Expanding the genetic code for photoclick chemistry in E. coli, Mammalian Cells, and A. thaliana

Article abstract of DOI:10.1002/anie.201303477

Breaking the (genetic) code: N-ε-acryl- lysine was genetically incorporated into proteins in bacterial cells, mammalian cells, and Arabidopsis thaliana by co-expressing a pyrrolysyl-tRNA mutant synthetase and its cognate tRNA in the model organism. A photoclick reaction driven by irradiation by 365 nm UV light or 405 nm laser light and allowed for site-specific protein labeling within minutes both in vitro and in vivo. Copyright

Full text of DOI:10.1002/anie.201303477

Angewandte
Chemie
Communications
Synthetic Biology
Breaking the (genetic) code: N-e-acryl-
lysine was genetically incorporated into
proteins in bacterial cells, mammalian
cells, and Arabidopsis thaliana by co-
expressing a pyrrolysyl-tRNA mutant
synthetase and its cognate tRNA in the
model organism. A photoclick reaction
driven by irradiation by 365 nm UV light
or 405 nm laser light and allowed for site-
specific protein labeling within minutes
both in vitro and in vivo.
F. H. Li, H. Zhang, Y. Sun, Y. C. Pan,
J. Z. Zhou, J. Y. Wang*
&&&& — &&&&
Expanding the Genetic Code for
Photoclick Chemistry in E. coli,
Mammalian Cells, and A. thaliana
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
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DOI: 10.1002/anie.201303477
Synthetic Biology
Expanding the Genetic Code for Photoclick Chemistry in E. coli,
Mammalian Cells, and A. thaliana**
Fahui Li, Hua Zhang, Yun Sun, Yanchao Pan, Juanzuo Zhou, and Jiangyun Wang*
Bioorthogonal chemical reactions together with techniques to
expand the genetic code have provided exciting new means
for protein labeling and visualization in living systems,[1] as
well as for optimizing the efficacy of therapeutic proteins.[2]
Toward these goals, amino acids with small bioorthogonal
functional groups, such as azide, alkyne, or cyclopropene
moieties,[3] as well as larger reactive bioorthogonal groups,[4]
such as cyclooctyne, norbornene, trans-cyclooctene, aryl-
tetrazole, or aryltetrazine, have been site-specifically incor-
porated into proteins, allowing for selective conjugation of
biophysical probes through azide–alkyne click chemistry
(AAC), tetrazole–alkene photoclick chemistry (TAP), and
reverse-electron demand Diels–Alder reactions.[5] The main
advantages of the photoclick reaction (Supporting Informa-
tion, Scheme S1) are: 1) its fast rate (up to 50mÀ1 sÀ1); 2) that
spatiotemporal control is initiated by a photo-induced
reaction; 3) that the photoclick reaction is fluorogenic,
allowing for high-contrast fluorescence imaging without
tedious washing steps. In previous studies, we reported the
site-specific incorporation of p-(2-tetrazole)phenylalanine
(p-Tpa)[4a] and N-e-(1-methylcycloprop-2-enecarboxamido)-
lysine (CpK)[3c] in E. coli and mammalian cells. Subsequent
photoirradiation of labeled proteins with UV light facilitates
selective conjugation with dimethyl fumarate or diaryltetra-
zole, respectively.
By expanding the genetic code and introducing photoclick
chemistry to plants, important problems in plant chemical
biology can be addressed,[6] such as photosynthesis and stress
response, which can only be studied at the organismal level.
Recently, expansion of the genetic code has been used to
optimize therapeutic proteins produced in bacteria and
mammalian cells.[2] Because plants offer an attractive alter-
native to microbial fermentation and animal cell cultures for
high-yield production of recombinant proteins on an agricul-
tural scale,[8] expanding the genetic code in plants would be
useful for producing recombinant therapeutic proteins and
enzymes with enhanced properties, better safety, and lower
costs.[8]
To fully realize the potential of photoclick reaction for
tracking fast cellular processes, it is desirable that the
unnatural amino acid (UAA) used has a small functional
group such that there is minimal perturbation of the target
protein, and a very brief exposure to long-wavelength UV
light or violet-blue light is used to drive the photoclick
reaction to minimize damage to cells and plants. Herein, we
addressed these issues by genetically incorporating N-e-
acryllysine (AcrK, Figure 1A), in response to an amber stop
codon (TAG) in bacterial cells, mammalian cells, and plants.
This new strategy was then used to efficiently label proteins
both in vitro and in vivo. In comparison to lysine, AcrK has
only four extra non-hydrogen atoms, which is significantly less
than other UAAs.[3,4] Replacing one lysine residue with AcrK
should cause only minimal perturbation to the target protein.
In addition, the electron-withdrawing amido group should
activate the terminal alkene group to achieve a higher
photoclick reaction rate. Indeed, the photoclick reaction
between tetrazole and acrylamide was found to proceed
nearly one hundred times faster than that of allylphenyl-
ether[9] and 1.5 times faster than that of cyclopropene.[3c] AcrK
was synthesized by reacting N-a-Boc-lysine with acryloyl
chloride in a basic ethyl acetate/water solution at 08C
(Figure 1A), followed by deprotection with HCl gas with an
overall yield of 72%, without the need for metal catalysts.
AcrK was found to be relatively stable in the presence of
glutathione, an abundant biomolecule both amine and thiol
groups; greater than 95% of AcrK remained following
incubation with 5 mm reduced glutathione in a buffer at
pH 7 for 24 hours (Figure S1). At neutral pH, most primary
amines are protonated and most thiols are neutral. Therefore,
glutathione could react slowly with the acrylamido group
through a Michael addition reaction. While AcrK has
comparable stability to CpK under physiological conditions,
the synthetic route for CpK requires six steps, expensive
heavy metal catalysis, with an overall yield of 15%.[3c]
Because a large amount of the UAA is required for modifying
plant proteins on an agricultural scale, it is essential that the
UAA is synthesized in an economically, without the use of
toxic heavy-metal catalysts.
[*] F. H. Li,[+] H. Zhang,[+] Y. Sun,[+] Y. C. Pan,[+] J. Z. Zhou,
Prof. Dr. J. Y. Wang
Laboratory of Non-coding RNA, Institute of Biophysics
Chinese Academy of Sciences
15 Datun Road, Chaoyang District, Beijing, 100101 (China)
E-mail: jwang@ibp.ac.cn
We chose diaryltetrazole 2 (Figure 1B) because it is highly
reactive for photoclick reactions[9] and is soluble in water.
Because the molar extinction coefficients of 2 at 365 nm and
405 nm are around 100mÀ1 cmÀ1 (Figure S2) and the quantum
yields for the photolysis of diaryltetrazoles are very high (0.5–
0.9),[5f] 2 should be efficiently activated by long-wavelength
UV light or violet light. An amine functional group provides
a convenient handle for further derivatization. Upon irradi-
[+] These authors contributed equally to this work.
[**] We gratefully acknowledge the Major State Basic Research Program
of China (2010CB912301, 2009CB825505), the National Science
Foundation of China (90913022, 31000364), and CAS grant (KSZD-
EW-Z-003). We thank Prof. Peter Schultz for providing the pEVOL
vector and Dr. Xiang Ding for help with mass spectrometry.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303477.
2
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pEVOL vector[10] and MbtRNACUA (pylT) into an ApaL1/
Xho1 restriction site to generate the pEVOL-AcrK vector
(Figure S3). An amber stop codon was substituted for Tyr151
in green fluorescent protein (GFP) that contained a C-
terminal six-histidine tag, affording the pET22b-GFP151TAG
vector. GFP expression was then carried out in the presence
of AcrKRS and MbtRNACUAPyl in E. coli grown in LB media
supplemented with 1 mm AcrK. As a negative control, protein
was also expressed in the absence of AcrK. Analysis of the
purified GFP by SDS-PAGE showed that full length GFP was
expressed only in the presence of AcrK (Figure 2A). The
yield of the mutant GFP was 20 mgLÀ1. In comparison, the
yield of wild-type GFP under similar conditions was
50 mgLÀ1. To the best of our knowledge, this is the highest
UAG codon suppression efficiency (40%) reported for UAA
Pyl
Pyl
incorporation using the PylRS/MbtRNACUA pair. ESI-MS
analysis of the mutant GFP showed an average mass of
27728 Da, consistent with the calculated mass of 27728 Da
(Figure 1B). No peaks corresponding to incorporation of any
of the 20 natural amino acids into the Tyr151 site of GFP were
observed. A small peak at 28035 Da was also found,
consistent with the formation of a Michael addition adduct
with glutathione (molecular weight = 307 Da). The relative
signal intensities of the 28035 Da peak versus the 27728 Da
peak indicates that less than 5% of the modified GFP reacted
with glutathione. To assess whether AcrK can serve as
a bioorthogonal reporter for protein labeling, we incubated
myoglobin-4-AcrK with tetrazole 3 in PBS buffer and
irradiated the sample at 365 nm for ten minutes. MS-MS
analysis of the product mixture revealed that the AcrK at the
fourth position was selectively and completely converted to
the pyrazoline product (Figures S4,S5).
To confirm whether AcrK can be used as a reactive
bioorthogonal group in living cells, we used a tubulin-like
bacterial cytoskeleton protein FtsZ as a model. Assembly of
FtsZ into ring structures is required for bacterial division and
small-molecule inhibitors of FtsZ have been shown to be
promising antibiotics.[11,12] We chose to incorporate 1 near the
N-terminus of FtsZ, at the third position, because this was
likely to cause the minimum perturbation to FtsZ assembly
and function.[12] An amber codon was substituted for the third
position of FtsZ, which also contained a C-terminal six-
histidine tag, affording the pET22b-FtsZ3TAG vector. Using
this and the pEVOL-AcrK vector, full length mutant FtsZ
protein with AcrK site-specifically incorporated at the third
position (FtsZ-3-AcrK) was expressed with a yield of
20 mgmLÀ1. As a control, we also expressed wild-type (wt)
FtsZ, which yielded 50 mgmLÀ1. Both wt FtsZ and FtsZ-3-
AcrK were then purified using Ni-NTA chromatography. Wt
FtsZ or FtsZ-3-AcrK (10 mm) were then incubated with
100 mm diaryltetrazole 2 in PBS buffer and irradiated at
365 nm for five minutes. Because the pyrazoline product is
fluorescent, we could easily follow this reaction using in-gel
fluorescence. A fluorescent FtsZ band was observed for FtsZ-
3-AcrK but not for wt FtsZ, and fluorescence was observed
for FtsZ-3-AcrK only after UV irradiation (Figure 2C).
Therefore, the photoclick reaction only occurred between
tetrazole 2 and AcrK but not the natural amino acids.
Photoclick reactions were then run under similar conditions,
Figure 1. A) Synthesis of AcrK 1. B) Photoclick reaction of diaryltetra-
zole 2 with acrylamide yields the fluorescent pyrazoline product 3
under 365 nm UV irradiation in PBS buffer. C) Absorption and emis-
sion spectra of pyrzoline 3 (pH 7.4, 100 mm sodium phosphate
buffer). Extinction coefficient=15000 at 380 nm with a quantum yield
of 0.27.
ation with 365 nm UV light (lamp: UVP TFML-20, 36 W), 2
reacted with acrylamide to generate a fluorescent pyrazoline
product 3 with absorption maxima at 360 nm and a broad
emission band at 400–700 nm (Figure 1C), which matches
well with the DAPI filter setting (lex = 365 nm, lem = 445 Æ
25 nm).
To evolve an orthogonal tRNA/aminoacyl-tRNA synthe-
tase pair that selectively charges AcrK in response to a TAG
amber codon, we performed three rounds of positive and two
rounds of negative selections with an MbPylRS library, as
previously described.[3c] An AcrK-specific PylRS mutant,
termed AcrKRS, was identified. Sequencing of this clone
revealed the following five mutations: D76G, L266M, L270I,
Y271F, L274A, and C313F. To test the efficiency and
selectively of AcrKRS, we first ligated AcrKRS sequentially
into SalI/BglII and Pst1/Nde1 restriction sites into the
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diaryltetrazole
2 in PBS buffer for
30 minutes at 378C and then irradiated
at 365 nm for one minute before fluo-
rescence imaging using a DAPI channel.
The fluorescent signal was only detected
in cells expressing FtsZ-3-AcrK, but not
wt FtsZ (Figure S7). Thus, our strategy
to genetically incorporate 1 into proteins
followed by a photoclick reaction was
found to be suitable for imaging in live
cells. Because this method requires the
mutation of only one amino acid and
diaryltetrazole 2 has a molecular weight
of only 338 Da, this method has minimal
perturbation on the native proteins for
visualization in living cells.
Because the pyrrolysyl tRNA syn-
thetase/tRNA pair is orthogonal in both
bacterial and mammalian cells,[1] we
tested
if
the
AcrKRS
and
Pyl
MbtRNACUA pair could facilitate the
genetic incorporation of 1 into mamma-
lian cells. We cloned AcrKRS into the
pCMV-NBK-1 vector,[4f] generating
a pCMV-AcrKRS vector, where the
expression of AcrKRS is driven by the
CMV promoter and the transcription of
Pyl
MbtRNACUA is under the control of
Figure 2. A) Coomassie Blue-stained SDS-PAGE of GFP-151-AcrK (arrow) expressed in the
presence (+) and absence (À) of 1 mm AcrK. B) ESI-MS spectra of GFP-151-AcrK. Top:
deconvoluted spectrum. Expected mass=27728 Da, found 27728 Da. Bottom: orgininal mass
spectra. C) Coomassie Blue-stained SDS-PAGE (top) and in-gel fluorescence image (bottom) of
purified wt FtsZ (WT) and FtsZ-3-AcrK (M), with or withoug 365 nm irradiation for 5 min in the
presence of 100 mm 2. D) In-gel fluorescence image (top) and SDS-PAGE (bottom) of FtsZ-3-
AcrK following 365 nm irradiation for 0–10 min in the presence of 100 mm 2. E) In-gel
fluorescence image (top) and SDS-PAGE (bottom) of crude cell lysates (WT=cells expressing
wt FtsZ; M=cells expressing FtsZ-3-AcrK), following 365 nm irradiation for 1–20 min in the
presence of 100 mm 2.
a human U6 promoter. The plasmids
pCMV-AcrKRS and pSwan-EGFP37-
TAG were cotransfected into Chinese
hamster ovary (CHO) cells. The cells
were then grown in the presence and
absence of 1 mm 1 for 36 hours. EGFP
fluorescence was observed only for cells
grown in the presence of 1, but not in its
absence (Figure S8), indicating that
1 was introduced into the 37th position
with 365 nm or 405 nm UV irradiation for 0–10 minutes. Only
30 seconds after UV irradiation, significant labeled FtsZ has
already formed as shown by in-gel fluorescence (Figure 2D),
and the reaction was complete within five minutes. Using
405 nm laser irradiation, the reaction was also complete
within five minutes (Figure S6).
To further test the utility of the photoclick reaction for
protein labeling inside living cells, we incubated E. coli cells
expressing either wt FtsZ or FtsZ-3-AcrK with 100 mm
tetrazole 2, followed by irradiation at 365 nm for 1–
20 minutes. The cells were then lysed and proteins were
analyzed by both SDS-PAGE and in-gel fluorescence. UV
irradiation for one minute was sufficient to observe the
photoclick labeling of FtsZ in vivo (Figure 2E). As expected,
the photoclick reaction occurred selectively only in cells
expressing FtsZ-3-AcrK, but not wt FtsZ (Figure 2E). This
indicates that there was no mis-incorporation of AcrK into
wild-type FtsZ or other cellular proteins in E. coli.
of EGFP through amber codon suppression in mammalian
cells using the AcrKRS/MbtRNACUA pair.
Pyl
Because many important plant processes, such as photo-
synthesis, light sensing, and growth control, can only be
studied at the organismal level, we tested whether unnatural
amino acids could be introduced into the genetic code of the
plant model organism Arabidopsis thaliana. We chose
A. thaliana because it is the first plant species to have its
genome sequenced, transformation of DNA into A. thaliana
using Agrobacterium is routine, high accumulation of thera-
peutic antibodies (35% of the total soluble protein) in A.
thaliana seeds has been achieved,[8] and it is a model organism
for plant biology much like Caenorhabditis elegans and
Drosophila melanogaster are for animal biology (expansion
of the genetic code in these two species was recently
reported)[13]
We first constructed
.
a
shuttle vector pARABZH-
AcrKRS that can replicate in Agrobacterium and deliver
We then tested if this new method could be applied to live
cells for fluorescent imaging. BL21 (DE3) cells expressing
either wt FtsZ or FtsZ-3-AcrK were incubated with 100 mm
genes into plant hosts. In this vector, AcrKRS expression was
driven by the CaMV 35S promoter, MbtRNACUA tran-
scription was driven by the 7L4 promoter, followed by the
Pyl
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length GFP-mCherry-HA fusion protein was not expressed.
No full-length GFP-mCherry-HA fusion protein was
Pyl
expressed in the absence of AcrKRS or MbtRNACUA
(Figure S10). These results indicate that no endogenous
Pyl
tRNA synthetase can charge MbtRNACUA with any amino
acids. By contrast, red fluorescence was observed in the
presence of 5 mm AcrK, indicating that the TAG amber
codon was efficiently suppressed, resulting in GFP-mCherry-
HA fusion protein expression (Figure 3B).
To further demonstrate that AcrK was selectively incor-
porated in response to the amber codon and its ability to
direct the photoclick reaction to a specific site in a protein, we
lysed seedlings grown in the presence or absence of 5 mm
AcrK, extracted the total protein, and performed western-
blots. Anti-HA western blots confirmed AcrK-dependent
production of GFP-mCherry-HA in seedlings (Figure S9).
The cell lysates were then reacted with 100 mm biotin-
tetrazole under 365 nm UV irradiation for ten minutes. The
products were then probed with streptavidin. Our results
show that the GFP-mCherry-HA fusion protein purified from
seedlings grown in the presence of AcrK was selectively
labeled with biotin-tetrazole through light-induced photoclick
reaction (Figure S9). No biotin labeling was observed in the
absence of AcrK. As a control, we also expressed GFP-
mCherry-HAusing a vector where the TAG amber codon was
mutated back to a sense codon, TCG, in the presence of
AcrK. We found that GFP-mCherry-HA expressed under
these conditions did not react with biotin-tetrazole and
therefore did not produce a positive streptavidin signal
(Figure S9). These results indicate that AcrK was not
incorporated into protein in response to a sense codon.
Taken together, the fluorescence imaging and western blot
Figure 3. A) DNA constructs for expanding the genetic code of A. thali-
Pyl
ana. B) Fluorescence images of A. thaliana expressing MbtRNACUA
,
AcrKRS, and GFP-TAG-mCherry-HA in the absence (top) or presence
(bottom) of 5 mm AcrK. DIC=differential interference contrast
microscopy.
7L4-3’UTR,[14] expression of the reporter GFP-TAG-
mCherry-HA was driven by the CaMV 35S promoter (Fig-
ure 3A). The 35S promoter drives strong expression of
AcrKRS and GFP in the plant, the HA tag allows for
convenient detection of fusion protein by anti-HA antibodies.
Pyl
results demonstrate that AcrKRS and MbtRNACUA direct
the site-specific incorporation of AcrK into proteins in
response to an UAG codon in A. thaliana.
In summary, we have demonstrated the site-specific
incorporation of AcrK into proteins in bacterial cells,
mammalian cells, and plants and the use of AcrK as
a bioorthogonal handle for directing rapid fluorescent label-
ing and ligation of chemical probes to the target protein. The
site-specific, photoclick protein labeling method described
herein has the following advantages: 1) it is spatiotemporally
controllable; 2) it causes less cellular damage through the use
of 365 nm UV or 405 nm violet light; 3) AcrK introduces
minimal perturbation to the target proteins; 4) its fast rate
allows for efficient bioconjugation; 5) AcrK can be easily
synthesized in two steps in bulk quantities using cheap
starting materials, without the need for metal catalysts. This
new method has great potential for investigating proteins that
assemble into large complexes, such as the cytoskeleton or
nucleosome, in which the introduction of a GFP fusion is
likely to perturb their functions. Importantly, because Agro-
bacterium is broadly applicable for plant genetic engineering
and crop improvement, our new method should be useful for
producing therapeutic proteins or enzymes with unnatural
amino acids at a specific site in well-establish plant hosts such
as tobacco, on an agricultural scale using only simple
nutrients, an inexpensive unnatural amino acid, water, and
sunlight.[7,8] Subsequent bioorthogonal chemical ligations
Pyl
We chose the 7L4 promoter to drive MbtRNACUA expres-
sion, because it is highly expressed in plants.[14] If the TAG
amber codon is efficiently suppressed, then full-length GFP-
mCherry fusion protein should be expressed. For efficient
amber codon read-through, it is necessary to minimize
nonsense-mediated mRNA decay (NMD), a surveillance
mechanism which eliminates mRNAs which contain prema-
ture termination codons (PTC). Previous studies showed that
the UP-Frameshift (UPF) protein UPF1 is essential for
normal NMD function in A. thaliana,[15] therefore we chose
a upf1-5 mutant lacking the UPF1 gene for these experiments.
The plasmid pARABZH-AcrKRS was first transformed
into Agrobacterium tumefaciens GV3101 competent cells
using electroporation. A. thaliana upf1-5 mutants were then
transformed by dipping their flowers into a broth of Agro-
bacterium carrying the pARABZH-AcrKRS plasmid. The T2
generation positive transgenic plants were screened on solid
MS medium containing 50 mgmLÀ1 hygromycin B, and one
line (upf1-5-4) was chosen for further investigation. The roots
of two-week upf1-5-4 seedlings were used for GFP and
mCherry fluorescence visualization under a confocal micro-
scope. In the absence of AcrK, green fluorescence, but not red
fluorescence, was observed (Figure 3B), indicating that full-
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would allow for the large-scale production of proteins with
enhanced properties.
Received: April 24, 2013
Revised: June 24, 2013
Published online: && &&, &&&&
Keywords: Arabidopsis thaliana · click chemistry ·
.
bioorthogonal groups · protein expression ·
unnatural amino acids
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