Site-specific covalent labeling of His-tag fused proteins with a reactive Ni(ii)-NTA probe

Article abstract of DOI:10.1039/b912025d

A new method for covalent labeling of a His-tag fused protein with a small reactive probe was developed; this method is based on the complementary interaction between the His-tag and Ni(ii)-NTA, which facilitates a nucleophilic reaction between a histidin

Full text of DOI:10.1039/b912025d

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Site-specific covalent labeling of His-tag fused proteins with a reactive
Ni(II)–NTA probew
Sho-hei Uchinomiya,a Hiroshi Nonaka,a Sho-hei Fujishima,a Shinya Tsukiji,a Akio Ojidaab
and Itaru Hamachi*ac
Received (in Cambridge, UK) 18th June 2009, Accepted 22nd July 2009
First published as an Advance Article on the web 13th August 2009
DOI: 10.1039/b912025d
A new method for covalent labeling of a His-tag fused protein
with a small reactive probe was developed; this method is based
on the complementary interaction between the His-tag and
Ni(II)–NTA, which facilitates a nucleophilic reaction between a
histidine residue of the tag and the electrophilic tosyl group of
the Ni(II)–NTA probe by the proximity effect.
The complementary recognition pair of an oligo-histidine tag
(His-tag) and
a Ni(II) complex of nitrilotriacetic acid
(Ni(II)–NTA), originally developed by the research group of
F. Hoffmann-La Roche,1 is now widely exploited as an
indispensable tool for protein research. A wide variety of
proteins are expressed as recombinants fused with a His-tag,
by which affinity handling, purification and surface
immobilization of proteins are largely facilitated.2,3 In recent
years, several groups reported an extension if the use of this
pair for bio-imaging studies of His-tag fused proteins with a
fluorescent Ni(II)–NTA probe, which further demonstrated its
versatility as a tool in protein functional analysis.4 These
successful examples fully exploit the selective and strong
binding properties of the His-tag–Ni(II)–NTA pair; however,
the reversibility of this labeling technique sometimes limits its
utility, particularly in post-labeling analyses such as
SDS-PAGE or Western blotting. Development of an irreversible
covalent labeling method should further extend the utility of
the His-tag to various biochemical analyses.5 Here, we report a
new system for covalent protein labeling by using the
His-tag–Ni(II)–NTA pair. The complementary interaction
between the His-tag and the Ni(II)–NTA probe selectively
facilitates a nucleophilic reaction between a histidine residue
of the tag and the reactive benzenesulfonyl (i.e., tosyl) group
incorporated in the probe (Fig. 1).6 This system enables us to
site-specifically introduce diverse functional molecules into a
His-tag site, allowing accurate post-labeling analysis and
functional modification of the recombinant proteins.
Fig. 1 (a) General design of the reactive Ni(II)–NTA probe (upper)
and its fluorescent coumarin derivatives (lower). (b) Schematic
illustration of the covalent modification of a His-tag fused protein
using the reactive Ni(II)–NTA probe driven by the proximity effect.
Fig. 1a. A tosyl unit was employed as a reactive linker between
Ni(II)–NTA, a recognition site, and a functional group that is
covalently attached to the His-tag. An advantageous point of
this labeling system is that a His-tag fused protein is labeled
with a small-sized functional unit, because the Ni(II)–NTA
moiety is concomitantly released upon the substitution reaction.
This is expected to minimize the problematic steric interference
of introduced molecules to protein functions. We initially
prepared a Ni(II)–NTA probe, 1, bearing a fluorescent coumarin
(Fig. 1) and evaluated its reactivity for the oligo-histidine
peptides (Trp–Ala–(His)n, n = 6 (His6) or 10 (His10)) in
neutral aqueous solution. The MALDI-TOF mass analysis
showed that the mono- and di-substituted peptides labeled
with the coumarin unit of 1 were detected at m/e 1470 and
1688, respectively, after 7 h of reaction, indicating that the
nucleophilic reaction of 1 with His6 peptide proceeded
(Fig. 2a). To enhance the reactivity of the probe towards rapid
labeling, we next prepared the halogen-substituted tosyl ester,
2 (Fig. 1), which should be more reactive owing to the
electron-withdrawing effect of the substituted F and Cl. As
the time trace plots of the labeling reactions monitored by
MALDI-TOF (Fig. 2b) indicate, indeed, the reaction of 2 with
His10 peptide proceeded most rapidly to yield the labeled
product in over 40% yield after 7 h. The covalent adduct of 2
The general design of the reactive Ni(II)–NTA probe and its
reaction mode with a His-tag fused protein are illustrated in
a Department of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto University, Katsura Campus,
Kyoto, 615-8510, Japan. E-mail: ihamachi@sbchem.kyoto-u.ac.jp
b JST, PRESTO (Life Phenomena and Measurement Analysis),
Sanbancho, Chiyodaku, Tokyo, 102-0075, Japan
c JST, CREST (Creation of Next-generation Nanosystems through
Process Integration), Sanbancho, Chiyodaku, Tokyo, 102-0075,
Japan
w Electronic supplementary information (ESI) available: Syntheses of
the probes, compound characterization and experimental details. See
DOI: 10.1039/b912025d
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Fig. 3 (a) SDS-PAGE analysis of the labeling reaction of 2 with
His10–EGFP (lanes 1–6) and control EGFP lacking a His10 tag
(lanes 7–12). Reaction conditions: 5 mM His10–EGFP or control
EGFP, 15 mM 2, 50 mM HEPES, 100 mM NaCl, pH 7.2, 37 1C.
(b) Time-trace plot of the labeling reaction of 2 with His10–EGFP (K)
and with control EGFP (’) based on in-gel fluorescence analysis. The
labeling yield (%) is defined as the amount of labeled coumarin units
per total amount of protein, and calculates based on the fluorescent
band intensity of the labeled protein relative to the authentic sample.
The reaction was performed in triplicate and the error bars represent
standard deviations.
Fig. 2 (a) MALDI-TOF mass analysis of the covalent adduct of His6
peptide with 1. Reaction conditions: 5 mM His6 peptide, 10 mM 1,
50 mM HEPES, 100 mM NaCl, pH 7.2, 37 1C, 7 h. (b) Time-trace plot
of the labeling reaction between His10 peptide and 2 (’), His10
peptide and 1 (K), His6 peptide and 2 (m), His6 peptide and 1 (&),
and N-Cbz-histidine and 2 (E) based on the MALDI-TOF mass
analysis. In the all reactions with the peptides, the mono-, and
di-substituted peptides were detected in MALDI-TOF mass analysis.
interaction with
a mM range of dissociation constants
(Kd, M).7z To further confirm the labeling site of the protein,
the labeled His10-tag fragment was selectively cleaved by
thrombin digestion from His10–EGFP, and the EGFP unit
was subjected to in-gel fluorescence analysis. Fluorescence was
scarcely observed at the band of the cleaved EGFP unit in
SDS-PAGE (ESI, Fig. S1w), whereas the mass peak assignable
to the labeled peptide with one coumarin unit was detected in
the cleaved His10 fragment by MALDI-TOF mass analysis
(ESI, Fig. S2w). These results clearly indicate that the labeling
reaction occurs at the His-tag site in a site-specific manner.
This covalent labeling system allows us to introduce a
variety of functional groups into a His-tag fused protein.
When the alkyne-tethered tosyl ester, 3, was used as a reactive
probe, the labeling reaction also proceeded to afford the
His10–EGFP tethered to a terminal acetylene unit, which is
a versatile reactive handle for Click chemistry.8 Indeed, the
labeling with 3, followed by the subsequent Huisgen reaction
with azide coumarin 4, afforded the coumarin-appended
EGFP, as shown by in-gel fluorescence analysis (Fig. 4a).
We also employed the biotin-appended tosyl ester, 5, for the
reaction with His10–EGFP, in which the covalent attachment
of the biotin unit was detected by chemical luminescence
analysis using HRP (horseradish peroxidase)–avidin conjugate
(Fig. 4b). These results demonstrated the general applicability
of the present reactive tag system for protein labeling.
was not detected in the reaction with a high concentration of a
monomeric N-Cbz-histidine (400 mM) (Fig. 2b), suggesting
that the reaction was effectively enhanced by the multivalent
binding sites of the oligo-histidine peptides. Interestingly, the
reaction with His10 peptide was significantly faster than with
His6 peptide in both cases of 1 and 2, probably due to the
stronger binding affinity and/or the increased reaction probability
of His10 peptide having more histidine residues than His6
peptide.7
The most reactive pair of 2 and His10-tag was subsequently
applied to the covalent labeling of a His-tag fused protein. The
labeling reaction was conducted between 15 mM 2 and 5 mM
EGFP (enhanced green fluorescent protein), tethering the
His10-tag at its N-terminal in neutral aqueous solution
(50 mM HEPES, 100 mM NaCl, pH 7.2, 37 1C). Fig. 3a shows
the fluorescence analysis of the labeling reaction by
SDS-PAGE (in-gel fluorescence analysis), in which the fluorescent
band of His10–EGFP, due to the labeled coumarin unit of 2,
gradually increased at the band of His10–EGFP (lanes 1–6),
indicative of the progress of the covalent labeling reaction in a
time-dependent manner. Fig. 3b shows a time-trace plot of the
reaction estimated from the in-gel fluorescence analysis,
showing that the labeling yield reached 80% after 12 h. In
contrast, the covalent labeling did not occur with control
EGFP lacking the His10-tag, even after 12 h (Fig. 3a, lanes
7–12, and Fig. 3b), suggesting that 2 effectively reacted at the
His10-tag site of His10–EGFP by assistance of the coordination
The specificity of this reactive tag system was evaluated in a
labeling experiment using a crude lysate of E. coli cells
expressing His10–EGFP. As shown in Fig. 5b, a single
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to each other in the covalent protein labeling, in spite of the
fact that similar nucleophilic reactions occur in both methods.
This sufficient orthogonality can be mainly ascribed to the
high recognition selectivity of each His10–Ni(II)–NTA and
D4–Zn(II)–DpaTyr pair.10
In summary, we have developed a new His-tag based reactive
tag–probe pair for site-specific covalent modification of proteins,
on the basis of our originally developed protein labeling method
using a reactive tosyl chemistry.6 This proximity-driven covalent
labeling method should be useful as a new tool for post-labeling
analysis, functional modification, as well as manipulation of
proteins of interest, which cannot be readily achieved by the
conventional His-tag–Ni(II)–NTA pair that relies on non-
covalent interactions. We have also demonstrated that the
present labeling method is orthogonal to the other reactive
tag–probe pair of CA6D4-tag–Zn(II)–DpaTyr. This makes us
envision that the combinational use of these orthogonal labeling
systems would be a promising strategy to elucidate complicated
biological events involving multiple proteins such as the
formation or de-formation of protein signaling complexes. We
believe that the present non-enzymatic labeling method would be
useful for bio-imaging studies as a complementary tool to the
existing enzyme-catalyzed protein labeling methods,11 though
further improvements might be needed for such living cell
applications. Our research is now ongoing along these lines.
Fig. 4 Covalent labeling His10–EGFP with alkyne ester 3 (a) and biotin
ester 5 (b). (a) In-gel fluorescence detection of the coumarin 4-appended
His10-EGFP, which was formed by the covalent protein labeling with 3
and the subsequent Huisgen reaction. (b) Chemical luminescence
analysis of the biotin-appended His10–EGFP using HRP (horse radish
peroxidase)–avidin conjugate. Lane
2 in (a) and (b) shows the
experimental result using control EGFP lacking a His10 tag.
Notes and references
z The labeling yield dramatically decreased to 11% (after 7 h) when the
reaction was carried out in the absence of Ni(II) ions. This result also
suggests that the coordination interaction between the Ni(II)–NTA site of
2 and the His10-tag works effectively to enhance the reaction.
Fig. 5 (a) Selective covalent labeling of His10-EGFP with 2 in crude
lysate of E. coli cells (lanes 1 and 2). Lanes 2 and 4 show the
experimental result using EGFP lacking the His10 tag. (b) Orthogonal
covalent labeling of His10–EGFP and CA6D4–MBP with the reactive
probes 2 and 6 in crude lysate of E. coli cells. The detection channels of
lanes 2 and 3 correspond to coumarin (480BP70) and rhodamine
(630BP30) emission, respectively. Details of the experimental
procedure are described in the experimental section in the ESI.w
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