use of organic co-solvents, unless the con-
centration of reactants is kept very low. Unfortunately,
the low concentration of reactants, together with the ten-
dency of the ligand to non-covalently interact with the
protein,11-14, 26 leads to low conjugation efficiency. Fur-
thermore, conjugation through protein carboxyl groups
is seldom used, likely because activated carboxyls
might react more readily with free amines within the
protein than with the ligand amines. Conjugation of
amphiphiles to hydrophobic ligands has been performed
in organic solvents to prevent the problems mentioned
above.27-29 However in most of these cases conjugation
is an extra step within the solid phase synthesis of the
amphiphile, so these methodologies do not transfer to
post-expression modification of a large amphiphile or
protein (usually obtained recombinantly). Furthermore,
ethyl-3-(3-dimethylaminopropyl)
carbodiimide
(EDC)/N-hydroxysulfosuccinimide (NHS) chemistry,
in aqueous buffers.25 Although this method is
ACS Paragon Plus Environment
2

Page 3 of 15
Bioconjugate Chemistry
recovery of unreacted ligand (desirable when expensive concentrations to low values, since a high ligand to pro-
1
2
3
4
5
6
7
8
or custom-made ligands are used) is rarely reported.
tein ratio is usually required. The sub-optimally low
concentration of reactants results in low conjugation ef-
ficiencies, even after pH optimization, co-solvent use,
and long incubation times. In the case of amphiphilic
polymer sEL, or any polymer/protein that undergoes
temperature dependent phase changes, an additional
hurdle is the need to perform the reaction at low temper-
ature, due to sEL coacervation at room temperature.7, 36,
Here we present a methodology for conjugation of
OPPV,30-32 triarylamine,33 and polypyridine-metal (Ru)
complexes,34 to variants of the polymer short elastin
(sEL, as exemplar amphiphile), in N-methyl-2-pyrroli-
done (NMP), through the polymer free amines or car-
boxyls (Figure 1). sEL is an elastin-like polymer (ELP)
and member of a broader class of stimuli responsive
‘‘smart’’ polymers that exhibit inverse temperature
phase transition behavior in response to changes in their
environment.35-38 The selected ligands are examples of:
a) substituted OPPV which upon aggregation and strong
dipole–dipole solute–solvent interactions exhibit strong
solvatochromism39 (4-[2-(2,5-Dimethoxy-4-styryl-phe-
nyl)-vinyl]-phenylamine, here called OPPVNH2, Figure
1E);40 b) electron-rich triarylamines known to be easily
oxidized while forming stable polarons and exhibiting
noticeable change of coloration33 (N-(4-Aminophenyl)-
N-phenyl-1-naphthalylamine, here called TAAy, Figure
1E); and c) polypyridine-metal complexes with high
photoluminescence and well-defined, tunable emission
spectra based on metal-to-ligand charge transfer,41
[Ru(2,2’-bipyridine)-2-(2,2’-bipyridine-4,4’-dicarbox-
ylic acid)](PF6)2, here called RuCOOH, Figure 1E; and
[Ru(2,2’-bipyridine)-2-(5-amino-1,10-phenanthro-
line)](PF6)2, here called RuNH2, Figure S1).42 Beyond
serving as exemplar hydrophobic ligands to develop our
conjugation methodology, the covalent conjugation of
these molecules to sEL can generate electrochromic and
optoelectronic materials with a wide spectrum of
applications.6, 31
56
9
Thus, for example, aqueous conjugation of terpyri-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
dine-metal complexes to ELP carboxyls typically re-
sulted in a conjugation yield of only about 45%.6 Fur-
thermore, when conjugation is performed in water we
have observed that even with the least hydrophobic lig-
and in our suite (RuNH2, Figure S1), a significant per-
centage of sEL is non-covalently bound to the ligand
(Figure 2). Driven by the expectation that a new strategy
for conjugation in organic solvents could solve the is-
sues described above, we investigated the solubility of
sEL in several organic solvents.
The simple conjugation methodology presented here
allows for facile and efficient derivatization of am-
phiphile amines or carboxyls, minimization of non-co-
valent interaction between amphiphile and hydrophobic
ligand, minimal protein multimerization, separation of
the product from non-covalently bound ligand, and re-
covery of unreacted ligand. Although we report on the
conjugation of sEL variants, our methodology could be
used for conjugation of any amphiphilic protein or pol-
ymer of interest, including: PEGylated antibodies43
(some of which are soluble in organic solvents44-46);
modified organosoluble enzyme conjugates such as
POXylated47, 48 and PEGylated enzymes;49, 50 a plethora
of amphiphilic peptides used as molecular building
blocks for nanostructures;51 food proteins used for drug
delivery (e.g. gelatin and casein);52,53 collagen;54 and
proteins such as hydrophobins (fungal derivatives used
for their unique surface-active and self-assembly prop-
erties).55
Figure 2. SDS-PAGE shows a very strong ligand-polymer non-
covalent interaction when amphiphile-hydrophobic ligand
conjugation is performed in water. The reaction of activated (re-
action) or non-activated (mock) sEL with RuNH2, in water, was ex-
amined by UV transillumination (left, the fluorescence observed is
due to the ligand) and by Coomassie blue staining of the protein
(right). The higher fluorescence associated with the sEL band in
the reaction lane vs the sEL band in the mock lane (left side), indi-
cates that there is covalent binding of RuNH2 to the activated sEL.
However, the significant amount of fluorescence associated with
the sEL band in the mock lane reveals a strong non-covalent inter-
action between protein and ligand, which withstands the denatur-
ing condition of the gel analysis. The fluorescence at the solvent
front reveals a large amount of ligand that could not be removed
after multiple ultrafiltration of the conjugation reaction mixture, and
that was most likely non-covalently bound to the protein prior to
separation on gel.
sEL behavior in organic solvents. The sEL variants
used in this work were WsEL, engineered to contain a
C-terminal tryptophan (W) for ease of characterization
(Figure S2A), and DsEL, containing an extra aspartate
(D) upstream from sEL, and (Figure S2B). WsEL con-
tains one primary amine (the N-terminus) available for
conjugation to carboxyl-containing ligands. DsEL con-
tains a total of three carboxyls (indicated in red in Figure
S2B) available for conjugation to amine-containing
RESULTS AND DISCUSSION
Conjugation of poorly water-soluble compounds to
amphiphilic proteins in aqueous conditions is difficult.
Even when the protein solubility is high, the poor solu-
bility of the ligand restricts the range of usable protein
ACS Paragon Plus Environment
3

Bioconjugate Chemistry
Page 4 of 15
ligands. Both DsEL and WsEL are very soluble (up to sEL does not coacervate at room temperature (as it does
1
2
3
4
5
6
7
50 mg/mL) in dipolar aprotic solvents dimethylforma-
mide (DMF), dimethylsulfoxide (DMSO) and NMP, but
insoluble in acetonitrile and dichloromethane.
in water) in the compatible organic solvents. Notably,
even after 24 h exposure of sEL to organic solvent, upon
removal of these solvents and resuspension of the pro-
tein in water, the reversible temperature-dependent co-
acervation typical of sEL7, 36, 56 is still observed (Figure
S3).
Conjugation strategies and conjugate purification.
WsEL was conjugated to RuCOOH (Figure 1A, mass
spectrum of WsEL Figure S4), while DsEL was conju-
gated to OPPVNH2 or TAAy (Figure 1B, mass spectrum
of DsEL Figure S5). Conjugation reactions were per-
formed in NMP, using EDC/NHS activation of carbox-
yls in the ligand (WsEL conjugation, Figure 1A) or in
the protein (DsEL conjugation, Figure 1B). Ten-fold ex-
cess of ligand with respect to the protein conjugation
sites was typically used, and the reaction was incubated
at 26 °C overnight. In our early conjugation attempts,
we observed a significant amount of DsEL multimeriza-
tion (Figure 3A) likely due to intermolecular reaction of
activated protein carboxyls with protein amines. Protein
multimerization was not observed in WsEL conjugation
possibly due to the lower carboxyl to EDC/NHS ratio
and to the higher availability of ligand carboxyls (free
and more mobile) versus protein carboxyls (protein-
bound and possibly buried) for reaction with protein
amines. To prevent DsEL multimerization, protein
amines were protected by acylation (mass spectrum,
Figure S5B). This pretreatment dramatically reduced
DsEL multimerization (Figure 3B). The removal of the
protecting reagent, acetyl chloride, necessary to avoid
acylation of the aminated ligand, was achieved very
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. SDS-PAGE analysis shows that activation of protein
carboxyls leads to uncontrollable protein multimerization
probably due to intermolecular reactions. Reaction between ac-
tivated (reaction) or non-activated (mock) DsEL and TAAy, were
analyzed by either UV transillumination (left panels of A and B) or
Coomassie Blue stain (right panels of A and B) of SDS-PAGE gels.
Amines in DsEL were either non-protected (A) or protected (B) by
acylation. When protection was not performed, we observed pro-
tein multimerization (A, reaction lanes). On the contrary, with pro-
tection only a miniscule amount of DsEL dimer was observed (B,
reaction lanes). The product of mock reaction (A, mock lanes) ap-
pears to be free of non-covalently bound TAAy, as revealed by the
absence of visible fluorescence associated with the DsEL band
and at the solvent front. However, analysis of the reaction product
reveals free TAAy at the solvent front, suggesting an interaction
between unreacted and covalently-bound ligand, disrupted during
gel electrophoresis.
Among the compatible solvents, we have chosen NMP
for the work presented here. We have also observed that
Figure 4. SDS-PAGE analysis shows that our methodology of amphiphile conjugation in organic solvent produces monomeric conju-
gates virtually free of non-covalently bound ligand. The purified product of reaction between activated (reaction) or non-activated (mock)
RuCOOH and WsEL (A), and between activated (reaction) or non-activated (mock) DsEL and either TAAy (B) or OPPVNH2 (C) were analyzed
by SDS-PAGE. Gels were examined by UV transillumination (left of each section) and Coomassie Blue staining (right of each section). The
fluorescence (derived from the ligand) is mainly associated with sEL (only minimally dimerized) in the reaction lanes. Negligible amount of ligand
non-covalently bound to the protein is observed in conjugation to TAAy or OPPVNH2, as shown by the minor amount of fluorescence associated
with the DsEL band in the mock lanes. Trace amount of ligand non-covalently bound to covalently-bound ligand (migrating with the front of the
solvent in denaturing conditions) was observed for DsEL-OPPVNH2 by UV transillumination captured with long exposure time (C).
ACS Paragon Plus Environment
4

Page 5 of 15
Bioconjugate Chemistry
conveniently by brief rotatory evaporation at low tem- Conjugate characterization. Successful chemical
1
perature (second step in Figure 1B). This step did not
result in any significant removal of NMP (a low volatil-
ity solvent), and it allowed proceeding to the next reac-
tion step within the same vessel. To monitor non-cova-
lent conjugation, mock reactions free of EDC and NHS
(i.e. containing non-activated protein or ligand) were al-
ways run in parallel with the conjugation reactions. At
the end of the reaction, addition of water caused precip-
itation of most unreacted ligand in DsEL conjugations
(Figure 1D). Extraction of the conjugated protein by
precipitation in acetonitrile (ACN) helped remove more
unreacted ligand in DsEL conjugation reactions (Figure
1D, second step) and was used as an initial purification
step for WsEL conjugation (Figure 1C, first step). Re-
sidual unreacted ligand in DsEL conjugation reactions
was removed by either acetonitrile wash of the protein
pellet (Figure 1D, third step) or by dichloromethane ex-
traction of solution obtained by resuspending the protein
pellet in water (Figure S6). For WsEL conjugation, re-
sidual unreacted ligand was removed by ultrafiltration
of the water-solubilized protein pellet obtained by ACN
precipitation (Figure 1C, second step). After the work
up procedure, conjugated sEL retained the thermal re-
sponsiveness of the unmodified sEL. Purified WsEL-
RuCOOH was completely free of non-covalently bound
ligand, as shown by UV transillumination of SDS-
PAGE gels (no fluorescence associated with the protein
band in the mock reaction nor detected at the solvent
front, Figure 4A) and by the UV/Vis analysis (no ligand
absorbance detected in the mock reaction). A minuscule
amount of ligand non-covalently bound to the protein
was observed in purified DsEL-TAAy and DsEL-
OPPVNH2, as shown by UV transillumination of SDS-
PAGE gels (minor amount of fluorescence associated
with the protein band was observed in the mock reac-
tions, Figure 4B and C) and by the UV/Vis analysis
(modest ligand absorbance in mock reactions, Figure
S7B and C). In the case of DsEL-OPPVNH2 negligible
amount of ligand non-covalently interacting with the co-
valently-bound OPPVNH2 was detected by SDS-PAGE
with UV transillumination at long exposure time (minor
amount of fluorescence was observed at the solvent
front in the reaction, Figure 4C, see further discussion
below).
conjugation of RuCOOH to WsEL was confirmed by
Electrospray Ionization Mass Spectrometry (ESI-MS)
and NMR (TOCSY and ROESY). ESI-MS revealed a
11,911 Da (m/z) molecule (Table S1 and Figure S4B)
consistent with the WsEL-RuCOOH conjugate. Further-
more, the de-convolved spectrum obtained for WsEL-
RuCOOH showed no trace of unbound WsEL. Since we
have observed that the unconjugated WsEL ionizes bet-
ter than the conjugated counterpart, this result suggested
high protein derivatization. TOCSY and ROESY NMR
analysis of free and conjugated WsEL further proved co-
valent conjugation (Figures 5, S8, and S9). Upon conju-
gation, the terminal amine of Ala1 becomes an amide
and shifts in the spectrum from 8.37 (Figure S8A), to
9.53 (Figure S8C) ppm while the Hα proton of Ala1
shifts from 4.26 to 4.54 ppm (Figure 5) unambiguously
indicating ligand coupling. This is further confirmed by
ROESY spectra showing cross-peaks between Ala1 NH
resonance and resonances of the ligand (Fig. S9).
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. TOCSY spectrum confirms ligand coupling to WsEL
Ala1. Upon conjugation of WsEL to RuCOOH the Hα proton of al-
anine at position 1 (Ala1) shifts from 4.26 to 4.54. Gray: WsEL;
Red: WsEL-RuCOOH.
Conjugation efficiency was quantified by UV/Vis spec-
trophotometry analysis (Figure S7A), which was per-
formed on the other two conjugates as well (Figure S7B
and C). Known concentrations of free ligand were used
to determine the equation describing the linear correla-
tion between absorbance and concentration (Beer-Lam-
bert law). This equation was then used to convert the
ligand absorbance of the purified protein obtained from
Table 1. Efficiency of protein conjugation
Conjugate
(protein-ligand)
WsEL-RuCOOH
DsEL-TAAy
[Protein] [Ligand] [non-covalently bound ligand]
Conjugation efficiency
(mM)a
39.7
12.1
58.7
(mM)b
38.8
12.7
73.7
(mM)
(% protein derivatization)e
None detected
0.974c
9.41c + 5.81d
97.7
96.9
99.7
DsEL-OPPVNH2
a concentration of protein from conjugation reaction determined by weight after extensive dialysis and lyophilization
b ligand associated with protein from conjugation reaction, as determined by UV/Vis spectrophotometry (red in Figure S7)
c ligand interacting non-covalently with the protein from conjugation mock, as determined by UV/Vis spectrophotometry (blue in Figure S7)
d ligand interacting non-covalently with the covalently-bound ligand in conjugation reaction, as determined by spot densitometry (Figure S13)
e monosubstitution
ACS Paragon Plus Environment
5

Bioconjugate Chemistry
Page 6 of 15
conjugation reaction or mock (extensively dialyzed, ly- in aqueous phase, even after insertion of extra amines,
1
2
3
4
5
6
7
8
ophilized and carefully weighed) to concentration or
protein-associated ligand. In the case of WsEL-
RuCOOH conjugation, there was no detectable non-co-
valently bound ligand, as demonstrated by the absence
of ligand absorbance in the purified protein from mock
conjugation, and by SDS-PAGE analysis of mock and
reaction (Figure 4A).The percentage of conjugation was
calculated using the ratio of the net ligand concentration
in the protein obtained from conjugation reaction (i.e.
[Ligand] minus [non-covalently bound ligand] in Table
1) to protein concentration ([Protein] in Table 1; ob-
tained by weight). Based on this method, derivatization
of the only amine (N-terminal) in WsEL was 97.7%
complete. We confirmed the conjugation quantification
using 700 MHz 1H NMR analysis of conjugated and free
WsEL (Figures 6 and S10). Resonance assignments
were obtained from TOCSY spectra (Figure S8). To cal-
culate the average ratio of elastin to ligand we used the
intensities of individual resonances of Trp132 and the
ligand in the aromatic region of the WsEL-RuCOOH
spectrum (blue trace in Figure 6), which are well re-
solved and quantifiable. The resulting estimate of con-
jugation efficiency was 95 ± 6%. This result corrobo-
rates the UV/Vis quantification. The good agreement of
the conjugation efficiencies obtained through NMR and
UV/Vis analyses renders the two methods interchange-
able. Both methods allow the reuse of the analyzed sam-
ple, however the UV/Vis method is preferable when
dealing with low scale reactions. The conjugation effi-
ciency obtained with WsEL is remarkable, since in other
reported attempts to conjugate ELPs through free
amines, at least one lysine was inserted in the se-
quence,57-62 indicating that the presence of the N -termi-
nal amine alone did not afford the desired level of con-
jugation. Furthermore, when reactions were performed
the ratio ligand:elastin ranged from 0.5 to 1, with 25 to
60
50% non-site selective amine derivatization.57,
Thanks to the high efficiency of our conjugation
method, only one amine is needed in the ELP to obtain
near complete derivatization of the polymer, also result-
ing in knowing the exact location of the derivatization.
Successful chemical conjugation of TAAy and
OPPVNH2 to DsEL was confirmed by Matrix Assisted
Laser Desorption/Ionization Time-of-Flight Mass Spec-
trometry (MALDI-TOF MS, Figure S11) and ESI-MS
(Figure S12), respectively (Table S1). The concentra-
tion of DsEL covalently linked to TAAy was calculated
as described for WsEL-RuCOOH (Figure S7B), except
that in this reaction a minuscule amount of non-cova-
lently bound ligand was detected in the mock reaction
(Figure 4B, mock lanes and Figure S7B). This ligand
was quantified by UV/Vis analysis and used to correct
the calculation. The resulting efficiency of conjugation
(as % of monosubstituted protein) was found to be
96.9% (Table 1). DsEL-OPPVNH2 also included a
small amount of non-covalently linked OPPVNH2
strongly associated to DsEL (Figure 4C, mock lanes).
This excess ligand was quantified by UV/Vis analysis
of the mock reaction and used for correcting the conju-
gation efficiency as for DsEL-TAAy (Figure S7C). An-
other fraction of the non-covalently linked OPPVNH2
(separated from the conjugated protein in denaturing
conditions and migrating with the solvent front),
seemed to be only associated to the covalently conju-
gated OPPVNH2 (Figure 4C, left panel, reaction lane,
and Figure S13A) and could not be detected by the
UV/Vis analysis of the mock reaction. The concentra-
tion of this ligand was therefore quantified by spot in-
tensity (Figure S13B) and used to further correct the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. NMR data reveal the conjugation efficiency to be 95 ± 6%. 1H NMR spectra (700 MHz, aromatic region) of free WsEL (black) and
WsEL-RuCOOH conjugate (blue) were used to determine conjugation efficiency. The individual Trp132 indole ring resonances and ligand res-
onances used for integration are labeled with colored dots also used to indicate the correspondent hydrogens in the structure of ligand
(RuCOOH, on the left) and Tryptophan (on the right).
ACS Paragon Plus Environment
6

Page 7 of 15
Bioconjugate Chemistry
concentration of covalently bound OPPVNH2 calcu- Conjugation is performed in organic solvent and puri-
1
2
3
4
5
6
7
8
lated by the UV/Vis method. The final efficiency of con-
jugation was 99.7% (Table 1) with virtually no multi-
merization. These results are noteworthy, since conju-
gation of amine-functionalized ligand to protein carbox-
yls with might be problematic Activated protein carbox-
yls tend to react with protein amines making protein
conjugation sites less available for reaction with ligand
and causing protein multimerization. In rare reports of
the successful use of this conjugation configuration,63, 64
the protein free amines were protected by lengthy pro-
cedures, and extensive dialysis was performed in be-
tween protection and conjugation. An additional report
states the failure to conjugate amine-containing ligand
to protein carboxyls, which prompted the thiolation of
the amine-containing ligand, and attachment of malei-
mide to the protein amines, leading to conjugation
through thiol–maleimide Michael addition.65 Compared
to these methods, our procedure, which includes amine
protection, carboxyl activation and conjugation to
amine moieties in “one pot”, is much nimbler and more
convenient. The availability of a viable alternative to the
most common ligand conjugation through protein
amines is very useful when trying to preserve protein
activity upon conjugation. For example, an antibody
might lose its immunoreactivity upon ligand conjuga-
tion through amines, but if the antibody binding site
lacks (or includes less critical) carboxyl groups, car-
boxyl-directed conjugation of an amine-functionalized
ligand is less likely to disturb the antibody-antigen in-
teraction.
fied conjugates are virtually free of non-covalent pro-
tein-ligand complexes, which are prevalent when reac-
tions are performed in water. Amine derivatization is
quantitative. Carboxyl derivatization, although not
quantitative, results in 98% protein derivatization when
three carboxyls are present. Furthermore, protein multi-
merization is avoided and unreacted ligand can be re-
covered efficiently in high purity.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL PROCEDURES
sEL production. The WsEL expression construct
was described previously.7 The DsEL gene was de-
signed based on the ELP-1 construct described previ-
ously.5-7 Addition of a carboxyl group to facilitate con-
jugation was achieved by PCR insertion of DNA encod-
ing the N-terminal sequence AGDGS (red in Figure
S15). Purified PCR product was subcloned into the
BsshII and NheI sites of the POE expression vector (Qi-
agen). ELP expression was achieved using a protocol
adapted from Hassouneh et al.66 and previously de-
scribed.5-7 Briefly, plasmids encoding WsEL or DsEL
were transformed into competent BL21(DE3) E. coli
(NEB). Transformed cells were plated on selective solid
medium (2xYT + agar + carbenicillin) overnight. A sin-
gle colony was transferred in 15 mL starter culture and
grown at 37 ºC at 300 rpm. Upon becoming visibly
cloudy (2-3 h), this culture was used to inoculate 1 L
2xYT. The culture was allowed to grow for 24 h at 37
ºC without induction. Cells were harvested by centrifu-
gation and the periplasmic fraction was obtained by cold
osmotic shock in 20% sucrose buffer. sELs were puri-
fied by inverse temperature cycling. Protein purity was
assessed by SDS-PAGE (Invitrogen, NP0343BOX),
and electrospray ionization (Synapt G2 ESI-MS, Wa-
ters, Figure S4A and Figure S5A). SDS-PAGE gels
were stained using the Coomassie-based stain GelCode
Blue (Pierce). Following purification, sELs were dia-
lyzed twice against 4 L of cold deionized (DI) water
(first time for 4 h, and second time for 12 h in fresh wa-
ter) at 4 ºC in a 10 KDa MWCO Slide-A-Lyzer dialysis
cassette (Pierce, 66383). Dialyzed proteins were lyoph-
ilized and stored dry at -20 ºC.
We verified the preservation of the characteristic ELP
reversible phase transition behavior in water following
our conjugation methodology. Investigators studying
proteins that may denature in organic solvents should
verify that the structure and/or function of the derivat-
ized protein is comparable to the structure and function
of the unmodified protein.
Recovery of unreacted ligand. Recovery of unre-
acted ligand might be desirable when custom-made, ra-
ther than inexpensive, commercially available com-
pounds, are used. When amine-containing ligands
TAAy and OPPVNH2 were reacted with the activated
protein carboxyls of DsEL, there was no ligand modifi-
cation prior to conjugation to protein and therefore the
unreacted ligand could be recovered intact. Exploiting
the low solubility of ligands in water and the insolubility
of sEL in acetonitrile, we were able to separate unre-
acted ligand from the protein conjugate. Except for re-
sidual NMP, the recovered TAAy and OPPVNH2 were
fairly pure as determined by 1H NMR (Figure S14).
RuCOOH and RuNH2 synthesis. The ruthenium
complexes
[Ru(2,2’-bipyridine)-2-(2,2’-bipyridine-
4,4’-dicarboxylic acid)](PF6)2, RuCOOH, and [Ru(2,2’-
bipyridine)-2-(5-amino-1,10-phenanthroline)](PF6)2,
RuNH2 were prepared and characterized via well-estab-
lished protocols.41, 42
TAAy synthesis. N-(4-Aminophenyl)- N-phenyl-1-
naphthalylamine (TAAy)39 was synthesized by Pd/C-
catalyzed hydrazine reduction of the dinitro compound,
which was prepared through the NaH-mediated nucleo-
philic aromatic substitution reaction of N-phenyl-1-
naphthalylamine with 4-fluoronitrobenzene.
CONCLUSION
We have developed and validated a versatile protocol
for derivatization of amines or carboxyls within am-
phiphilic proteins/polymers, with hydrophobic ligands
containing carboxyls or amines respectively.
OPPVNH2 synthesis. 4-[2-(2,5-Dimethoxy-4-styryl-
phenyl)-vinyl]-phenylamine
(OPPVNH2)
was
ACS Paragon Plus Environment
7

Bioconjugate Chemistry
Page 8 of 15
synthesized by stannous chloride-assisted reduction of DsELconjugation to OPPVNH2 or TAAy. Conjuga-
1
2
3
4
5
6
7
8
the nitro-compound, which was achieved via the
Horner-Wadsworth-Emmons method by reacting
tion of OPPVNH2 or TAAy with ELP was performed by
EDC/NHS coupling, preceded by acetyl chloride (AcCl,
Sigma-Aldrich, 00990) protection in the presence of
diisopropylethylamine (DIEA, Sigma-Aldrich, 03440)
(Figure 1B). A standard reaction utilized 1 to 2 mg of
DsEL. Typically, 40 µL of a 2.25 mM solution of DsEL
in NMP was mixed with 80 µL of a 5.6 mM solution of
Diisopropylethylamine (DIEA) and AcCl in NMP (5-
fold excess of DsEL amines). The solution was incu-
bated on ice for 1 h and 45 min. AcCl, and some DIEA,
were aspirated by either rotavaping on ice for 10 min or
placing the frozen solution in a lyophilizer for 5 min.
The acetylation product was characterized by ESI-MS
(Figure S5B). Removal of AcCl was necessary to avoid
acetylation of the ligand added in the subsequent step.
Half of the protected protein (60 µL) was removed and
mixed with 60 µL NMP (mock reaction). The remaining
60 µL of the protected protein was mixed with 60 µL of
a 22.5 mM EDC and 16.8 mM NHS solution in NMP
(reaction, 10-fold EDC and 7.5-fold NHS in excess of
DsEL carboxyls). Immediately after this addition, mock
and reaction solutions both received 2 µL of a 675 mM
solution of OPPVNH2 or TAAy in NMP (10-fold in ex-
cess of DsEL carboxyls) and 2 µL of a 13.5 mM DIEA
solution in NMP (to facilitate activation of DsEL car-
boxyls in the reaction solution). Both solutions were in-
cubated at 26 °C for 12 h, with shaking (1300 rpm). A
small fraction of reaction or mock solution (2 µL) was
mixed with PBS (10 µL), causing the excess unreacted
OPPVNH2 or TAAy to precipitate. This step was
needed to avoid excessive fluorescence coming from the
unreacted ligand, and clear visualization of the fluores-
cence associated with DsEL, in the subsequent SDS-
PAGE analysis. Upon centrifugation (15,000 rpm, 10
min, 4 °C), supernatants were mixed 1:1 with loading
buffer, and analyzed by SDS-PAGE. This analysis was
needed to verify covalent conjugation in the reaction
and lack of covalent conjugation in the mock before
subsequent workup. Unreacted ligand was precipitated
from the remainder of reaction or mock solutions by
adding 5 volumes of cold water and then centrifuging
the solution. This step allows for recovery of pure unre-
acted OPPVNH2 or TAAy (in the precipitate). Superna-
tants were frozen and concentrated in a lyophilizer for
roughly 15 min (wherein the volume was reduced from
120 µL to 40 µL). DsEL was precipitated by addition of
40 µL ACN. In order to remove residual non-covalently
bound ligand, the precipitant was either washed 3 times
with ACN, or resuspended in water and extracted 3
times with the same volume of CH2Cl2 (VWR, UN
1593). Washed precipitate (if using ACN wash), or pro-
tein film between phases (if using CH2Cl2 extraction),
was resuspended in water and dialyzed twice against 4
L of cold water (first time for 4 h, second time for 12 h
in fresh water) in a 10,000 MWCO Slide-A-Lyzer. The
dialyzed and purified product of mocks and reactions
phosphonate
carbanions
of
[4-(diethoxy-
phosphorylmethyl)-2,5-dimethoxybenzyl]-phosphonic
acid diethyl ester with subsequent benzaldehyde and
then 4-nitrobenzaldehyde.5, 40
WsEL conjugation with RuCOOH. The required
amount of WsEL protein, RuCOOH ligand, EDC,
(Thermo Scientific 22980), NHS, (Thermo Scientific
24510) and triethylamine (TEA, Alpha Aesar 12391)
were weighed ahead of time into separate small glass
vials before combining. 340 µL of NMP (Sigma-Al-
drich, 328634) was used to solubilize 6.8 mg of WsEL
protein (0.6 µmol). The solution was added to 10-fold
molar excess of RuCOOH with respect to WsEL (3.9
mg, 6 µmol). Once RuCOOH was fully solubilized
within the protein solution, 10-fold molar excess of
EDC and NHS with respect to WsEL (1.2 mg and 1.3
mg respectively, or 6 µmol of each) was added. Finally,
TEA (a base used to facilitate ligand COOH activation)
was added to the solution in 1.5-fold molar excess with
respect to WsEL (12.5 µL of 72 mM TEA stock). The
solution was sealed and incubated overnight while mix-
ing (1300 rpm, TAITEC BioShaker N-BR-022UP). The
mock reaction was prepared in a similar fashion, except
that no EDC nor NHS was present. As a preliminary pu-
rification step, acetonitrile (ACN, Fisher Scientific,
A955-212), was added to the reaction solution (~4.5:1
volume ratio ACN:reaction). The solution mixture was
centrifuged and the supernatant was removed. Since
RuCOOH, EDC, and NHS are soluble in ACN, but the
protein polymer is not, this step allowed to separate the
protein (in the precipitate) from the unreacted reagents
(in the supernatant). Sterile-filtered cold water (4 ºC)
was added to the precipitate to re-solubilize the conju-
gated protein. The cold solution was then transferred to
a prewashed and cooled 15 mL 10 kDa MWCO centrif-
ugal filter (EMD Millipore) and cold water was added
to further dilute the solution. The solution was mixed by
pipetting up and down before centrifugation at ~15K
rpm, at 4 ºC. The centrifugation progression was
checked periodically (every ten minutes) to avoid exten-
sive concentration on top of the filter, thus leading to
sticking of conjugated product to centrifuge filter mem-
branes and loss of product. Sequential addition of 4 ºC
cold water to the centrifuge filter, re-mixing, and centri-
fuging was carried out until the flow through was color-
less. The conjugated product was collected and dialyzed
twice against 4 L of cold DI water (first time for 4 h, and
second time for 12 h in fresh water) at 4 ºC in a 10 KDa
MWCO Slide-A-Lyzer dialysis cassette. Finally, the
product was collected and lyophilized overnight (Lab-
cono, Freezone). Conjugated protein was stored dry at -
20 ºC until needed and it was characterized by SDS-
PAGE, ESI-MS, UV/Vis and NMR.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
8

Page 9 of 15
Bioconjugate Chemistry
were lyophilized overnight and a cottony solid was ob- OPPVNH2). Measurement was performed on a Waters
1
2
3
4
5
6
7
8
tained. Conjugates were stored dry at -20 ºC and char-
acterized by SDS-PAGE, ESI-MS or MALDI-TOF MS,
and UV/Vis.
Instrument (Synapt G2), using MassLynx software. For
WsEL-RuCOOH and WsEL the instrument settings
were: Capillary kV = 5; Sampling Cone = 25; Extraction
cone = 1.2; Source T = 80 °C; Desolvation T = 200 °C;
Cone gas flow = 32 (L/h); Desolvation gas flow = 500
(L/h). For DsEL the settings were the same as above,
except that the Cone gas and the Desolvation gas flow
rates were 200 and 700 L/h respectively. For DsEL-
OPPVNH2 and protected DsEL the instrument settings
were: Capillary kV = 5; Sampling Cone = 40; Extraction
cone = 0.3; Source T = 80 °C; Desolvation T = 100 °C;
Cone gas = 97 (L/h); Desolvation gas flow = 648 (L/h).
Data were processed using the MaxEnt1 tool of Mass-
Lynx. All measurements were conducted using a freshly
cleaned cone.
UV/Vis spectrophotometry analysis. The amount of
lyophilized protein (reaction or mock) needed to obtain
200 µL solution in double digit micromolar range was
weighed and dissolved in DMSO. The absorbance max-
imum (at 461, 308 and 390 nm for WsEL-RuCOOH,
DsEL-TAAy, and DsEL-OPPVNH2, respectively) was
measured and recorded. A set of solutions of known
concentration of ligand spanning from single to double-
digit micromolar concentration were analyzed by
UV/Vis, and their absorbance maxima were plotted vs
the corresponding concentrations. The equation defin-
ing the linear correlation between absorbance and con-
centration (Beer-Lambert law) was used to determine
the concentration of ligand present in the protein solu-
tion (reaction or mock), using the measured absorbance
(A) and the following equation: [ligand] = (A-y axis in-
tercept)/ε (extinction coefficient and slope of the line).
For WsEL conjugation, there was no ligand absorption
in the mock reaction, therefore ligand concentration in
the reaction was actually concentration of covalently-
bound ligand. A correction was needed for DsEL-TAAy
and DsEL-OPPVNH2. In the case of DsEL-TAAy the
concentration of covalently bound TAAy was calculated
using the following equation [ligand] = (A reaction-A
mock)/ε. The same calculation was used for DsEL-
OPPVNH2 but the resulting concentration was further
corrected using the spot intensity quantification de-
scribed next.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Matrix Assisted Laser Desorption/Ionization Time-
of-Flight Mass Spectrometry (MALDI-TOF MS)
analysis. Samples were mixed in a 10:1 ratio (ma-
trix:sample) with 10 mg/mL of ultrapure 2,5-Dihy-
droxybenzoic acid (DHB) (Protea, Morgantown, West
Virginia). Prior to sample analysis, the mass spectrom-
eter was externally calibrated with a TOF/TOF Calibra-
tion peptide mixture of des-Arg-Bradykinin (1.0
pmol/µL), Angiotensin I (2.0 pmol/µL, Glu-Fibrinopep-
tide B (1.3 pmol/µL), and adrenocorticotropic hormone
(ACTH), (1-17 clip-2.0 pmol/µL), (18-39 clip-
1.5pmol/µL), (7-38 clip-3.0 pmol/µL). The 4800 Plus
MALDI TOF/TOF Analyzer (ABSciex, Foster City,
CA, USA) was used in positive reflector mode. Data
were collected in manual mode and utilizing random
sampling over the entire sample spot. The mass spec-
trometer is equipped with a 200-Hz frequency Nd:YAG
laser, operating at a wavelength of 355 nm. Twenty five
sub-spectra for each of 100 randomized positions within
the spot (2,500 spectra/spot) were collected in MS-TOF
mode and presented as one main spectrum. Fixed laser
intensity in MS-TOF reflector positive mode, 3600 (ar-
bitrary units), final detector voltage set at 1.883 KV.
Spectral Mass range 1000-2500 m/z, focus mass 1800
m/z. Data were exported to Data Explorer™ Software,
Version 4.9 (ABSciex, Foster City, CA, USA) and in-
terpreted using the centroid function of the software.
NMR analysis. All spectra of WsEL and WsEL-
RuCOOH conjugate were obtained on a Bruker Avance
Spot intensity quantification of non-covalently
bound ligand. SDS-PAGE analysis of purified DsEL-
OPPVNH2 reaction revealed a small amount of
OPPVNH2 interacting non-covalently with the cova-
lently bound ligand (see buffer front at the bottom of
Figure 4C, “reaction lane”). In order to quantify this
OPPVNH2 a series of standard OPPVNH2 solutions of
known concentration were run on the same gel (Figure
S13A). The intensity of standard OPPVNH2 spots was
determined by using GeneTools analysis software
(Syngene) and plotted against the corresponding con-
centrations to obtain a calibration curve. The concentra-
tion associated with the spot intensity of OPPVNH2
freed from the covalently conjugated protein was deter-
mined using the following equation [ligand] = (SI-y axis
intercept)/ε, where SI is the intensity of the spot corre-
sponding to the freed OPPVNH2 (Figure S13B). This
piece of data was used to further correct the conjugation
quantification obtained by UV/Vis spectrophotometry
analysis.
1
III NMR spectrometer operating at H frequency of
700.13 MHz in DMSO-d6. One dimensional spectra
used for conjugation efficiency quantification were ob-
tained using recycle delay of 12 s (D1=10s, AQ=2s) to
allow for complete magnetization recovery between
scans. 512 scans per sample were collected, resulting in
acquisition time of 1.7 h per spectrum. Spectra were
apodized with 1Hz exponential function, Fourier trans-
formed and baseline corrected using a polynomial func-
tion. 1H-1H TOCSY spectra were obtained using
DIPSI2 mixing sequence67 for spin-lock (40 and 60 ms
mixing time), with 2048 data points in t2 and 512 points
in t1 and relaxation delay of 3 s. Quadrature detection in
Electrospray Ionization Mass Spectrometry (ESI-
MS) analysis. A continuous spray ESI-MS configura-
tion was used to analyze a 2 ng/µL conjugate solution in
1:1 H2O:ACN, 1% formic acid (for WsEL-RuCOOH)
or in 50 mM ammonium acetate pH 7.0 (for DsEL-
ACS Paragon Plus Environment
9

Bioconjugate Chemistry
Page 10 of 15
t1 was achieved using States-TPPI scheme. Data were Dimethylformamide; DMSO, Dimethyl sulfoxide;
apodized using a cos2 function, zero filled to a
2048x1024 matrix and Fourier transformed. 1H-1H
ROESY spectra were obtained using a standard pulse
sequence with cw spin-lock68, with mixing time of 300
or 400 ms. 2048 data points were collected in t2 and 512
points in t1 and relaxation delay was set to 3 s. Quadra-
ture detection in t1 was achieved using States-TPPI
scheme. Data were apodized using a cos2 function, zero
filled to a 2048x1024 matrix and Fourier transformed.
DsEL, short elastin with extra Aspartate (D); EDC, 1-
ethyl-3-(-3-dimethylaminopropyl) carbodiimide; ELP,
elastin-like polypeptide; ESI-MS, ElectroSpray Ioniza-
tion Mass Spectrometry; MALDI, Matrix Assisted La-
ser Desorption/Ionization Time-of-Flight Mass Spec-
trometry; NHS, N-hydroxysuccinimide; NMP, N-Me-
thyl-2-pyrrolidone; NMR, nuclear magnetic resonance;
OPPV, p-phenylene vinylene oligomers; PBS, phos-
phate buffer saline; sEL, short elastin; SDS-PAGE, so-
dium dodecyl sulfate poly acrylamide gel electrophore-
sis; TAAy, triarylamine; TEA, triethylamine; W, Tryp-
tophan (W); WsEL, short elastin with C-terminal Tryp-
tophan (W).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information.
Structure of RuNH2; sEL variants sequences; sEL be-
havior in organic solvents and water; SDS-PAGE
analysis of reaction workup; Calculation of conjuga-
tion efficiency by UV/Vis absorbance; TOCSY spec-
tra of WsEL and WsEL-RuCOOH; ROESY spectrum
REFERENCES
(1)
Spicer, C. D., and Davis, B. G. (2014)
Selective chemical protein modification.
Nat Commun 5, 4740.
1
of conjugated WsEL; H NMR spectra of free and
(2)
Stephanopoulos, N., and Francis, M. B.
(2011) Choosing an effective protein
bioconjugation strategy. Nat. Chem. Biol.
7, 876-884.
Alley, S. C., Okeley, N. M., and Senter,
P. D. (2010) Antibody–drug conjugates:
targeted drug delivery for cancer.
Current opinion in chemical biology 14,
529-537.
Dadachova, E., and Casadevall, A.
(2014) Radiolabeled antibodies for
therapy of infectious diseases. Microbiol.
Spectr. 2, 0023.
Balog, E. R. M., Ghosh, K., Park, Y. I.,
Hartung, V., Sista, P., Rocha, R. C.,
Wang, H. L., and Martinez, J. S. (2016)
conjugated WsEL; Quantification of non-covalently
bound ligand by SDS-PAGE and spot intensity; H-
NMR of recovered ligands; Primer for DsEL cloning;
Table of calculated and found masses.
1
The Supporting Information is available free of charge
on the ACS Publications website (PDF).
(3)
AUTHOR INFORMATION
Corresponding Author
*Address: Bikini Atoll Rd, MS-K771, Los Alamos, NM,
87545; E-mail: jenm@lanl.gov; Phone: 505-665-0045
(4)
(5)
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
The authors acknowledge financial support by the La-
boratory Directed Research and Development (LDRD)
program for synthesis of conjugated oligomers and se-
lection of emerging and adaptive polymers. This work
was performed at the Center for Integrated Nanotech-
nologies, an Office of Science User Facility operated for
the U.S. Department of Energy (DOE) Office of Sci-
ence. Los Alamos National Laboratory, an affirmative
action equal opportunity employer, is operated by Los
Alamos National Security, LLC, for the National Nu-
clear Security Administration of the U.S. Department of
Energy under contract DE-AC52-06NA25396. We
thank Tatyana Elkin for the critical reading of the man-
uscript.
Stimuli-Responsive
Genetically
Hydrogel
Optical
Engineered
Demonstrates
Polymer
Emergent
Responses. ACS Biomater. Sci. Eng. 2,
1135-1142.
Ghosh, K., Balog, E. R. M., Kahn, J. L.,
Shepherd, D. P., Martinez, J. S., and
Rocha, R. C. (2015) Multicolor
Luminescence from Conjugates of
(6)
(7)
Genetically
Encoded
Elastin‐like
Polymers and Terpyridine‐Lanthanides.
Macromol. Chem. Phys. 216, 1856-1861.
Ghosh, K., Balog, E. R. M., Sista, P.,
Williams, D. J., Kelly, D., Martinez, J. S.,
and Rocha, R. C. (2014) Temperature-
dependent morphology of hybrid
ABBREVIATIONS
2xYT, 2x yeast extract tryptone; AcCl, acetyl chloride;
D, Aspartate; CH2Cl2, Dichloromethane; DI, deionized;
DIEA,
diisopropylethylamine;
DMF,
ACS Paragon Plus Environment
10

Page 11 of 15
Bioconjugate Chemistry
nanoflowers
from
elastin-like
(17) Kim, W., Xiao, J., and Chaikof, E. L.
(2011) Recombinant amphiphilic protein
micelles for drug delivery. Langmuir 27,
14329-14334.
1
2
3
4
5
6
7
8
9
polypeptides. Apl Materials 2, 021101.
Berndt, P., Fields, G. B., and Tirrell, M.
(1995) Synthetic Lipidation of Peptides
and Amino-Acids - Monolayer Structure
and Properties. J. Am. Chem. Soc. 117,
9515-9522.
(8)
(18) Gonzalez-Toro,
D.
C.,
and
Thayumanavan, S. (2013) Advances in
Polymer and Polymeric Nanostructures
for Protein Conjugation. Eur. Polym. J.
49, 2906-2918.
(9)
Jain, R. K., Gupta, C. M., and Anand, N.
(1981)
Synthesis
of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Peptidylglycophospholipids,
Derivatives of Muramyl-Dipeptide.
Tetrahedron Lett. 22, 2317-2320.
Novel
(19) Qi, Y., and Chilkoti, A. (2015) Protein-
polymer conjugation-moving beyond
PEGylation. Curr. Opin. Chem. Biol. 28,
181-193.
(20) Acar, H., Ting, J. M., Srivastava, S.,
LaBelle, J. L., and Tirrell, M. V. (2017)
Molecular engineering solutions for
therapeutic peptide delivery. Chem. Soc.
Rev. 46, 6553-6569.
(10) Thompson, N. L., Brian, A. A., and
McConnell, H. M. (1984) Covalent
linkage of a synthetic peptide to a
fluorescent phospholipid and its
incorporation
into
supported
phospholipid monolayers. Biochim.
Biophys. Acta 772, 10-19.
(21) Zhang, L., Gu, F. X., Chan, J. M., Wang,
A. Z., Langer, R. S., and Farokhzad, O.
C. (2008) Nanoparticles in medicine:
(11) Chakrabarti, A., Kelkar, D. A., and
Chattopadhyay, A. (2006) Spectrin
organization and dynamics: new insights.
Biosci. Rep. 26, 369-386.
(12) Wang, K., Xiao, Y., Wang, Y., Feng, Y.,
Chen, C., Zhang, J., Zhang, Q., Meng, S.,
Wang, Z., and Yang, H. (2016) Self-
assembled hydrophobin for producing
water-soluble and membrane permeable
fluorescent dye. Sci. Rep. 6, 23061.
therapeutic
applications
and
developments. Clin. Pharmacol. Ther.
83, 761-769.
(22) Kim, Y., Lillo, A. M., Steiniger, S. C.,
Liu, Y., Ballatore, C., Anichini, A.,
Mortarini, R., Kaufmann, G. F., Zhou, B.,
Felding-Habermann, B., and Janda, K. D.
(2006) Targeting heat shock proteins on
cancer cells: selection, characterization,
and cell-penetrating properties of a
peptidic GRP78 ligand. Biochemistry 45,
9434-9444.
(23) Lillo, A. M., Sun, C., Gao, C., Ditzel, H.,
Parrish, J., Gauss, C. M., Moss, J.,
Felding-Habermann, B., Wirsching, P.,
Boger, D. L., and Janda, K. D. (2004) A
human single-chain antibody specific for
integrin alpha3beta1 capable of cell
internalization and delivery of antitumor
agents. Chem. Biol. 11, 897-906.
(13) Chakrabarti, A. (1996) Fluorescence of
spectrin-bound
prodan.
Biochem.
Biophys. Res. Commun. 226, 495-497.
(14) Xia, X. X., Wang, M., Lin, Y., Xu, Q.,
and Kaplan, D. L. (2014) Hydrophobic
drug-triggered
self-assembly
of
nanoparticles from silk-elastin-like
protein polymers for drug delivery.
Biomacromolecules 15, 908-914.
(15) Green, M., Manikhas, G., Orlov, S.,
Afanasyev, B., Makhson, A., Bhar, P.,
and Hawkins, M. (2006) Abraxane®, a
novel Cremophor®-free, albumin-bound
particle form of paclitaxel for the
treatment of advanced non-small-cell
lung cancer. Ann. Oncol. 17, 1263-1268.
(16) Kataoka, K., Harada, A., and Nagasaki,
Y. (2001) Block copolymer micelles for
drug delivery: design, characterization
and biological significance. Adv Drug
Deliv Rev 47, 113-131.
(24) Lin, E. W., Boehnke, N., and Maynard,
H.
D.
(2014)
Protein-polymer
conjugation via ligand affinity and
photoactivation of glutathione S-
transferase. Bioconjug. Chem. 25, 1902-
1909.
(25) Anderson, G. W., Zimmerman, J. E., and
Callahan,
F.
M.
(1963)
N-
hydroxysuccinimide esters in peptide
synthesis. J. Am. Chem. Soc. 85, 3039.
ACS Paragon Plus Environment
11

Bioconjugate Chemistry
Page 12 of 15
(26) Zanetti-Domingues, L. C., Tynan, C. J.,
properties. J. Polym. Sci., Part A: Polym.
Chem. 48, 1469-1476.
1
2
3
4
5
6
7
8
Rolfe, D. J., Clarke, D. T., and Martin-
Fernandez, M. (2013) Hydrophobic
Fluorescent Probes Introduce Artifacts
(34) Binnemans, K. (2009) Lanthanide-based
luminescent hybrid materials. Chem. Rev.
109, 4283-4374.
(35) Urry, D. W. (1997) Physical chemistry of
biological free energy transduction as
demonstrated by elastic protein-based
polymers. J. Phys. Chem. B 101, 11007-
11028.
(36) Meyer, D. E., and Chilkoti, A. (2004)
Quantification of the effects of chain
length and concentration on the thermal
behavior of elastin-like polypeptides.
Biomacromolecules 5, 846-851.
(37) Lopez, G., Chilkoti, A., Atanassov, P.,
and Goparaju, V. R. (2004) Stimuli-
responsive hybrid materials containing
into
Single
Molecule
Tracking
Experiments Due to Non-Specific
Binding. PLoS One 8, e74200.
(27) Janosch, S., Nicolini, C., Ludolph, B.,
Peters, C., Volkert, M., Hazlet, T. L.,
Gratton, E., Waldmann, H., and Winter,
R. (2004) Partitioning of dual-lipidated
peptides into membrane microdomains:
Lipid sorting vs peptide aggregation. J.
Am. Chem. Soc. 126, 7496-7503.
(28) Bull, S. R., Guler, M. O., Bras, R. E.,
Meade, T. J., and Stupp, S. I. (2005) Self-
assembled peptide amphiphile nanofibers
conjugated to MRI contrast agents. Nano
Lett. 5, 1-4.
(29) Yokoyama, M., Inoue, S., Kataoka, K.,
Yui, N., and Sakurai, Y. (1987)
Preparation of adriamycin‐conjugated
poly (ethylene glycol)‐poly (aspartic
acid) block copolymer. A new type of
polymeric anticancer agent. Macromol.
Rapid Commun. 8, 431-435.
(30) Brouwer, H., Hilberer, A., Krasnikov, V.,
Werts, M., Wildeman, J., and
Hadziioannou, G. (1997) LEDs based on
conjugated PPV block copolymers.
Synth. Met. 84, 881-882.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
molecular
actuators
and
their
applications, University of New Mexico,
STC. UNM, USA. US6755621B2.
(38) Li, L., Li, N. K., Tu, Q., Im, O., Mo, C.-
K., Han, W., Fuss, W. H., Carroll, N. J.,
Chilkoti, A., and Yingling, Y. G. (2017)
Functional Modification of Silica
through Enhanced Adsorption of Elastin-
Like Polypeptide Block Copolymers.
Biomacromolecules 19, 298-306.
(39) Park, Y. I., Kuo, C. Y., Martinez, J. S.,
Park, Y. S., Postupna, O., Zhugayevych,
A., Kim, S., Park, J., Tretiak, S., and
Wang, H. L. (2013) Tailored electronic
structure and optical properties of
conjugated systems through aggregates
and dipole-dipole interactions. ACS Appl
Mater Interfaces 5, 4685-4695.
(31) Friend, R. H., Gymer, R. W., Holmes, A.
B., Burroughes, J. H., Marks, R. N.,
Taliani, C., Bradley, D. D. C., Dos
Santos, D. A., Bredas, J. L., Logdlund,
M., and Salaneck, W. R. (1999)
Electroluminescence in conjugated
polymers. Nature 397, 121-128.
(40) Park, Y. I., Postupna, O., Zhugayevych,
A., Shin, H., Park, Y. S., Kim, B., Yen,
H. J., Cheruku, P., Martinez, J. S., Park,
J. W., Tretiak, S., and Wang, H. L. (2015)
A new pH sensitive fluorescent and white
(32) Jørgensen, M., and Krebs, F. C. (2004)
Stepwise and directional synthesis of
end-functionalized
single-oligomer
light
emissive
material
through
OPVs and their application in organic
solar cells. J. Org. Chem. 69, 6688-6699.
(33) Wu, H. Y., Wang, K. L., Liaw, D. J., Lee,
K. R., and Lai, J. Y. (2010)
Electrochromic material containing
unsymmetrical substituted N, N, N′, N′‐
controlled intermolecular charge transfer.
Chem. Sci. 6, 789-797.
(41) Terpetschnig, E., Szmacinski, H., Malak,
H., and Lakowicz, J. R. (1995) Metal-
ligand complexes as a new class of long-
lived
fluorophores
for
protein
tetraaryl‐1,
Synthesis
4‐phenylenediamine:
and their optical,
hydrodynamics. Biophys. J. 68, 342-350.
(42) Ellis, C. D., Margerum, L. D., Murray, R.
W., and Meyer, T. J. (1983) Oxidative
electrochemical, and electrochromic
ACS Paragon Plus Environment
12

Page 13 of 15
Bioconjugate Chemistry
Electropolymerization of Polypyridyl
Complexes of Ruthenium. Inorg. Chem.
22, 1283-1291.
(51) Cavalli, S., Albericio, F., and Kros, A.
(2010) Amphiphilic peptides and their
cross-disciplinary role as building blocks
for nanoscience. Chem. Soc. Rev. 39,
241-263.
(52) Kuijpers, A. J., van Wachem, P. B., van
Luyn, M. J., Brouwer, L. A., Engbers, G.
H., Krijgsveld, J., Zaat, S. A., Dankert, J.,
and Feijen, J. (2000) In vitro and in vivo
1
2
3
4
5
6
7
8
9
(43) Chapman, A. P. (2002) PEGylated
antibodies and antibody fragments for
improved therapy: a review. Adv Drug
Deliv Rev 54, 531-545.
(44) Tada, S., Abe, H., and Ito, Y. (2013)
PEGylated Antibodies and DNA in
Organic Media and Genetic PEGylation,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
evaluation
of
gelatin-chondroitin
in
Green
Polymer
Chemistry:
sulphate hydrogels for controlled release
of antibacterial proteins. Biomaterials 21,
1763-1772.
Biocatalysis and Materials II. Volume
1144 (Cheng, H. N., Gross, R. A., Smith,
P. B., Ed.) pp 223-233, Chapter 16, ACS
Publications.
(53) Latha, M. S., Lal, A. V., Kumary, T. V.,
Sreekumar, R., and Jayakrishnan, A.
(2000) Progesterone release from
(45) Liu, M., Xu, M., Loh, X. J., Abe, H.,
Tsumuraya, T., Fujii, I., Li, J., Son, T. I.,
and Ito, Y. (2011) PEGylated antibody in
organic media. J Biosci Bioeng 111, 564-
568.
(46) Sasaki, S., Tokitsu, Y., Ikebukuro, K.,
Yokoyama, K., Masuda, Y., and Karube,
I. (1997) Biosensing of a herbicide using
a chemically modified antibody in
organic solvent. Anal. Lett. 30, 429-443.
(47) Konieczny, S., Krumm, C., Doert, D.,
Neufeld, K., and Tiller, J. C. (2014)
Investigations on the activity of poly (2-
oxazoline) enzyme conjugates dissolved
in organic solvents. J. Biotechnol. 181,
55-63.
glutaraldehyde
cross-linked
casein
microspheres: in vitro studies and in vivo
response in rabbits. Contraception 61,
329-334.
(54) Swatschek, D., Schatton, W., Muller, W.,
and Kreuter, J. (2002) Microparticles
derived from marine sponge collagen
(SCMPs): preparation, characterization
and suitability for dermal delivery of all-
trans retinol. Eur. J. Pharm. Biopharm.
54, 125-133.
(55) Hektor, H. J., and Scholtmeijer, K.
(2005) Hydrophobins: proteins with
potential. Curr. Opin. Biotechnol. 16,
434-439.
(48) Konieczny, S., Leurs, M., and Tiller, J. C.
(2015) Polymer enzyme conjugates as
(56) Urry, D. W. (1976) On the molecular
mechanisms of elastin coacervation and
chiral
ligands
for
sharpless
coacervate
calcification.
Faraday
dihydroxylation of alkenes in organic
solvents. Chembiochem 16, 83-90.
Discuss. Chem. Soc. 61, 205-212.
(57) Meyer, D. E., Kong, G. A., Dewhirst, M.
W., Zalutsky, M. R., and Chilkoti, A.
(49) Mabrouk, P. (1997) The use of poly
(ethylene
nonaqueous
Poly(ethylene glycol). Volume
(Harris, J. M., Zalipsky, S., Ed.) pp 118-
133, Chapter 8, ACS.
glycol)-enzymes
enzymology,
in
in
680
(2001)
Targeting
a
genetically
engineered elastin-like polypeptide to
solid tumors by local hyperthermia.
Cancer Res. 61, 1548-1554.
(58) Chilkoti, A., Dreher, M. R., and Meyer,
D. E. (2002) Design of thermally
responsive, recombinant polypeptide
carriers for targeted drug delivery. Adv
Drug Deliv Rev 54, 1093-1111.
(59) Dreher, M. R., Raucher, D., Balu, N.,
Michael Colvin, O., Ludeman, S. M., and
Chilkoti, A. (2003) Evaluation of an
(50) Matsushima, A., Nishimura, H.,
Ashihara, Y., Yokota, Y., and Inada, Y.
(1980) Modification of E. coli
asparaginase with 2, 4-bis (O-
methoxypolyethylene glycol)-6-chloro-
S-triazine
(activated
PEG2);
disappearance of binding ability towards
anti-serum and retention of enzymic
activity. Chem. Lett. 9, 773-776.
elastin-like
polypeptide-doxorubicin
ACS Paragon Plus Environment
13

Bioconjugate Chemistry
Page 14 of 15
conjugate for cancer therapy. J. Control.
Release 91, 31-43.
(60) Meyer, D. E., Shin, B. C., Kong, G. A.,
Dewhirst, M. W., and Chilkoti, A. (2001)
(64) Kaufmann, D., Fiedler, A., Junger, A.,
1
2
3
4
5
6
7
8
Auernheimer, J., Kessler, H., and
Weberskirch, R. (2008) Chemical
conjugation of linear and cyclic RGD
moieties to a recombinant elastin-
Drug
responsive
targeting
using
thermally
and local
polymers
mimetic
polypeptide--a
versatile
hyperthermia. J. Controlled Release 74,
213-224.
approach towards bioactive protein
hydrogels. Macromol. Biosci. 8, 577-
588.
9
(61) Na, K., Lee, S. A., Jung, S. H., Hyun, J.,
and Shin, B. C. (2012) Elastin-like
polypeptide modified liposomes for
enhancing cellular uptake into tumor
cells. Colloids and Surfaces B-
Biointerfaces 91, 130-136.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(65) Uludag, H., Kousinioris, N., Gao, T. J.,
and Kantoci, D. (2000) Bisphosphonate
conjugation to proteins as a means to
impart bone affinity. Biotechnol. Progr.
16, 258-267.
(66) Hassouneh, W., Christensen, T., and
Chilkoti, A. (2010) Elastin‐Like
Polypeptides as a Purification Tag for
Recombinant Proteins. Curr. Protoc.
Protein Sci., 6.11. 1-6.11. 16.
(62) Kaufmann, D., and Weberskirch, R.
(2006) Efficient Synthesis of Protein‐
Drug
Conjugates
Using
a
Functionalizable Recombinant Elastin‐
Mimetic Polypeptide. Macromol. Biosci.
6, 952-958.
(67) Shaka, A. J., Lee, C. J., and Pines, A.
(1988) Iterative Schemes for Bilinear
(63) Trouet, A., Masquelier, M., Baurain, R.,
and Deprez-De Campeneere, D. (1982) A
covalent linkage between daunorubicin
and proteins that is stable in serum and
reversible by lysosomal hydrolases, as
required for a lysosomotropic drug-
carrier conjugate: in vitro and in vivo
studies. Proc. Natl. Acad. Sci. U. S. A. 79,
626-629.
Operators
-
Application to Spin
Decoupling. J. Magn. Reson. 77, 274-
293.
(68) Bax, A., and Davis, D. G. (1985)
Practical Aspects of Two-Dimensional
Transverse Noe Spectroscopy. J. Magn.
Reson. 63, 207-213.
TABLE OF CONTENTS
ACS Paragon Plus Environment
14

Page 15 of 15
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
15