C O M M U N I C A T I O N S
In conclusion, we have created a general method for the
quantitative, site-specific incorporation of Nε-methyl-L-lysine in
recombinant proteins. The method has two steps: first, an amino
acid containing an auxiliary group is used to differentiate Nε-methyl-
L-lysine from L-lysine and to provide a good substrate for the
pyrrolysyl synthetase; second, the auxiliary group is removed to
reveal Nε-methyl-L-lysine. We have demonstrated the utility of the
method by site-specifically installing Nε-methyl-L-lysine into full-
length histone H3 and demonstrated that the modified H3 specif-
ically recruits HP1.21 We are currently extending our approach to
installing other modifications implicated in the histone code and
epigenetic inheritance to understand how combinations of post-
translational modifications program cellular outcomes.
Figure 2. 2 can be site-specifically incorporated into recombinant proteins
in response to an amber codon and quantitatively, post-translationally
converted to 3. (A) Myoglobin-His6 is purified from E. coli containing
pMyo4TAGPylT-his6, and pBKPylS in the presence of amino acids 1 or 2.
(B) Synthesis of H3K9me1, lane 3, His6 H3 incorporating 2 in place of K9
and deprotected with 2% TFA, lanes 4 and 5 are postcleavage of the
N-terminal His6 tag with TEV protease. (C) HP1 specifically recognizes
H3K9me1. HP1 was used to immunoprecipitate H3 or H3K9me1. The
immunoprecipitation was probed for H3 using an anti H3 antibody. Input:
2% of total Histone H3. PD “pull down”. Mock: no HP1 added.
Acknowledgment. We are grateful to Daniela Rhodes, Andrew
Routh, and Lynda Chapman for reagents and discussions and to
Sew Peak-Chew for assistance acquiring MS/MS spectra. This work
was supported by The Medical Research Council, UK, and grants
from the European Research Council & the Human Frontiers of
Science Program to J.W.C.
To specifically and efficiently introduce 2 in a histone at a
physiologically relevant site, we transformed E. coli BL21(DE3)
with pBKPylS and pCDF-PylT-H3K9TAG (a vector which encodes
MbtRNACUA and a N-terminally hexahistidine tagged histone H3
gene in which the codon for lysine 9 is replaced with an amber
codon).15 We grew the cells in the presence of 2 mM 2 and
expressed and purified the recombinant histone in good yield
(2 mg per liter of culture). ESI-MS analysis of the purified histone
confirms the incorporation of 2 into histone H3 (Supplementary
Figure 2B).
To demonstrate that the tert-butyl-oxycarbonyl group can be
quantitatively removed from the histone under mild conditions, the
purified H3K9-2 was treated with a solution of 2% trifluoroacetic
acid (TFA) for 4 h at 37 °C. Western blots with an anti-H3K9me1
antibody against unmodified H3, H3 bearing 2 at position 9 (H3K9-
2), and the TFA treated H3K9-2 confirmed the presence of methyl-
L-lysine at position 9 in the deprotected sample (Figure 2B, lane
3). The ESI-MS spectra of the deprotected H3K9-2 sample
(Supplementary Figure 2C) demonstrates that the auxiliary is
quantitatively removed under these conditions to reveal Nε-methyl-
L-lysine. MS/MS protein sequencing (Supplementary Figure 2D)
further confirms that the site of lysine methylation is as genetically
encoded. H3K9me1 can be assembled into nucleosomes in vitro
with a comparable efficiency to unmodified H3 (Supplementary
Figure 3).
Supporting Information Available: Experimental protocols and
supplementary figures. This material is available free of charge via the
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