One-pot native chemical ligation of peptide hydrazides enables total synthesis of modified histones

Article abstract of DOI:10.1039/c4ob00715h

One of the rising demands in the field of protein chemical synthesis is the development of facile strategies that yield the protein in workable quantities and homogeneity, with fewer handling steps. Although the native chemical ligation of peptide hydrazides has recently been shown to be useful for the chemical synthesis of proteins carrying acid-sensitive modification groups, previous hydrazide-based protein synthesis studies have used sequential ligation strategies. Here, we report a practical method for a one-pot native chemical ligation of peptide hydrazides that would circumvent the need for the isolation of the intermediate products. This method employed a fast and selective arylboronate oxidation reaction mediated by H₂O ₂, which draws attention to the potential applications of the thus far under-exploited boron-based functionalities in protein chemical synthesis. To demonstrate the practicality and efficiency of the new one-pot method, we report its application to a scalable total synthesis of modified histones (with five analogues of H3 and H4 as examples) on a multi-milligram scale, with good homogeneity. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00715h

Organic &
Biomolecular Chemistry
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One-pot native chemical ligation of peptide
hydrazides enables total synthesis of modified
histones†
Cite this: Org. Biomol. Chem., 2014,
12, 5435
Jiabin Li,‡a Yuanyuan Li,‡a Qiaoqiao He,a,b Yiming Li,*a,b Haitao Lia and Lei Liu*a
One of the rising demands in the field of protein chemical synthesis is the development of facile strategies
that yield the protein in workable quantities and homogeneity, with fewer handling steps. Although the
native chemical ligation of peptide hydrazides has recently been shown to be useful for the chemical
synthesis of proteins carrying acid-sensitive modification groups, previous hydrazide-based protein syn-
thesis studies have used sequential ligation strategies. Here, we report a practical method for a “one-pot”
native chemical ligation of peptide hydrazides that would circumvent the need for the isolation of the
intermediate products. This method employed a fast and selective arylboronate oxidation reaction
mediated by H2O2, which draws attention to the potential applications of the thus far under-exploited
boron-based functionalities in protein chemical synthesis. To demonstrate the practicality and efficiency
of the new one-pot method, we report its application to a scalable total synthesis of modified histones
(with five analogues of H3 and H4 as examples) on a multi-milligram scale, with good homogeneity.
Received 4th April 2014,
Accepted 9th May 2014
DOI: 10.1039/c4ob00715h
www.rsc.org/obc
or peptides. These semi-synthetic strategies have been success-
fully used to produce many modified histones, enabling
Introduction
Histone post-translational modifications (PTMs, e.g. acety- elegant structural and functional studies.3–5 Nonetheless, the
lation, methylation and phosphorylation) play a central role in total chemical synthesis of histones is needed for the gen-
regulating the structure and dynamics of chromatin, as well as eration of histones with multiple, different modifications at
DNA-driven cellular processes.1 An understanding of how both the terminal and middle regions.
histone PTMs translate into diverse cellular events is impor-
In this context, Ottesen and coworkers described the first
tant from both the fundamental and therapeutic perspectives. total synthesis of histone H3, through sequential native chemi-
Studies for this purpose need access to homogenously modi- cal ligations.7 This important, pioneering work enabled the
fied histones that unfortunately cannot be easily isolated production of a fully synthetic H3 with 2–7% overall yields. A
from natural sources, or obtained by classical biochemical potential limitation is that the peptide segments were prepared
methods.2 To solve this problem four strategies have been using Boc SPPS (solid-phase peptide synthesis), whose HF
used to make modified histones: (1) amber suppression muta- cleavage conditions were unsuitable for some PTMs, such as
genesis,3 (2) Cys-directed protein modification,4 (3) expressed phosphorylation. To overcome this problem Ottesen et al.
protein ligation5 and (4) total chemical protein synthesis.6 In tested the N-acylurea approach for the Fmoc SPPS of histone
the first three approaches the main structures of histones are peptide thioesters.8 Meanwhile, Aimoto and coworkers accom-
produced recombinantly, whereas the modification is intro- plished the first Fmoc-based total synthesis of H3 by using the
duced later by chemoselective reactions with small molecules Cys-Pro ester autoactivating unit as a thioester precursor.9
Notwithstanding these key advances, to meet the escalating
needs in the studies of histone PTMs it is helpful to develop a
more scalable and cost-effective method for the total synthesis
of modified histones on a multi-milligram scale.
aTsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus
Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry,
MOE Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life
We recently described the native chemical ligation of
peptide hydrazides,10 which can be readily converted to thio-
esters via NaNO2 activation and subsequent thiolysis. We
expected that this approach may allow an expedient synthesis
of modified histones, as peptide hydrazides are easily synthe-
sizable through automated Fmoc SPPS. Indeed, with the help
Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
E-mail: lliu@mail.tsinghua.edu.cn
bSchool of Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009,
China. E-mail: lym2007@mail.ustc.edu.cn
†Electronic supplementary information (ESI) available: Experimental details.
See DOI: 10.1039/c4ob00715h
‡These authors contributed equally to this work.
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Fig. 1 One-pot ligation of peptide hydrazides.
of peptide hydrazides, Brik et al. accomplished the first total
synthesis of Lys34-ubiquitinated H2B.11 However, in our syn-
thesis of H3 and H4 using the previous sequential ligation/
purification method,10b we encountered difficulty in separating
the intermediate products, which not only complicated the
experiments, but also reduced the yield. To overcome this
challenge we sought to use a “one-pot” strategy that would
need a single final purification step.12 However, our own tests
revealed that the previously developed one-pot procedures
were not readily applicable to peptide hydrazides.13
In the present study, we describe a one-pot method for the
ligation of peptide hydrazides (Fig. 1). We also explore the
possible use of the thus far under-exploited boron-based func-
tionalities in the chemical synthesis of proteins. The practical-
ity and efficiency of the method was demonstrated by the
multi-milligram-scale synthesis of modified histone H3 and
H4. Thus, in terms of the concept as well as the technological
advances, this work extends the hydrazide-based approach of
native chemical ligation.
Fig. 2 Synthesis of H3K4me3 by using N-to-C sequential ligations. (a)
Sequence of H3. (b) Procedure for the N-to-C sequential ligations. (c)
Analytical HPLC traces (λ = 214 nm) for the ligation between 1 and 2
after 0 min and 24 h. “$” denotes solvent fronts. (d) Analytical HPLC
trace (λ = 214 nm) for the final, purified H3K4me3 and its ESI-MS:
observed mass 15 240.2 0.7 Da, calc. 15 239.62 Da (average isotopes).
With 1–3 in hand, we first condensed 1 and 2 with the help
of MPAA (4-mercaptophenylacetic acid) to produce an inter-
mediate, 4. After the purification of 4, we conducted the
second ligation between 4 and 3 to obtain the full-length
peptide. Finally, the desired protein (i.e. H3K4me3) was gener-
ated through a free radical desulfurization reaction initiated,
by VA-044 (2,2′-azobis[2-(2-imidazolin-2-yl)-propane] dihydro-
chloride)16 followed by Acm deprotection. As shown in Fig. 2c,
a tricky problem encountered in the above synthesis was that
the retention times of 2 and 4 were almost identical in the
reverse-phase HPLC under the conditions used. This rendered
the monitoring of the first ligation reaction difficult. The puri-
fication of 4 was also challenging, which seriously increased
the time cost of the synthesis while reducing the yield.
Results and discussion
One-pot ligation of peptide hydrazides: model test
Problem of sequential ligations
To overcome the problem of the intermediate separation, we
Before we describe the “one-pot” method, we wish to show, decided to use a one-pot ligation approach12 to the native
with experimental data, why a one-pot method would some- chemical ligation of peptide hydrazides. This strategy has been
times be practically advantageous, if not obligatory for the successfully used in many previous studies to reduce the hand-
native chemical ligation of peptide hydrazides. For the syn- ling time and cost and increase the yields in protein chemical
thesis of histone H3 trimethyl Lys4 (H3K4me3) we started with synthesis.12 For the ligation of peptide hydrazides, the one-pot
three synthetic peptide segments (Fig. 2), i.e. H3K4me3[Ala1- method must be compatible with the NaNO2 activation step.13
Val46]-NHNH2 (1, 46-mer), H3K4me3[Cys47-Gly90]-NHNH2 Although we previously developed the sequential procedure for
(2, 44-mer), and H3K4me3[Cys91-Ala135] (3, 45-mer). Both the the ligation of peptide hydrazides,10b the one-pot ligation of
Ala47 and Ala91 residues were replaced by Cys to facilitate the peptide hydrazides has not been accomplished. A critical
ligations, which would be converted back to Ala after the full- requirement is the development of a readily reversible protec-
length peptide was produced.14 Furthermore, the side chain tion of the N-terminal Cys residue. To compromise the oper-
thiol of Cys110 was protected by the Acm (acetamidomethyl) ational ease and reaction efficiency, we were interested in the
group to enable the selective desulfurization of Ala47 and use of the p-boronobenzyloxycarbonyl (Dobz) group developed
Ala91 in the full-length polypeptide product.15
by Kemp and Roberts.18 We anticipated that the Dobz-
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protected Cys should remain un-reactive under native chemical
ligation conditions, unless it was activated by the addition of
H2O2.17 In the previous studies on Dobz,18 only small peptides
(up to a pentamer, synthesized by the solution-phase coupling
method) were tested. Thus, for the use of Dobz to accomplish
the one-pot native ligation of peptide hydrazides in protein
total synthesis, we needed to examine a number of important
issues regarding the synthesis, stability and handling of the
Dobz peptides.
First, we synthesized the pDobz protected cysteine
(i.e. pDobz-Cys(Trt)-OH) in which the boronic acid was pro-
tected as a pinacol ester (Fig. 3b). We then tested the use of
pDobz-Cys(Trt)-OH in standard Fmoc SPPS followed by regular
trifluoroacetic acid (TFA) cleavage (Fig. 3c). The desired
peptide (5) was obtained smoothly, in which the free boronic
acid group was generated. The next test was the native chemi-
cal ligation of the Dobz-Cys-peptide-NHNH2 (5) under the pre-
viously described NaNO2 activation and MPAA thiolysis
conditions (Fig. 3d).10 The expected peptide thioester inter-
mediate (6) was formed cleanly, which reacted in situ with the
subsequently added Cys to produce the ligation product 7, in
an almost quantitative yield after 60 min (Fig. 3e). Some minor
products, due to formation of a thiolactone with the internal
Cys and lactam with the C-terminal Lys,11 were observed. To
further confirm the stability of the Dobz-amino protecting
group to the NaNO2 oxidation conditions, another test of the
Dobz-Cys-peptide-NHNH2 was performed (Fig. 3f) in which Ala
was the ligation site and the middle Cys was eliminated.
In this test, the ligation proceeded more cleanly without the
formation of any thiolactone or lactam by-products (Fig. 3g).
Collectively, these results showed that the Dobz group was
compatible with the modern Fmoc SPPS and the native
chemical ligation of peptide hydrazides.
A critical test for the Dobz group was its selective removal
by H2O2 oxidation, followed by a spontaneous hydrolysis. For
this purpose, we dissolved 5 in a ligation buffer containing 65
equivalents of MPAA (pH 6.5–7.0) to simulate the conditions
needed by the one-pot ligation (Fig. 3h). To test whether or not
the H2O2 treatment may cause any damage to the peptide, we
incorporated all the redox sensitive amino acids (i.e. Cys, Met,
Trp, Tyr, His) into 5. When 1 equivalent of H2O2 (as 1 M
Fig. 3 Tests for the use of Dobz-peptides. (a) Structure of Dobz. (b) The
synthesis of pDobz-Cys(Trt)-OH. (c) Fmoc SPPS of Dobz-Cys-peptide-
aqueous solution) was added to 5 in the ligation buffer, we NHNH2. (d) Conversion of Dobz-Cys-peptide-NHNH2 to a thioester and
its ligation with Cys. (e) Analytical HPLC traces (λ = 214 nm) for purified
5, crude reaction mixture of 5 with NaNO2 and MPAA, and crude ligation
product with Cys. “$” denotes solvent fronts. (f) Ligation of 8 with 9. (g)
Analytical HPLC traces (λ = 214 nm) for 8, ligation mixture of 8 and 9
observed no change in 5 after 50 min, indicating that MPAA
was the most susceptible to H2O2 oxidation in the system
(Fig. 3i). To fully consume the MPAA and oxidize 5, we tested
the addition of 33.5 equivalents of H2O2. Gratifyingly, under
after 10 min and 3 h. (h) Treatment of 5 with H2O2 in the ligation buffer.
this condition we observed a complete and clean conversion of (i) Analytical HPLC traces (λ = 214 nm) after treatment of 5 (in the pres-
ence of 65 equiv. MPAA) with 1 equiv. H2O2 (50 min), 33.5 equiv. H2O2
(10 min), and 66 equiv. H2O2 (10 min).
the Dobz-protected 5 to the deprotected peptide product 11, in
10 min. A further increase of H2O2 to 50 equivalents (data not
shown) and 66 equivalents was found to generate a by-product,
whose mass was 16 Da higher than that of 11. According to
remove the Dobz group, selectively. The Met would be oxidized
if excessive H2O2 was added, but this side reaction could be
prevented by using an accurately calculated quantity of H2O2.19
Furthermore, use of norleucine to replace Met,20 which has
the previous study by Schultz et al. on BoPhe-incorporated pro-
teins, this by-product was assigned as peptide 12, with the Met
oxidized.17 Nonetheless, even 66 equivalents of H2O2 did not
fully oxidize 11 to 12. Collectively, the above results suggested
that H2O2 should oxidize MPAA first and then oxidize and
been
a
frequently employed tactic in protein chemical
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synthesis, would completely circumvent the Met oxidation 13 (Fig. S2 and S11†), we concluded that the low yield of 13
problem. It is important to emphasize that except for Met (and was due to the nature of the peptide sequence, but not due to
Cys that forms disulfides), we did not observe the reaction of the introduction of the Dobz group. The segments were con-
H2O2 with any other amino acid residues (in particular, Tyr or densed in a one-pot fashion as follows: firstly, 13 (21 mg) was
Trp) under the conditions used to remove the Dobz group.19
ligated with 3 (20 mg) at pH 6.8 for 1 hour; secondly, the reac-
tion mixture was treated with 115 μL of 1 M aqueous H2O2 for
10 minutes and finally, 1 (37 mg, pre-activated by NaNO2 and
thiolyzed with MPAA) was added to the reaction mixture.
Because the second ligation occurred at a Val-Cys junction, it
was allowed to proceed for 48 hours. The full-length peptide
was obtained through an HPLC purification process in 12 mg,
corresponding to an isolated yield of 21% for the one-pot reac-
tions (Fig. 4b). The HPLC traces of this one-pot transformation
were relatively clean, indicating that the intended reactions
proceeded smoothly (For an explanation of every peak in
Fig. 4b, please refer to Fig. S12†). Subsequently we carried out
a desulfurization and Acm deprotection on the HPLC purified
materials, to generate 8.3 mg of the target product, H3K4me3,
corresponding to a 14.4% overall isolated yield. A repeated
experiment using 14 mg 13, 13 mg 3, and 25 mg 1 afforded
7.7 mg of H3K4me3, corresponding to a 19.8% overall isolated
yield. Through analysis of the MALDI-TOF spectra (Fig. S14†)
and every peak in the ESI-MS (data are not shown), we con-
cluded that the Met120 in H3K4me3 was not oxidized in the
H2O2 treatment. Thus, our experiments showed that modified
H3 could be practically synthesized on a multi-milligram scale.
To further characterize the final synthetic H3K4me3, we
conducted an isotope envelope analysis (Fig. 5a and Fig. S15,
Table S1†). The results showed that the observed isotope envel-
ope was fully consistent with the theoretically predicted
pattern of the target H3K4me3, but not the oxidized side
product (+16 Da). Although there were peaks in the region of
the predicted oxidized product (Fig. S15b†), we could not
Total synthesis of modified histones
The efficient and selective oxidation of the Dobz group by
H2O2 and its removal from the N-terminal Cys allowed for the
design of a H2O2-controlled one-pot ligation method suitable
for peptide hydrazides (Fig. 1). From an operational perspec-
tive, the H2O2 treatment did not need any pH change and it
released water as the only by-product. With this key chemistry
for the one-pot native chemical ligation of peptide hydrazides
in hand, we carried out a new synthesis of H3K4me3.
The three peptide segments (i.e. 1, 13, and 3, Fig. 4a) were
prepared by using automated Fmoc SPPS and sufficient puri-
fied materials were obtained. However, the key segment 13 was
synthesized with a low yield. After analysis of the crude 2 and
Fig. 4 Synthesis of H3K4me3 by using H2O2-controlled one-pot native
chemical ligation of peptide hydrazides. (a) The overall procedure. (b)
Analytical HPLC traces (λ = 214 nm) for the different steps in the one-
pot ligation. Black: beginning of the whole process; Red: ligation
mixture of 3 and 13 after 1 h; Olive: ligation mixture after the H2O2 treat-
ment; Blue: the final ligation mixture. “$” denotes solvent fronts. For
more explanation of each peak, please refer to Fig. S12.† (c) Analytical
HPLC trace (λ = 214 nm) for the final, purified H3K4me3. (d) its ESI-MS:
observed mass 15 238.3 1.6 Da, calc. 15 239.62 Da (average isotopes).
Fig. 5 Isotope envelopes and MS-MS analysis of synthetic H3K4me3.
(a) The observed isotope envelopes. (b) The MS-MS analysis data with
the observed fragments marked. For more details please refer to Fig. S15
and S16.†
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exclude that these were caused by baseline noise. Collectively, (27-mer). Both Ala38 and Ala76 were changed to Cys to facilitate
from the above results of the MALDI-TOF and ESI-MS, we had the ligations, which would be converted back to Ala after the
reason to believe that the oxidation of Met was almost pre- full-length peptide was produced.14 Again, these three peptide
vented, albeit not completely. In the MS-MS analysis (Fig. 5b segments were prepared by using automated Fmoc SPPS
and Fig. S16†), the observed fragments closely matched the (Fig. 6b). The middle segment was also difficult to synthesize,
sequence of H3K4me3, especially that the ligation junction due to its hydrophobicity (Fig. S18†). Comparing the HPLC
(Gly-Ala) was observed. These experimental results confirmed trace of the crude peptide H4K16ac[Cys38-His75]-NHNH2 with
that the synthetic H3K4me3 was highly homogeneous.
an unprotected Cys at the N terminal (data are not shown), we
For the application of the H2O2-controlled one-pot ligation confirmed that the low yield was not caused by the Dobz
method to the total synthesis of a modified H4 (i.e. H4K16ac), group.
we also divided it into three peptide segments (Fig. 6a). They
were H4K16ac[Ser1-Leu37]-NHNH2 (37-mer), Dobz-H4K16ac- pot fashion from 28, 16, and 11 mg of the starting materials
[Cys38-His75]-NHNH2 (38-mer), and H4K16ac[Cys76-Gly102
The three segments of H4K16ac were condensed in a one-
]
with the control of 106 μL of 1 M aqueous H2O2. The full-
length peptide was then obtained through HPLC purification
in 11 mg, corresponding to an isolated yield of 27.4% for the
one-pot ligation (Fig. 6c). Subsequently, we carried out a desul-
furization procedure to generate the target product, H4K16ac,
in 7.3 mg, corresponding to an 18.3% overall isolated yield. A
repeated experiment using 14 mg, 8 mg, and 5.5 mg of the
starting materials afforded 4.0 mg of H4K16ac, corresponding
to a 20.1% overall isolated yield. Again, the HPLC traces of this
one-pot transformation (Fig. 6c) were relatively clean, indicat-
ing that the designed reactions proceeded smoothly. (For an
explanation of every peak in Fig. 6c, please refer to Fig. S20†).
Note that Met84 in H4K16ac was not oxidized in the H2O2 treat-
ment (Fig. 6e and Fig. S21†). Additionally, in the synthesis we
observed that ca. 30% of Dobz-H4K16ac[Cys38-His75]-NHNH2
was hydrolyzed during the ligation (Fig. 6c). The hydrolysis by-
product was almost inseparable from the product of the first
ligation (i.e. Dobz-H4K16ac[Cys38-Gly102]. Thus, the one-pot lig-
ation also circumvented the intermediate separation problem
that would be expected in the stepwise synthesis of H4K16ac.
Furthermore,the synthetic H4K16ac was successfully character-
ized by the isotope envelope and MS-MS analysis (Fig. 7 and
Fig. S22, S23 and Table S2†). Collectively, the above results of
Fig. 6 Synthesis of H4K16ac by using H2O2-controlled one-pot ligation
of peptide hydrazides. (a) Sequence of H4. (b) The overall procedure. (c)
Analytical HPLC traces (λ = 214 nm) for the different steps in the one-
pot ligation. Black: beginning of the first ligation; Red: reaction mixture
after the first ligation; Olive: ligation mixture after the H2O2 treatment;
Blue: the final ligation mixture. # correspond to the hydrolysis by-
product. Red: Dobz-[Cys38-His75]-OH; Olive: [Cys38-His75]-OH; Blue:
[Ser1-His75]-OH.“$” denotes solvent fronts. For explanation of other
peaks, please refer to Fig. S20.† (d) Analytical HPLC trace (λ = 214 nm)
for the final, purified H4K16ac. (e) ESI-MS: observed mass 11 277.2
0.7 Da, calc. 11 278.04 Da (average isotopes).
Fig. 7 Isotope envelopes and MS-MS analysis of synthetic H4K16ac. (a)
The observed isotope envelopes. (b) The MS-MS analysis with the
observed fragments marked. For more details, please see Fig. S22
and S23.†
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Fig. 9 Histone octamer reconstitution of synthetic H3K4me3–H4K16ac
tetramer with H2A–H2B dimer. (a) Gel filtration profile of histone
octamer eluted over a Superdex 200 10/300 column. Histone octamer
was eluted at 12.8 ml; excess H2A–H2B dimer was eluted at 15.2 ml. (b)
SDS–PAGE (left) and Western blotting (right) analysis of the reconsti-
tuted histone octamer. CBB: coomassie brilliant blue; WB: Western
blotting.
Fig. 8 Histone tetramer formation of synthetic H3K4me3 and H4K16ac.
Blue curve: gel filtration profile of histone H3–H4 tetramer eluted over a
Superdex 200 10/300 column. The peak of H3–H4 tetramer was eluted
at 13.6 ml. Left to the peak: SDS-PAGE of the H3–H4 tetramer peak
stained by coomassie brilliant blue.
the characterization confirmed that the synthetic H4K16ac was
highly homogenous and the potential side reaction of oxi-
dation was not a problem.
use of a novel application of the Dobz protecting group, which
was removable under mild conditions by a fast and selective
arylboronate oxidation reaction mediated by H2O2, thus
drawing attention to the interesting potential of using boron-
based functionalities in protein chemical synthesis. Our
experiments showed that the H2O2-based deprotection, when
carefully controlled, was fast, clean and compatible with all
the proteinaceous amino acids including Cys, Met, Trp, and
Tyr. Thus, the present one-pot approach adds a new tool to the
arsenal for the total chemical synthesis of small to medium
sized proteins, which may find a complementary or unique
usefulness to certain protein molecules.
By using the new one-pot method we accomplished the
total chemical synthesis of the modified histone proteins
H3K4me3, H4K16ac, and several others22 on a multi-milligram
scale and with good homogeneity. Our results showed that
such a synthesis was fast (time cost = ca. 2 weeks for each
protein) and scalable with regular research laboratory equip-
ment. Given the fact that the hydrazide-based ligation method
is compatible with many post-translational modifications,
including phosphorylation, ubiquitination and glycosylation,
we expect that the present method may find applications in
the studies on histone post-translational modifications. The
application of the one-pot approach to the total synthesis of
histone H2A and H2B, as well as the use of fully synthetic
histones to study the biology of epigenetics, are ongoing in our
laboratory.
Histone octamer reconstitution
The purity and identity of the synthetic H3K4me3 and
H4K16ac were successfully characterized by the HPLC, MALDI-
TOF and ESI-MS analyses, as well as the isotope envelope and
MS-MS analyses (Fig. 4–7). To conduct biophysical characteri-
zations, we dissolved the synthetic H3K4me3 and H4K16ac in
an unfolding buffer containing 6 M guanidine hydrochloride,
5 mM dithiothreitol, and 20 mM Tris-HCl, pH 8.0. After
2 hours, the solution was subjected to four rounds of dialysis
against a refolding buffer containing 2.0 M sodium chloride,
1 mM Na-EDTA, 5 mM dithiothreitol, and 20 mM Tris-HCl, pH
8.0 at 4 °C for about 48 hours. Gratifyingly, we identified and
isolated the refolded H3K4me3–H4K16ac tetramer through gel
filtration chromatography (Fig. 8).
With this tetramer in hand, we next mixed it with a pre-
prepared recombinant histone H2A–H2B dimer at a molar
ratio of 1 : 2.5. The mixture was then dialyzed against the
refolding buffer over four rounds, for 48 hours at 4 °C.
Through gel filtration chromatography over a Superdex 200
10/300 column (GE Healthcare), we successfully identified and
isolated the histone octamer as a single mono-dispersed peak
eluted at 12.8 ml (Fig. 9a), a retention volume characteristic
for the histone octamer.21 Furthermore, the identities of the
H3K4me3 and H4K16ac in the reconstituted histone octamer
were confirmed by Western blotting, using rabbit anti-
H3K4me3 and anti-H4K16ac antibodies (Fig. 9b). Collectively,
these experiments verified that our synthetic H3K4me3 and
H4K16ac had the correct biophysical activities that could
form the histone octamer smoothly and be recognized by
antibodies.
Acknowledgements
This work was supported by the national “973” grants from the
Ministry of Science and Technology (2011CB965300 for L. Liu
and H. Li), NSFC (91313301 and 21225207 for L. Liu and
31270763 for H. Li), and the Specialized Research Fund for
Conclusion
Here, we report a practical method for the one-pot native the Doctoral Program of Higher Education (grant no.
chemical ligation of peptide hydrazides. This method made 20120002130004).
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(Please see ESI†).
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