C O M M U N I C A T I O N S
peutics that are more efficacious. The labeling method is compatible
with and orthogonal to cysteine labeling and will prove useful for
introducing two distinct labels into a single protein for fluorescence
resonance energy transfer (FRET) experiments to probe protein
function, structure, and dynamic behavior. Moreover, since this
synthetase tRNA pair is functional and orthogonal in eukaryotic
1
2
cells, it will be possible to extend the approach reported here to
the labeling of proteins produced in, and displayed on, eukaryotic
cells.
Figure 2. (A) Efficient and specific labeling of genetically encoded 2 with
azido probes. (Left) The biotin azide 5 labeling reaction was performed on
myoglobin containing 4 or 2 at position 4 (myo-4his6-4 and myo-4his6-2).
Proteins were probed for biotin (top). (Right) By3 labeling with 6 was
imaged directly. Coomassie stained protein gels (bottom) demonstrate equal
protein recovery in the samples. (B) ESI-MS of the myoglobin-his6
containing 2 labeled with biotin azide 5 (Found: 19199.5 ( 1.5 Da, expected:
The alkyne 2 and azide 3 are incorporated using a synthetase
and tRNA pair that is mutually orthogonal in its aminoacylation
specificity to the MjTyrRS/tRNACUA pair (JWC unpublished) that
has been used to incorporate a range of aromatic unnatural amino
4
acids. This suggests it will be possible to incorporate 2 or 3 in
combination with genetically encoded aromatic amino acids,
1
9198.2 Da).
5
7
including previously incorporated azides and alkynes, at distinct
sites in recombinant proteins using suitably altered combinations
bin-his6 bearing 2 at position 4 was treated with the biotin azide 5
or a fluorophore (By3) azide 6 (Supplementary Figure 3), in the
17
of synthetase/tRNA pairs and evolved orthogonal ribosomes. This
will enable the formation of directional intramolecular cross-links
to constrain protein structure and may allow for the genetic selection
of enhanced protein stability and function.
presence of CuSO
disodium salt, and ascorbate in sodium phosphate buffer (pH 8.3).
4
, 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid
15
In control experiments myoglobin-his6 bearing 4 at position 4 was
treated identically. After 18 h, the purified labeling reactions were
probed and analyzed by SDS-PAGE (Figure 2A). These experi-
ments demonstrate the specific labeling of the alkyne containing
protein.
Acknowledgment. A.D. acknowledges support by North Caro-
lina State University and is a Beckman Young Investigator and
Cottrell Scholar. J.W.C. acknowledges the MRC and European
Research Council for support.
Previous work has visualized protein labeling by gel-based
7,11,14
methods alone,
which does not allow quantification of labeling
Supporting Information Available: . Experimental protocols and
supplementary figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
efficiency. ESI-MS of our purified labeling reaction (Figure 2B)
demonstrates a labeling efficiency of 90-100%. Quantification of
the ratio of biotin or By3 to protein in purified samples provides
independent confirmation of the labeling efficiency (Supplementary
Methods).
References
(
1) Hermanson, G. T. Bioconjugate Techniques; Academic Press: 1996.
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(
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In contrast to previous work
the amino acids can be synthesized
in just two steps in excellent yield and site-specifically incorporated.
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(
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(
(
(
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efficiently labeled with azides that introduce biotin or fluorescent
4
0, 2004–2021.
7,11,14
(14) Fekner, T.; Li, X.; Lee, M. M.; Chan, M. K. Angew. Chem. 2009, 121,
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we have explicitly
1
661–1663.
demonstrated and quantified the efficient conjugation of probes to
the genetically encoded amino acid. Since many protein therapeutics
are conjugated in a residue specific manner to polyethylene glycols
(15) Schoffelen, S.; Lambermon, M. H.; van Eldijk, M. B.; van Hest, J. C.
Bioconjugate Chem. 2008, 19, 1127–31.
(
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1
6
through lysine, the method reported may provide a direct route
to discovering site-specifically modified versions of these thera-
JA900553W
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