An iminodiacetate-modified conjugated polyelectrolyte for fluorescent labeling of histidine-tagged proteins

Article abstract of DOI:10.1039/c7cc00850c

The iminodiacetate-Ni²⁺-hexahistidine system is extensively used in protein purification. In this study, an anionic conjugated polyelectrolyte (CPE) with a poly(p-phenylene ethynylene) backbone and iminodiacetate side chains, named PPEIDA, was designed and synthesized. Recognition and visualization of hexahistidine-tagged (His-tagged) proteins using PPEIDA was demonstrated.

Full text of DOI:10.1039/c7cc00850c

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
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DOI: 10.1039/C7CC00850C
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Iminodiacetate-modified conjugated polyelectrolyte for
fluorescent labeling of histidine-tagged proteins
Received 00th January 20xx,
Accepted 00th January 20xx
†
ab
†ab
ab
ab
ab
ab
ab
Kai Cai, Ying Tan, Chunyan Tan,* Jiatao Wu, Pan Wu, Jiamei Liang, Shuwen Liu, Bibo
ab
bc
Zhang, and Yuyang Jiang
DOI: 10.1039/x0xx00000x
www.rsc.org/
2
+
The iminodiacetate-Ni -hexahistidine system is extensively used
in protein purification. In this study, an anionic conjugated
polyelectrolyte (CPE) with a poly(p-pheynylene ethynylene)
backbone and iminodiacetate side chains, named PPEIDA, was
designed and synthesized. Recognition and visualization of
hexahistidine-tagged (His-tagged) proteins using PPEIDA was
demonstrated.
Conjugated polyelectrolytes (CPEs), which consist of electron-
delocalized conjugated backbones with charged side groups,
have been extensively studied due to their strong light
absorption, high fluorescence quantum yields, and signal
2+
Scheme 1 Schematic illustration of PPEIDA-Ni complex coupled to His-tagged protein.
Iminodiacetate (IDA) and nitrilotriacetate (NTA) are
commonly known as complexes with Ni , which consequently
bind to the hexahistidine tag (His-tag).
tag and NTA-Ni -His-tag systems have been seen in small
molecular probes as well as surface-modified nanoparticles to
immobilize and purify recombinant proteins in biological
2+
1-5
amplification properties. Many commonly used charged side
moieties, such as carboxylate, sulfonate, phosphate, alkyl
ammonium groups, etc., have been reported to enhance the
23, 24 2+
Thus, IDA-Ni -His-
2+
4, 6-10
water-solubility and biocompatibility of CPEs.
Over the
past decades, the introduction of pendant charged groups
enable CPEs to interact with species with opposite charges
through electrostatic attractions, enhancing their applicability
as potential fluorescent chemosensors and biosensors as well
2
5-34
studies.
CPEs have emerged as a new material platform in
biological studies and applications due to their strong
fluorescence, good bio-compatibility, low cytotoxicity, and
excellent photostability. Herein, we report a novel CPE with
IDA side chains pendant on a PPE backbone, named as PPEIDA,
which can be used for the recognition of His-tagged proteins
through fluorescence anisotropy (FA) and Förster resonance
1-5, 11-13
as fluorescent cell imaging materials.
However, non-
specific electrostatic interactions have been reported as
perturbations, sacrificing the sensitivity and selectivity of CPEs
1
4, 15
in recognition of targets.
Therefore, there is a significant
2+
energy transfer (FRET). The proposed PPEIDA-Ni complex
could find its potential applications in the development of an
6-18 alternative to the conventional Western blotting method, as
well as specific cell imaging.
challenge and demand to develop novel CPEs with specific
target recognition elements in order to practically use CPEs in
1
biological studies. Some CPEs grafting with folic acid,
1
9
20, 21
22
lipid, antibodies,
and peptides have been reported to
35
PPEIDA was synthesized via Sonogashira protocol.
recognize specific receptors or proteins. For example, folate-
substituted poly(p-phenyleneethynylene) (PPE) was efficiently
used in cancer cell imaging due to the specific interaction
between folate and folate-receptors overexpressed in some
Generally speaking, the ester precursor PPE-iminodiacetate
ester was synthesized first with the two building blocks of
iminodiacetic ester-substituted diiodobenzene and 1,4-
diethynylbenzene using Pd(PPh ) and CuI as catalysts. The
1
6-18
3
4
tumor cells.
following hydrolysis of the ester polymer gave the final
polymer, PPEIDA (Scheme S1, ESI†). The molecular weight
(
MW) of PPEIDA was determined by Maldi-Tof mass
a.
തതതതത
spectrometry. The number average MW ܯ݊, value was 22.5
Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China.
The State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical
Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055,
P. R. China. Email: tancy@sz.tsinghua.edu.cn; Tel: 86-755-26036533.
School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, P. R.
China.
b.
തതതതത
kD, while the weight average MW ݓܯ , was 45.6 kD,
corresponding to the polydispersity index (PDI) at 2.04. As
shown in the structure in Scheme 1, IDA groups on the
polymer side chains contributed two chelating ligands to each
repeating unit and were proposed to coordinate with metal
c.
†
These authors contributed equally to this work. Electronic Supplementary
Information (ESI) available: Experimental including materials, synthesis,
instrumentation and some supplementary figures. See DOI:10.1039/x0xx00000x
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Fig. 1 Fluorescence emission quenching spectra of 1.125 μM PPEIDA with different
2+
2 0
concentrations of Ni in (A) H O and (B) MeOH. (C) Quenching efficiency of PPEIDA. I
2+
and I are the fluorescence intensities of PPEIDA in the absence and presence of Ni ,
respectively.
Fig. 2 Fluorescence anisotropy of PPEIDA in response to varying concentrations of Bcl-
xL or His-tagged Bcl-xL in the presence or absence of Ni ; schematic diagram of
fluorescence anisotropy variation.
2+
2+
ions, such as Ni . Thus, protein recognition was achieved by
2
+
the multivalent chelation between Ni
and His-tagged with the His-tagged Bcl-xL. As a distinctive comparison, other
2+
2+
2+
proteins. The UV-Vis absorption and fluorescence emission divalent metal cations, such as Zn , Mg and Ca , didn’t
spectra of PPEIDA showed typical characteristics of PPE bridge the binding between PPEIDA and His-tagged proteins
polymers with broad absorption from 350 to 450 nm and (Figure. S3, ESI†). The plot of anisotropy versus protein
emission from 400 to 600 nm (Fig. S1, ESI†).
Numerous studies have shown that fluorescence quenching the binding constant between PPEIDA-Ni and the protein was
concentrations was fitted into a single-site binding model and
2+
6
-1
of CPEs could be induced by either electron transfer or energy determined to be 1.02 × 10 M . Compared to commonly used
3
6-38
transfer mechanism.
causes fluorescence emission to decrease.
properties of PPEIDA under the effect of different metal ions, tagged proteins, without complicated operation in either
In addition, CPE aggregation also protein fluorescent labeling techniques, our method provides
6, 39, 40
Fluorescence a feasible and convenient approach to label recombinant His-
2+
2+
2+
2+
including Ni , Zn , Mg , and Ca , in methanol and aqueous chemical synthesis or genetically encoding of fluorescent
solutions were characterized. As shown in Fig. 1, the proteins.
fluorescence emission of PPEIDA decreased more efficiently by
FRET is another fluorescent technique commonly used in
adding Ni in methanol compared to that in aqueous solution. biological research, which takes place through dipole-dipole
2+
2+
There was almost 83.6% quenching by Ni
at the coupling when a donor fluorophore and an acceptor have
concentration of 7 μM in methanol solution, while only about good spectral overlap, parallel transition dipole orientation,
2+
43
1
3.5% quenching of PPEIDA by Ni at the concentration of 20 and are in close proximity, typically 10-100 Å. Therefore,
μM in aqueous solution. Several other metal ions, including FRET is a powerful tool, also termed as a “molecular ruler”, to
2+
2+
2+
Zn , Mg , and Ca , also showed stronger quenching in measure the distance between two sites comparable to the
methanol than in aqueous solution (Fig. S2, ESI†). These dimensions of biological macromolecules. FRET experiments
interesting results might be due to polymer aggregation were conducted using
a recombinant His-tagged red
induced by the complexation of the IDA ligand with metal ions fluorescent protein (RFP). The schematic representation of
in methanol solution. Due to the strong interaction between FRET from PPEIDA to His-tagged RFP is shown in Fig. 3A. RFP
2+
PPEIDA and Ni , this system was further explored for His- absorbs visible light at λmax = 550 nm and emits at λmax = 580
tagged proteins recognition using different spectroscopic nm. It is weakly fluorescent in water when excited at 380 nm
strategies.
(Fig. S4, ESI†). Due to the good overlap of the fluorescence
As an important spectroscopic tool, fluorescence anisotropy emission of PPEIDA and the absorption of RFP (Fig. S5, ESI†), it
FA) is widely used in study of molecular interactions, is feasible that the FRET occurs from PPEIDA to RFP, when they
(
4
1, 42
especially in biological systems.
Using this method, protein
binding to the fluorescent PPEIDA to form a complex of
2+
PPEIDA-Ni -His-tagged protein can be detected by the FA
signal enhancement resulting from increases in complex
volume and/or decreases in PPEIDA flexibility. IDA chelated
2+
Ni with three valencies, while the other 3 valencies were
used for binding His-tagged residues. Fig. 2 shows the principle
2+
for FA sensing of the PPEIDA-Ni complex with and without
the His-tagged protein. As shown in Fig. 2, FA increased from
2
+
0
.25 to 0.35 in the presence of PPEIDA, Ni , and His-tagged
2+
Bcl-xL. In the presence of 200 μM Ni , the FA signal reached a
plateau when the concentration of His-tagged Bcl-xL increased
to 11 μM. However, as a distinctive comparison, all three
control experiments, including PPEIDA with non-tagged Bcl-xL,
PPEIDA with Ni and non-tagged Bcl-xL, and PPEIDA with His-
tagged Bcl-xL in the absence of Ni , did not show any obvious
FA signal change. Overall, the addition of Bcl-xL without His-tag,
2+
Fig. 3 (A) Schematic representation of FRET from PPEIDA to His-RFP. (B) Normalized
2+
2+
fluorescence spectra (λex = 380 nm) of PPEIDA-Ni complex upon addition of His-RFP
(0-277.5 nM) in aqueous solution. Arrows show direction of change of the bands with
2+
increasing [His-RFP]. (C) Ratio of intensities at 580 and 433 nm of PPEIDA or PPEIDA-
or without Ni , had no effect on FA, which highly suggests that
Ni acts as a bridge ligand for the complexation of PPEIDA-Ni
2+
2+
2+
Ni complex after addition of RFP or His-RFP, respectively, at various concentrations in
aqueous solution.
2
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2+
Fig. 4 Procedure comparison of (B) our method to the (A) conventional Western blot analysis. (C) Results of PPEIDA-Ni complex-based Western blot analysis for His-tagged
protein detection. Protein quantities over a range from 100 to 6000 ng.
are reasonably close to each other. The Förster radius, R
0
,
research, a simplified Western blot method would be a
between the donor polymer and the acceptor RFP was valuable alternative for the detection of recombinant proteins.
calculated as 4.83 nm (Table S1, ESI†). Fig. 3B illustrates the The conventional method requires at least 7 steps and 17~21.5
changes in fluorescence that occurred concomitant with h to complete the procedure. As shown in Fig. 4, our method
addition of His-tagged RFP to an aqueous buffer solution of (Fig. 4B) dramatically reduced the number of steps, time, and
2+
PPEIDA with the presence of Ni . The fluorescence of PPEIDA costs compared to those in conventional Western blot analysis
at 433 nm was quenched, while a new emission band emerged (Fig. 4A). To the best of our knowledge, there have been no
2+
at 580 nm from His-RFP. Importantly, under the same reports on the direct use of fluorescent CPE-Ni complex,
excitation conditions, the fluorescence from His-tagged RFP which eliminated the laborious primary and secondary
alone is negligible (Fig. S4, ESI†), which confirms the antibody binding and wash steps, in a Western blotting
occurrence of the FRET process.
application. The time spent on Western blot using our method
The FRET efficiency is evaluated by the ratio of the is even comparable to some automatic Western blot
fluorescence intensity at 580 and 433 nm (Fig. 3C). Three instruments, which cost about 4 hours. Another advantage is
2+
systems were compared, including PPEIDA-Ni -RFP (non His- that our method provided high-quality and reproducible
2+
tagged), PPEIDA-His-tagged RFP (without Ni ) and PPEIDA- fluorescent protein bands. As shown in Fig. 4C, fluorescence
2+
2+
Ni -His-tagged RFP. PPEIDA-Ni -His-tagged RFP system signals are proportional to protein quantities over a range
appeared to have the most responsive FRET, which increased from 100 to 6000 ng. This method thus provides a quantitative
to 0.67 as the protein concentration increased from 0 to 7.5 analysis of Western blots more easily and more reliable.
2+
μ
g/mL. In the absence of Ni , I580/I433 of PPEIDA-His-tagged Moreover, owing to the extraordinary optical properties of
RFP complex still showed a certain increase (Fig. S6A, ESI†) up CPEs, our system can harness high brightness and extreme
to 0.20, which is likely due to the electrostatic attraction stability, which showed minimal fluorescence decline in blots
between the positively charged histidine residues and even after exposure to the air after one year on the shelf.
negatively charged iminodiacetates that brings His-tagged RFP
The MTT assay was utilized to evaluate the cell cytotoxicity
and PPEIDA closer. The calculated donor-acceptor distances of PPEIDA (Fig. S8, ESI†). PPEIDA treated HeLa cells showed cell
2+
are 4.76 nm and 10.39 nm, for PPEIDA-Ni -His-tagged RFP and viability about 95% at the highest concentration of 200 μM.
PPEIDA-His-tagged RFP, respectively, when [His-tagged RFP] = The low cytotoxicity of PPEIDA makes it a good material for
2
+
2
77.5 nM (Table S1, ESI†). These results suggest that Ni can vivo cell imaging applications. In-situ fluorescent labeling of
2+
bring the polymer and the protein to a closer proximity for an His-tagged proteins was carried out using PPEIDA-Ni on cell
2+
efficient FRET in the case of PPEIDA-Ni -His-RFP. When RFP membranes under live cell conditions. Bradykinin receptor
has no His-tag, I580/I433 showed minimum increase during type2 (B2R) was tethered with a His-tag at the exoplasmic N-
2+
the addition of protein (Fig. S6B, ESI†). When either Ni was terminus and employed as the target protein expressed on cell
2+
2+
2+
replaced by other metal ions, including Zn , Mg , and Ca , or surfaces. In cell imaging experiments, HeLa cells expressing
no metal ions was present, no obvious FRET was observed, His-tagged B2R were incubated with PPEIDA-Ni PBS solution
indicating that Ni played an important role in connecting the (3.25 μM PPEIDA, 200 μM Ni ) for 3 min and then washed by
polymer probe and the target protein (Fig. S7, ESI†).
2+
2+
2+
Both FA and FRET studies encouraged us to further explore
2+
the possibility of using this novel PPEIDA-Ni -His-tagged
protein system in fluorescent labeling applications. Western
blot analysis is a widely used analytical technique in molecular
biology to detect and quantify specific proteins in a
homogenate or extracted sample from tissue or cells. The
main principle behind Western blot analysis is to utilize an
44, 45
antigen-antibody immune reaction.
However, this
technique is time-consuming and laborious. It was expected
2+
that using the PPEIDA-Ni complex to fluorescently label His-
tagged proteins would provide a simple, time-saving, and Fig. 5 Visualization of the His-B2R in Hela cells. HeLa cells expressing His-B2R were
2+
sensitive method for Western blot analysis. Due to the incubated with (A) PPEIDA-Ni PBS solution and (B) PPEIDA PBS solution. Cells were
extensive use of recombinant His-tagged proteins in biological washed before imaging. Scale bar: (A) 30 μm; (B) 20 μm.
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