Protein Labeling
Application Note NT-MO-29
One-step, purification-free and site-specific labeling of
polyhistidine-tagged proteins for MST
Nuska Tschammer*, Stefanie Galinec*, Sebastian Weigert#, Yves Muller#, Changjiang You§,
Jacob Piehler§, Dennis Breitsprecher*
*NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich
#
Division of Biotechnology, Department of Biology, University Erlangen-Nürnberg, Henkestr. 91, 91052 Erlangen
Division of Biophysics, Department of Biology, University Osnabrück, Barbarastr. 11, 49076 Osnabrück
§
Abstract
NanoTemper Technologies uses the popular
polyhistidine-tag
as
target
for
an
unparalleled one step, purification-free
labeling of proteins for MST experiments.
The labeling strategy employs a high affinity
multivalent nitrilotriacetic acid (NTA)
derivative conjugated with the MSToptimized NT-647 dye (RED-tris-NTA). As the
hexahistidine tag (His6) provides binding
sites for three NTA moieties, RED-tris-NTA
is perfectly suited for non-covalent, stable,
highly selective labeling with a 1:1
stoichiometric ratio. The labeling can be
performed with minute amounts of either
purified biomolecules or directly in the cell
lysate.
NTA moiety binds His-tagged proteins sitespecifically in stoichiometrically defined and
stable manner. Moreover, because of its small
size, binding of tris-NTA has minimum effect on
biochemical and physicochemical properties of
the protein.
Introduction
Nitrilotriacetic acid (NTA) and its derivatives
have broad applications in the manipulation of
polyhistidine-tagged (His-tagged) proteins such
as in immobilized metal affinity chromatography
(IMAC) (Hochuli, Dobeli et al., 1987, Ueda,
Gout et al., 2003) and surface immobilization
(Gershon & Khilko, 1995, You & Piehler, 2014).
Design of various multivalent NTA led to the
discovery of tris-NTA, a powerful tool to modify
His-tagged proteins with low nanomolar affinity
toward hexahistidine tags (Huang, Hwang et al.,
2009, Lata, Reichel et al., 2005). When coupled
to a fluorophore or biotin, tris-NTA efficiently
labels His-tagged proteins even in complex
cellular systems (Kim, Jeyakumar et al., 2007,
Lata, Gavutis et al., 2006). This advanced tris-
Figure 1: Chemical structure of the NT-647
conjugated tris-NTA (RED-tris-NTA) and schematic
representation of RED-tris-NTA – protein interaction.
The conjugate is shown loaded with nickel(II) ions as
used for the site-specific labeling of His-tagged
proteins. Two remaining coordination sites of the
NTA-complexed nickel(II) ions are occupied by
general ligands X, and can interact with His moieties
of His-tags.
Results and Discussion
The basic mechanisms of the interaction
between the NTA-based label and histidines is
governed by the intrinsic property of the
imidazole group present in the histidine to
chelate nickel(II) nitrilotriacetate (Ni(II)-NTA)
(Terpe, 2003). The hexahistidine tag (His6)
provides six binding sites precisely matching
the three Ni(II) ions complex of the NTA
moieties (Figure 1) (Lata et al., 2005). Tris-NTA
is thus perfectly suited for in situ non-covalent,
stable, highly selective labeling of biomolecules
carrying the His6 tag in a stoichiometric ratio of
1:1.
With the intention to exploit the use of His-tags
for site-specific protein labeling in MST
experiments, the tris-NTA scaffold was
conjugated with the MST-optimized NT-647 dye
(RED-tris-NTA). To demonstrate the versatility
and superiority of the novel NanoTemper
Technologies RED-tris-NTA dye, an array of
MST experiments was performed on different
proteins, which were either purified or present in
crude cell lysate.
Labeling
The versatility, robustness and specificity of
the unique RED-tris-NTA dye were tested
with a peptide as well as proteins in
different labeling buffers, crude cell lysate
and in the presence of various potential
interfering compounds and additives. The
affinity of the dye towards a His6 peptide, a
cancer-associated isocitrate dehydrogenase point-mutant (His6-IDH R132H)
and mitogen-activated protein kinase 14
(p38α) was determined by MST (Figure 2).
The His6 peptide, His6-IDH R132H and
His6 p38α were titrated against 10 nM of
RED-tris-NTA in PBS-T buffer. The
observed Kd values were 1.3 ± 0.2 nM for
the His6 peptide, 0.6 ± 0.3 nM for IDH
R132H and 2.4 ± 1.1 nM for p38α. The
minor deviation in the Kd between different
His-tags are most likely due differences in
the accessibility and the local electrostatic
potential within the respective proteins, or
due to slight inaccuracies in the
determination of the protein stock
concentrations.
Figure 2: MST traces (top) and dose-response curves (bottom) of His6 peptide (A), His6-IDH R132H (B) and His6p38α (C) protein towards RED-tris-NTA. The resulting dose-response curves were fitted to a one-site binding model
to extract Kd values. MST experiments were performed at a LED and MST power of 40%. Fnorm = normalized
fluorescence
The reversibility of the classical Ni(II)-monoNTA
interaction
is
advantageous
for
applications such as immobilized metal affinity
chromatography and surface immobilization,
because it enables full recovery of the
immobilized His-tagged biomolecule by adding
a competitor such as imidazole that disrupts the
Ni(II)-NTA -His-tag interaction. His-tagged
proteins are e.g., eluted from Ni-NTA columns
with buffers containing high concentrations of
imidazole. Therefore, the reversibility of labeling
using RED-tris-NTA needs to be considered
working at low concentrations, since it might
result in dissociation of the dye. Thus, we
analyzed in detail whether common buffer
components interfere with the RED-tris-NTA
labeling of the His-tagged protein. We found
that buffer components like Mg2+, Ca2+, bovine
serum albumin and other proteins without Histag do not interfere with the labeling procedure.
A summary of common buffer components and
their maximum allowed concentration for the
RED-tris-NTA labeling is presented in the
Table 1.
Table 1: List of common buffer components and
their maximum allowed concentration
Compound
Maximum allowed
concentration
Histidine
1 mM
Imidazole
1 mM
EDTA
0.5 mM
TCEP
0.5 mM
DTT
5 mM
β-mercapto-ethanol
1 mM
GSH
10 mM
GTP, GDP
1 mM
AMP, ADP, ATP
5 mM
Glycerol
10 %
His- tagged ligand
None
Overall, the RED-tris-NTA labeling procedure
exhibits low sensitivity towards an array of
different components, which are often used in
storage buffers. Even competitors like histidine
and imidazole only interfere with the labeling at
concentration higher than 1 mM. Reducing
agents, which are not tolerated during standard
NHS labeling, also do not significantly interfere
with RED-tris-NTA labeling. This means that in
most cases direct labeling of the protein in its
storage buffer is possible, and no buffer
exchange is required prior to labeling with REDtris-NTA. Interestingly, we in addition observed
that RED-tris-NTA dye exhibits a much lower
degree of photobleaching when compared to
the standard RED dye, which is likely caused by
the presence of nickel(II) in the complex
(Glembockyte, Lincoln et al., 2015).
Binding assays
Next we tested whether RED-tris-NTA labeled
His-tagged proteins can be used for interaction
quantification by MST. We first tested proteinsmall molecule interactions, using purified His6IDH R132H and His6-p38α. Figure 3 shows that
clear binding events with excellent signal-tonoise ratios could be measured by MST for both
systems. The affinity of PD169316 for p38α was
124 ± 8 nM and the affinity of C35 for IDH
R132H was 100 ± 14 nM, which is in excellent
agreement with published data (Nordin,
Jungnelius et al., 2005, Rohle, Popovici-Muller
et al., 2013). Determined Kd values for the
PD169316-p38α
interaction
were
also
comparable to data obtained with traditional
RED-NHS labeling. Interestingly, the IDH1
R132H-C35 interaction yielded much better
dose-response curves than approaches with
standard RED-NHS labeling, suggesting that
His-tag labeling by Tris-NTA is the method of
choice for labeling of fragile proteins which are
sensitive towards the respective labeling buffer
conditions or covalent modifications.
Figure 3: The MST traces (top) and dose-response (bottom) curves for the binding interactions between the REDtris-NTA labeled His6-IDH R132H (A), His6-p38α (B) and their ligands. Measurements were performed at LED
power 50 % and MST power 80 %. Fnorm = normalized fluorescence
Interaction measurements in crude bacterial
cell lysate
The broad application range of the RED-trisNTA labeling was demonstrated by direct
labeling of a target protein, His6-pUL53, in
crude E. coli lysate followed by the
measurement of the interaction between the
labeled target protein and its binding partner
pUL50. Both proteins pUL53 and pUL50 form
the core nuclear egress complex of human
cytomegalovirus (HCMV) (Walzer, EgererSieber et al., 2015). His6-pUL53 could be
efficiently labeled with RED-tris-NTA directly in
the lysate as shown in Figure 4. The dilutions
series of the lysate containing His6-pUL53 was
prepared in mock lysate which was void of any
His-tagged protein. The concentration of His6pUL53 was estimated based on the previous
experiences with the purification yields. To
determine the binding affinity of pUL50 towards
His6-pUL53, His6-pUL53 was labeled either
directly in the lysate or after purification in
buffer. For this experiment, PBS-T was used as
labeling buffer and the dilution series was
prepared in HEPES buffer. For the purified
His6-pUL53 a Kd value of 1.20 ± 0.45 M was
determined. The measurement in lysate yielded
a Kd value of 1.78 ± 0.24 M. Determined Kd
values did not differ significantly between both
experimental approaches, highlighting the
robustness and reproducibility of the RED-trisNTA labeling system.
Conclusion
The novel NanoTemper Technologies RED-trisNTA dye provides a versatile tool for efficient
and site-specific in situ labeling of His-tagged
proteins. The RED-tris-NTA labeling procedure
is robust towards a variety of different buffer
conditions and components which are often
used in storage buffers. High affinity and
selectivity of the dye for His-tags enables the
labeling of target proteins even in crude cell
lysates. Only very small amounts (picogram) of
protein are needed for the labeling and the
protocol is optimized to label only as much
protein as needed for an MST experiment.
Thus, even very sensitive and low abundant
proteins can be labeled and directly analyzed by
MST without any waste of material.
Figure 4: MST traces (top) and dose-response curves (bottom) of His6-pUL53 for RED-tris-NTA (A) in the E. coli
lysate and (B) the comparison of the binding affinity of pUL50 toward His6-pUL53 measured either with purified
His6-pUL53 or His6-pUL53 in crude lysate. Measurements were performed at LED power 40% and MST power
60 %. Fnorm = normalized fluorescence
Material and Methods
Labeling of His-tagged
proteins in buffer
peptides
and
The His6- and His10-tagged peptides and
proteins (His6-IDH R132H, His6-pUL53, and
His6-p38) were diluted to 200 nM in PBS-T
buffer (137 mM NaCl, 2.5 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4, pH 7.4; 0.05 %
Tween-20). The RED-tris-NTA dye was diluted
in PBS-T to 100 nM. Protein and the dye were
mixed in 1:1 volume ratio and incubated for
30 min at room temperature.
Production and Labeling of His6-pUL53 in
the E. coli lysate
The protein His6-pUL53(50–292) was produced
in E. coli BL21(DE3) (Walzer et al., 2015).
Bacterial cells were grown in TB medium in the
presence of 45 g/ml kanamycin at 37 °C to an
A600 of 0.6 before the temperature was lowered
to 20 °C, and protein expression was induced
with
0.25 mM
isopropyl-D-thiogalactopyranoside overnight. Bacterial cells were
harvested by centrifugation, resuspended in
lysis buffer (50 mM phosphate buffer, pH 7.4,
300 mM NaCl) containing protease inhibitors,
and
disrupted
by
high
pressure
homogenization. In the same manner, lysis of
bacterial cells containing no His6-pUL53 was
produced for the dilution series in buffer. The
lysate containing His6-pUL53 was diluted 1:10
in PBS-T and the RED-tris-NTA dye added at
the final concentration of 50 nM. The mixture
was incubated for 30 min at the room
temperature. The ligand buffer was HEPES
buffer (200 mM, 25 mM HEPES, 1 mM TCEP,
pH 8.0).
MST experiment
The interactions with the dye or ligands with the
peptide, His6-p38 and His6-pUL53 were
measured in Standard treated capillaries,
His6-IDH was measured in MST Premium
coated capillaries. The measurements were
performed in the PBS-T buffer. Before the MST
measurements all samples were centrifuged for
10 min at 4 C and 14000 g.
For the binding studies His6-p38 and His6-IDH
R132H were labeled with the RED-tris-NTA dye
in PBS-T buffer in the ratio 1:2 (50 nM dye,
100 nM protein) for 30 min at the room
temperature. The ligand dilution series (1:1)
were prepared in PBS-T. The ligands for the
binding studies were dissolved in PBS-T at twofold concentration as indicated in the figures.
7
Instrumentation and data analysis
The measurements were performed on a
NanoTemper Monolith™NT.115 instrument.
Final dye concentration of 25 nM yielded the
fluorescence intensity of around 300 counts at
a LED power of 50 %. The samples were
measured at MST power between 40 % and
60 % with a laser-on time of 30 s and a laser-off
time of 5 s. The data were analyzed by MO
Affinity Analysis Software.
References
1
2
3
4
5
6
Ueda, E. K., Gout, P. W. & Morganti,
L. Current and prospective
applications of metal ion-protein
binding. J Chromatogr A 988, 1-23
(2003).
Hochuli, E., Dobeli, H. & Schacher, A.
New metal chelate adsorbent selective
for proteins and peptides containing
neighbouring histidine residues. J
Chromatogr 411, 177-184 (1987).
Gershon, P. D. & Khilko, S. Stable
chelating linkage for reversible
immobilization of oligohistidine tagged
proteins in the BIAcore surface
plasmon resonance detector. J
Immunol Methods 183, 65-76 (1995).
You, C. & Piehler, J. Multivalent
chelators for spatially and temporally
controlled protein functionalization.
Anal Bioanal Chem 406, 3345-3357,
doi:10.1007/s00216-014-7803-y
(2014).
Huang, Z., Hwang, P., Watson, D. S.,
Cao, L. & Szoka Jr, F. C. Trisnitrilotriacetic acids of subnanomolar
affinity toward hexahistidine tagged
molecules. Bioconjugate chemistry 20,
1667-1672 (2009).
Lata, S., Reichel, A., Brock, R.,
Tampé, R. & Piehler, J. High-affinity
adaptors for switchable recognition of
histidine-tagged proteins. Journal of
the American Chemical Society 127,
10205-10215 (2005).
8
9
10
11
12
13
Kim, S. H., Jeyakumar, M. &
Katzenellenbogen, J. A. Dual-mode
fluorophore-doped nickel nitrilotriacetic
acid-modified silica nanoparticles
combine histidine-tagged protein
purification with site-specific
fluorophore labeling. Journal of the
American Chemical Society 129,
13254-13264 (2007).
Lata, S., Gavutis, M., Tampé, R. &
Piehler, J. Specific and stable
fluorescence labeling of histidinetagged proteins for dissecting multiprotein complex formation. Journal of
the American Chemical Society 128,
2365-2372 (2006).
Terpe, K. Overview of tag protein
fusions: from molecular and
biochemical fundamentals to
commercial systems. Applied
microbiology and biotechnology 60,
523-533 (2003).
Glembockyte, V., Lincoln, R. & Cosa,
G. Cy3 Photoprotection Mediated by
Ni2+ for Extended Single-Molecule
Imaging: Old Tricks for New
Techniques. Journal of the American
Chemical Society 137, 1116-1122,
doi:10.1021/ja509923e (2015).
Nordin, H., Jungnelius, M., Karlsson,
R. & Karlsson, O. P. Kinetic studies of
small molecule interactions with
protein kinases using biosensor
technology. Analytical biochemistry
340, 359-368,
doi:10.1016/j.ab.2005.02.027 (2005).
Rohle, D. et al. An inhibitor of mutant
IDH1 delays growth and promotes
differentiation of glioma cells. Science
340, 626-630,
doi:10.1126/science.1236062 (2013).
Walzer, S. A. et al. Crystal Structure of
the Human Cytomegalovirus pUL50pUL53 Core Nuclear Egress Complex
Provides Insight into a Unique
Assembly Scaffold for Virus-Host
Protein Interactions. Journal of
Biological Chemistry 290, 2745227458, doi:10.1074/jbc.C115.686527
(2015).
NOTES
Contact
NanoTemper Technologies
GmbH
Floessergasse 4
81369 Munich
Germany
Phone: +49 (0)89 4522895 0
Fax: +49 (0)89 4522895 60
[email protected]
http://www.nanotemper-technologies.com
© 2016 NanoTemper Technologies GmbH