FLAG-tag selective covalent protein labeling via a binding-induced acyl-transfer reaction

Article abstract of DOI:10.1016/j.bmcl.2009.09.122

A FLAG tag selective protein labeling method is newly developed in this study. Coupling of the selective binding between synthetic Ni-complex probe and FLAG tag with the acyl transfer reaction enables the site-selective covalent modification of FLAG pepti

Full text of DOI:10.1016/j.bmcl.2009.09.122

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6696–6699
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
FLAG-tag selective covalent protein labeling via a binding-induced
acyl-transfer reaction
Hiroshi Nonaka a, Sho-hei Fujishima a, Sho-hei Uchinomiya a, Akio Ojida a,b, Itaru Hamachi a,c,
*
a Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan
b JST, PRESTO (Life Phenomena and Measurement Analysis), Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
c CREST (Creation of Next-generation Nanosystems through Process Integration), Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A FLAG tag selective protein labeling method is newly developed in this study. Coupling of the selective
binding between synthetic Ni-complex probe and FLAG tag with the acyl transfer reaction enables the
site-selective covalent modification of FLAG peptide and FLAG-tag fused protein.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 26 August 2009
Revised 29 September 2009
Accepted 30 September 2009
Available online 3 October 2009
Keywords:
FLAG tag
Protein labeling
Molecular recognition
Acyl-transfer
Specific covalent labeling of a protein with a small molecular
probe is a powerful technique for elucidation of protein functions.
Among various labeling methods, the modification using a short
peptide tag incorporated into a recombinant protein is a recently
developing useful technique.1 In recent years, several enzymatic2
or non-enzymatic3 methods have been reported for the covalent
labeling of tag-fused proteins. It is still strongly desirable to expand
the repertory of the labeling techniques, which is available for mul-
ti-color protein labeling or in a complementary way with other
labeling techniques.4
FLAG tag (DYKDDDDK), a genetically encodable short peptide
tag, has been widely used in protein research, especially in Wes-
tern blotting and affinity purification of recombinant proteins.5
Such analyses only relies on the FLAG tag specific antibody
(FLAG-antibody). However, non-specific binding and the large size
of expensive FLAG-antibody often limit the utility of FLAG tag. The
small molecular probe that is able to specifically label FLAG tag is
expected to largely expand the utility of FLAG tag in protein re-
search. We recently proposed that the binding-induced chemical
reaction for a peptide tag is a powerful strategy for selective pro-
tein labeling.3a,b Here we extend this chemical strategy for the
covalent labeling of the FLAG tag system. The selective binding of
the probe to a FLAG tag facilitated a chemical reaction in the bind-
ing complex, allowing the covalent modification of FLAG tag fused
proteins with synthetic markers. This covalent labeling system
may partially serve as an alternative to FLAG-antibodies.
Figure 1 shows our strategy for the selective covalent modifica-
tionof a FLAGtagfused protein. Werecentlyfound thatthebinuclear
Ni(II) complex of DpaTyr 1-2Ni(II) binds with an oligo-aspartate tag
(DDD or DDDD) with a strong affinity (Kapp >105 MÀ1) under neutral
aqueous conditions.6 Containing a tetra-aspartate sequence in FLAG
tag (DYKDDDDK) made us expect that Ni(II)–DpaTyr could interact
with FLAG tag. We further envisioned that the proximity of the tag
and the probe in the binding complex enhances a nucleophilic reac-
tion of amino acid residues of FLAG tag towards a Ni(II)–DpaTyr
probe having an electrophilic counterpart. Among various electro-
philes, thioester is selected because of its biocompatibility and mod-
erate reactivity under biological conditions.7 Thus, we designed
Ni(II)–DpaTyr probe appended with a thioester group (2-2Ni(II)),
which can potentially undergo an acyl-transfer reaction with some
amino acid residues (e.g., Lys) of FLAG tag to form the covalent bond
of the tag-fused protein. The reactive thioester-type of probe 2-
2Ni(II) possessing a coumarin fluorophore was synthesized as
shown in Scheme 1.
The labeling reaction was initially examined using 2-2Ni(II) and
a FLAG peptide (Ac-DYKDDDDK-NH2) under slightly basic condi-
tions (50 mM HEPES, pH 8.0). MALDI-TOF mass analysis (negative
mode) showed a new peak at 1353 (m/z), which corresponds to
the FLAG tag peptide labeled with one coumarin unit of the probe,
together with a small peak observed at 1653 (m/z), which was
assignable as the FLAG peptide modified with two coumarin units
(Fig. 2).
* Corresponding author.
E-mail address: ihamachi@sbchem.kyoto-u.ac.jp (I. Hamachi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.122

H. Nonaka et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6696–6699
6697
N
N
N
N
N
N
Ni
N
Ni
N
Ni
O
Ni
N
O
N
• 3Cl -
BocHN
• 3Cl -
O
N
N
N
O
O
O
O
H
N
OMe
OMe
S
N
O
H
O
1-2Ni(II)
2-2Ni(II)
Figure 1. (Upper) Selective covalent labeling of a FLAG tag fused protein via an acyl-transfer reaction. (Lower) Structure of the Ni(II)-DpaTyr probes.
N
N
N
N
N
N
N
N
O
OH
OH
N
N
N
N
S
OH
i) P(nBu)3, CH2Cl2, rt
2
O
ii) BOP, DIEA, DMF, rt
O
EDCI, HOBt,
OMe
OMe
DIEA, DMF, rt
H2N
S
N
H
O
O
(95%)
OH
2
N
H
O
3
4
Et2N
O
O
N
N
5
N
N
(37% in 2 steps)
OH
N
N
2 equiv NiCl2
(quant.)
Et2N
O
O
O
O
2-2Ni(II)
H
N
OMe
S
N
H
O
O
2
Scheme 1. Synthesis of 2-2Ni(II).
In the HPLC analysis of the reaction mixture (Fig. 3a), three new
peaks (peak 1, 2, and 3 in Fig. 3a) appeared, all of which were
assignable by MALDI-TOF mass analysis (positive mode) as the
FLAG tag peptides labeled with one coumarin unit, which had a
calculated mass number of 1355 (m/z). The sequence analysis by
the PSD mode of tandem mass identified that these labeled pep-
tides were modified with one coumarin unit at Tyr2 (peak 3),
Lys3 (peak 2), or Lys8 (peak 1) (Fig. S1), as a result of the acyl-
transfer reaction (for the labeling ratio, 1:4:2 = Tyr2: Lys3: Lys8
in Fig. 3b).
showed the gradual increase of the fluorescent band intensity over
8 h, indicative of the progress of the acyl transfer reaction in a
time-dependent manner (Fig. 4a). The reaction was also monitored
by MALDI-TOF mass analysis, in which a new peak corresponding
to FLAG-Ubi (10,054, m/z) labeled with one coumarin unit of 2-
2Ni(II) was observed at 10,357 (m/z), along with a small peak at
10,659 (m/z) corresponding to FLAG-Ubi labeled with two couma-
rin units (Fig. S2). The time-trace plot of the labeling reaction based
on the in-gel fluorescence analysis indicated that ca. 30% of FLAG-
Ubi is labeled for 8 h (Fig. 4b). In a good contrast, ubiquitin lacking
FLAG tag (control-Ubi) was negligibly labeled even after 8 h
( Fig. 4b, Fig. S2), demonstrating the binding-induced selective
labeling of FLAG-Ubi. The labeling site was determined by the
selective cleavage of the FLAG-tag part from FLAG-Ubi at the single
Met16 site by the treatment of CNBr.8 In-gel fluorescence analysis
The present acyl-transfer type of labeling was next examined
using ubiquitin protein (Ubi) fused with FLAG tag at its N-terminal
(FLAG-Ubi). The reaction was performed with 5
the presence of 40 M of 2-2Ni(II) in aqueous solution (50 mM
HEPES buffer, 100 mM NaCl, pH 8.0). In-gel fluorescence analysis
lM of FLAG-Ubi in
l

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H. Nonaka et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6696–6699
showed that the fluorescence due to the coumarin unit was not
detectable in the cleaved ubiquitin part (Fig. S3). The MALDI-TOF
mass analysis further confirmed that the main mass peak detected
at 8487 (m/z) corresponded to the cleaved Ubi without any couma-
rin units, whereas the FLAG tag labeled with one coumarin unit
was indentified in the lower mass range at 1908 (m/z) as another
main peak (Fig. S4). These results clearly indicate that the labeling
reaction takes place site-selectively at the FLAG tag. To confirm the
protein selectivity of this covalent labeling method, the labeling
experiment was then carried out under the conditions of a protein
mixture including carbonic anhydrase (CA), avidin (AV), ribonucle-
ase A (RNase), cytochrome C (CyC), and FLAG-Ubi (Fig. 5). In-gel
fluorescence analysis showed only one fluorescent band from
FLAG-Ubi, demonstrating that the acyl transfer reaction selectively
proceeded in FLAG tag-fused Ubi.
FLAG tag peptide
FLAG tag peptide
+ 1 coumarin unit
FLAG tag peptide
+ 2 coumarin unit
In conclusion, we succeeded in the selective covalent labeling of
FLAG tag and tag-fused protein on the basis of the binding-induced
acyl-transfer reaction. In addition, we showed a potential utility of
the present labeling method under the biologically complicated
conditions. Further improvement of this method in the labeling
rate and yield would be anticipated to provide a useful molecular
tool as an alternative to the conventional FLAG-antibody. We
envision further applications of this acyl-transfer based labeling
(m/z)
1,700
1,000 1,100 1,200 1,300 1,400
1,500 1,600
Figure 2. MALDI-TOF mass analysis of the acyl-transfer reaction of 2-2Ni(II) with
FLAG tag peptide. Reaction conditions; 20 M of FLAG peptide, 40 M of 2-2Ni(II),
l
l
50 mM HEPES, 100 mM NaCl, pH 8.0, 37 °C, 4 h.
a
b
Peak 2
O
O
O
N
O
NH
O
O
H
N
N
H
N
H
Peak 1
Lys3 or Lys8
*
O
H
N
N
Peak 3
H
O
O
N
O
O
H
N
O
Tyr2
O
50
60
(min)
Figure 3. (a) HPLC analysis of the acyl-transfer reaction of 2-2Ni(II) with FLAG tag peptide. Reaction conditions; 20 lM of FLAG peptide, 40 lM of 2-2Ni(II), 50 mM HEPES,
100 mM NaCl, pH 8.0, 37 °C, 4 hr. The peaks were detected by UV absorbance change at 430 nm. The peak marked with z was identified as the hydrolysis fragment of 2-
2Ni(II). (c) Structures of the covalent adducts of FLAG tag peptide formed by the acyl-transfer reaction at Lys3, Ly8, or Tyr2 with 2-2Ni(II).
Figure 4. (a) SDS–PAGE analysis of the labeling reaction of 2-2Ni(II) with FLAG tag fused ubiquitin (FLAG-Ubi, lanes 1–4) and control-Ubi lacking a FLAG tag (lanes 5–8).
Reaction conditions; 5 lM of FLAG-Ubi or control-Ubi, 40 lM of 2-2Ni(II), 50 mM HEPES, 100 mM NaCl, pH 8.0, 25 °C. (b) Time-trace plot of the labeling reaction of 2-2Ni(II)
with FLAG-Ubi (j) and control-Ubi (d) based on in-gel fluorescence analysis. The labeling yield (%) is defined as the labeled coumarin unit per total amount of the ubiquitin,
and calculated based on the fluorescence band intensity of each sample relative to the authentic sample.

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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.122.
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