Genetically encoded alkenes in yeast

Article abstract of DOI:10.1002/anie.200905590

(Figure Presented) Olefin-containing amino acids have been genetically introduced into proteins in Saccharomyces cerevisiae by orthogonal tRNA/aminoacyl-tRNA synthetase pairs. These nonnatural amino acids can be used for selective protein modification thr

Full text of DOI:10.1002/anie.200905590

Angewandte
Chemie
DOI: 10.1002/anie.200905590
Expanded Genetic Code
Genetically Encoded Alkenes in Yeast**
Hui-wang Ai, Weijun Shen, Eric Brustad, and Peter G. Schultz*
Several bioorthogonal chemical reactions have been explored
for the selective modification of proteins,[1,2] including the
coupling of alkoxyamines and hydrazides to ketones or
aldehydes,[3] the Staudinger ligation of azides to modified
phosphines,[4] and click reactions between azides and
alkynes.[5] Recently, alkene moieties have also been exploited
as uniquely reactive chemical handles. Examples include, the
photoaddition of a diaryl tetrazole to alkenes;[6,7] a Diels–
Alder reaction between tetrazines and trans-cyclooctenes;[8]
the cross-metathesis of olefins with allyl thioether modified
proteins;[9,10] the copolymerization of alkene-containing pro-
teins and acrylamide;[11] and the coupling of two alkene-
containing residues in peptides, resulting in improved stability
and pharmacological properties.[12–15]
In addition to chemical semisynthesis, a number of in vitro
and in vivo methods have been developed to incorporate the
bioorthogonal alkene groups into proteins. For example,
Scheme 1. Structures of nonnatural amino acids described herein.
Davis and co-workers developed a variety of in vitro chemical
methods to convert cysteine residues in proteins into S-
allylcysteine (1a), a reactive cross-metathesis substrate;[10,16]
methionyl-tRNA synthetase has been used to incorporate
homoallylglycine in a methionine auxotroph E. coli strain;[17]
and pyrrolysyl-tRNA synthetase has been used to charge the
alkene-containing nonnatural amino acid (UAA) 6-N-ally-
loxycarbonyl-l-lysine onto its cognate tRNA.[18] In addition,
we have genetically encoded O-allyltyrosine[19] and phenyl-
selenocysteine (which can be converted to dehydroalanine by
oxidative elimination)[20] in E. coli with engineered orthogo-
nal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs. Here we
report the site-specific incorporation of several alkene-con-
taining UAAs (Scheme 1) into proteins in eukaryotic cells
with orthogonal tRNA/aaRS pairs evolved in Saccharomyces
cerevisiae, and their subsequent application to protein modi-
fication.
Our first attempt to genetically encode 1a in S. cerevisiae
made use of promiscuous aaRS mutants that incorporate
amino acids with long aliphatic side chains, such as 1b.[21] 1a
resembles 1b in structure, so we directly tested these mutants
for their ability to incorporate 1a. The yeast strain
MAV203:pGAD-Gal4(2TAG),[22] in which suppression of
the amber codon (TAG) results in the expression of the
reporter gene ura3, was transformed with plasmids encoding
individual synthetases that aminoacylate 1b. The resulting
cells were then cultured on uracil-deficient (ÀUra) agar
plates in the presence of 1 mm 1a. Since the gene ura3
encodes an enzyme for uracil biosynthesis, TAG suppression
is necessary for cell growth. Cells harbouring the plasmid
encoding the aaRS Cap2X grew faster than cells containing
other plasmids (Supporting Information, Figure S1-a);[21]
therefore, Cap2X was further investigated. Additional experi-
ments showed that Cap2X incorporated 1a in response to the
amber codon in human superoxide dismutase (hSOD-
Trp33TAG) in SCY4 yeast (Figure S1-b). We then tested
Cap2X in a yeast strain deficient in nonsense-mediated
mRNA decay (Dupf1) and expressing a significantly higher
level of the amber suppressor tRNA.[23] This new system was
reported to increase protein production by 300 ꢀ relative to
SCY4. However, the expression of GFP-Tyr39TAG in the
presence of Cap2X and 2 mm 1a in Dupf1 yeast resulted in
heterogeneous GFP, which indicated that multiple cell-
endogenous amino acids were incorporated at residue 39 of
GFP (Figure S4-a).
[*] Dr. H. W. Ai, Dr. W. Shen, Dr. E. Brustad,[+] Prof. Dr. P. G. Schultz
Department of Chemistry, The Scripps Research Institute
10550 N. Torrey Pines Rd., La Jolla, CA 92037 (USA)
Fax: (+1)858-784-9440
E-mail: schultz@scripps.edu
[+] Present address: Division of Chemistry and Chemical Engineering
California Institute of Technology, Pasadena, CA 91125 (USA)
[**] We thank Prof. Benjamin Davis, Justin Chalker, and Yuya Lin
(University of Oxford) for helpful discussion; Prof. Karol Grela and
Lukasz Gulajski (University of Warsaw), and Prof. Lei Wang (Salk
Institute) for providing reagents; and Dr. Chang Liu and Emily
Remba for manuscript preparation. This work is supported by the
US Department of Energy, Division of Materials Sciences, under
Award No. DE-FG03-00ER46051 and the Skaggs Institute for
Chemical Biology.
In EcLRS (the aminoacyl-tRNA synthetase from which
the promiscuous synthetases were derived), the additional
CP1 editing domain corrects mischarged amino acids,[24,25]
leading to the incorporation of leucine with high fidelity.
Therefore, we hypothesized that the fidelity of these synthe-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905590.
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Communications
Table 1: Observed molecular weights (by ESI-MS) and calculated
molecular weights of GFP and cpVenus (expressed with AK-1 in the
presence of 2 mm UAAs).
tases might be improved by mutations in the CP1 domain. To
test this notion, we generated a new library in which the active
site of the synthetase CP1 editing domain (residues Thr247,
Thr248, and Thr252) was fully randomized. This library also
contains some recombined sequences of the aforementioned
promiscuous synthetases, since a mixture of plasmids encod-
ing these aaRSs was used as the template for library
construction and recombination can occur in the overlap
polymerase chain reaction (PCR) (Figure S2). We then
subjected the new aaRS library to four positive and three
negative rounds of selection as previously described.[22] We
observed convergence to two independent sequences, desig-
nated AK-1 and AK-2, respectively (Table S1; the sequences
of synthetase AK-1 and AK-2 can be found under GenBank
accession numbers GU059871 and GU059870). Both were
tested for expression of the model protein hSOD using the
plasmid hSOD-Trp33TAG. As expected, we observed high
hSOD production in the presence of 1 mm 1a, and little
hSOD in the absence of 1a. (Figure S1-b). We next attempted
to express GFP-Tyr39TAG in Dupf1 yeast in the presence of
2 mm 1a; approximately 5 mg protein could be purified from
a 1 L culture using either the AK-1 or AK-2 synthetase. The
resulting proteins were then subjected to ESI-MS character-
ization (Figure S4-b). Although a single protein peak was
detected, the molecular weight did not match the calculated
theoretical number (Table 1). We reasoned that 1a is unstable
in S. cerevisiae, and is oxidized to alliin (1c). In fact, when 1a
was replaced with 2 mm 1c during protein expression, we
detected GFP with the same molecular weight.
Amino acid
Obsd
[Da]
Calcd[a]
[Da]
Yield
[mgLÀ1
]
GFP-Y39TAG
1a
1d
1e
1 f
1g
28752
28719
28731
28733
28745
28734
28716
28730
28732
28746
5.0
3.3
4.8
6.3
5.5
cpVenus-2TAG
1g (before reaction)
1g (after reaction)
29446
29391
29451
29395
0.3
[a] GFP and cpVenus were N-acetylated in yeast. Listed are average
molecular weights of acetylated mature peptide (GFP loses 20 Da upon
chromophore maturation).[26]
Previous studies have suggested that heteroatoms (e.g.,
oxygen) at the allylic positions of alkenes can facilitate
olefin metathesis reactions; and that the substituted alkyl-
idene, which is the propagating catalytic species resulting
from 1 f, is more stable in water than the methylidene
resulting from terminal olefins.[10,27] Circularly permutated
yellow fluorescent protein (cpVenus-2TAG) with 1 f incorpo-
rated at two spatially adjacent residues (Arg168 and Leu178
in Venus[28]) was expressed and purified in Dupf1 yeast. The
resulting protein was subjected to olefin metathesis catalyzed
by the 2nd generation Hoveyda–Grubbs catalyst in aqueous
solution containing 30% tert-butanol (Supporting Informa-
tion). The reaction mixture was directly analyzed by LC-ESI-
MS, and near-complete conversion was observed in 5 h. The
observed mass loss was consistent with the formation of a new
The 1a analogues, 1d–1g, are chemically resistant to
oxidation, so we next tested whether AK-1 or AK-2 could
incorporate 1d–1g. Dupf1 yeast cells harbouring the GFP-
Tyr39TAG gene, the corresponding tRNA
gene, and synthetase AK-1 or AK-2 genes
were cultured with 2 mm of each UAA. Cells
cultured with 1d–1g were all highly fluores-
cent. Suppression with AK-1 showed better
contrast between the presence and absence
of UAAs (Figure S3); thus, AK-1 was chosen
for further characterization. Yields of
alkene-containing GFPs produced with
AK-1 were between 3.3 mgLÀ1 and
6.3 mgLÀ1 in the presence of 2 mm 1d–1g,
and molecular weights of purified GFPs
agreed well with the corresponding calcu-
lated numbers (Table 1 and Figures S4–S5).
The yield of protein production by AK-1 is
comparable to other previously reported
tRNA/aaRS pairs in yeast.[23] In addition,
although there was noticeable basal amber
suppression in the absence of UAAs, there
was no apparent incorporation of endoge-
nous amino acids detectable when 2 mm
UAAs were present to compete with cell-
endogenous amino acids as determined by
ESI-MS (Figures S4–S5).
Next, we investigated the ability of
proteins containing O-crotylserine (1 f) to
Figure 1. ESI-MS of cpVenus double O-crotylserine mutant, a) before and b) after olefin
be selectively modified by olefin metathesis.
metathesis.
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In summary, we have developed new tRNA/aaRS pairs
that make possible the genetic incorporation of a set of
alkenes (1c–1g) into proteins in S. cerevisiae. These alkene
moieties have flexible and relatively long side chains, and are
useful bioorthogonal handles for various protein modification
reactions. In one example, we demonstrated that protein side-
chain bridges could be formed from intramolecular olefin
metathesis of two alkene-containing residues. Similar reac-
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Keywords: nonnatural amino acids · olefin metathesis ·
protein modification
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