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809
Incorporation of non-natural amino acids into proteins
Takahiro Hohsaka and Masahiko Sisido*
Chemical and biological diversity of protein structures and
functions can be widely expanded by position-specific
incorporation of non-natural amino acids carrying a variety of
specialty side groups. After the pioneering works of Schultz’s
group and Chamberlin’s group in 1989, noticeable progress
has been made in expanding types of amino acids, in finding
novel methods of tRNA aminoacylation and in extending
genetic codes for directing the positions. Aminoacylation of
tRNA with non-natural amino acids has been achieved by
directed evolution of aminoacyl-tRNA synthetases or some
ribozymes. Codons have been extended to include four-base
codons or non-natural base pairs. Multiple incorporation of
different non-natural amino acids has been achieved by the use
of a different four-base codon for each tRNA. The combination
of these novel techniques has opened the possibility of
synthesising non-natural mutant proteins in living cells.
codons, only the amber stop codon can be used for the
suppression, multiple incorporation of non-natural amino
acids into single proteins cannot be expected [4,5•].
Addresses
Department of Bioscience and Biotechnology, Okayama University,
3-1-1 Tsushimanaka, Okayama 700-8530, Japan
*e-mail: [email protected]
Extension of codon–anticodon pairs
Current Opinion in Chemical Biology 2002, 6:809–815
1367-5931/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Published online 18 October 2002
Abbreviations
aaRS
aminoacyl-tRNA synthetase
aa-tRNA aminoacyl-tRNA
DHFR dihydrofolate reductase
Introduction
Amino acid substitution of proteins by genetic engineering
had been restricted to the 20 naturally occurring amino
acids. In 1989, the amber suppression method for introducing non-natural amino acids into specific positions of
proteins was developed, opening the way to expanding
protein functions [1,2]. In this method, amber suppressor
tRNAs are aminoacylated with desired non-natural amino
acids through chemical aminoacylation (i.e. an enzymatic
ligation of an aminoacyl-dinucleotide with a tRNA lacking
the terminal dinucleotide unit) [3]. The resulting aminoacyltRNAs (aa-tRNAs) are added to an in vitro translation system
or Xenopus oocytes together with a mRNA or DNA of interest
containing an amber stop codon (UAG) at a desired position.
The amber codon is suppressed by the added aa-tRNA,
resulting in the incorporation of the non-natural amino acid
into the directed position of the target protein.
This method, however, suffers from several disadvantages.
First, stop codons are under tough competition with the
release factors that terminate peptide elongation. Thus,
the release factors interfere with the incorporation of nonnatural amino acids. Second, because among three stop
Preparation of aa-tRNAs is another issue to be improved,
especially for a large-scale expression of non-natural
proteins. Once an aa-tRNA is used for translation or is
hydrolyzed at an aminoacyl bond, the deacylated tRNA
will not be aminoacylated again in the cell-free translation
systems or Xenopus oocytes. This disadvantage significantly
decreases the yield of non-natural proteins.
This review focuses on recent improvements of non-natural
amino acid mutagenesis addressed to these problems.
Applications of non-natural mutagenesis to create proteins
with specialty functions are also introduced.
The genetic code consists of 61 codons for assigning
20 naturally occurring amino acids and 3 codons as stop
signals. To fit non-natural amino acids into the existing
genetic code system, it is essential to generate new codons
that are specific to them. The use of an amber stop codon
(Figure 1a) is one of the solutions without large alteration
of the existing codon system. Alternatively, four-base
codons have been developed (Figure 1b) [5•,6]. In an
Escherichia coli in vitro translation system, various kinds
of four-base codons — AGGU, CGGU, CGCU, CGAU,
CCCU, CUCU, CUAU, GGGU, and their fourth-letter
variants — were able to assign non-natural amino acids
within the framework of the existing three-base codon
system. Among them, GGGU gave the most efficient
result, in which p-nitrophenylalanine (11, Figure 2) was
introduced into streptavidin in 86% yield relative to a wildtype streptavidin. Because most of the four-base codons
listed above are orthogonal to each other, double incorporation of two different non-natural amino acids into
individual positions of streptavidin has been achieved by
using efficient sets of the four-base codons such as GGGU
and CGGG [5•]. Surprisingly, codons could be further
extended to five-base codons such as CGGN1N2, where
N1 and N2 indicate one of four nucleotides. Indeed, the
five-base codons were decoded by aa-tRNAs containing
the corresponding five-base anticodons [7•]. In addition,
we have found that several kinds of four-base codons were
also highly effective in a rabbit reticulocyte lysate
(Hohsaka T, Sisido M, unpublished data). Magliery et al.
undertook an in vivo selection of efficient four-base codons
by a library method [8,9], and four four-base codons,
AGGA, CUAG, UAGU and CCCU, were identified [8].
Another strategy for the extension of the genetic code has
been proposed by using non-natural base pairs that are
orthogonal to the naturally occurring four types. In 1991,
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Biopolymers
Figure 1
(a) Amber codon
Non-natural amino acids discussed in the text.
(b) Four-base codon
A
U
U
A
G
C
C
C
G
C
G
G
C
G
(c) Non-natural codon
N
N
S
H
N
N
Ribose
s
H
s
N
N
H
y
U
y
H
A
G
O
C
Ribose
Current Opinion in Chemical Biology
Bain et al. [10] showed that an isoC and isoG base pair
is correctly formed when it is incorporated into codon
and anticodon, respectively. Although the isoC–isoG pair
works effectively in the translation process, the mRNA and
tRNA containing the non-natural bases should be synthesized by chemical methods. Recently, novel non-natural
base pairs that fit into the replication and transcription
processes have been designed. Hirao et al. [11••,12] reported
that a non-natural base, pyridine-2-one (y), was specifically
introduced into a mRNA corresponding to a 2-amino-6-(2thienyl)purine (s) in a DNA template. The non-natural
codon yAG was successfully translated by a tRNA containing
the corresponding non-natural anticodon CUs (Figure 1c)
and aminoacylated with m-chlorotyrosine (22). Although
the anticodon could not be introduced into the tRNA
through transcription in this report, the result clearly
demonstrates the feasibility of using a non-natural base
pair to assign non-natural amino acids in in vitro and in vivo
Figure 2 legend
Codons that can be assigned to non-natural amino acids. (a) Amber codon.
An amber codon UAG is decoded by aa-tRNA containing a CUA anticodon.
(b) Four-base codon. CGGG is shown as an example of four-base codons.
The CGGG codon is decoded by aa-tRNA containing the corresponding
anticodon CCCG. (c) Non-natural codon. A recently developed non-natural
codon-anticodon pair yAG-CUs and structures of y and s are shown. The
yAG codon makes a pair with the CUs anticodon exclusively through a cellfree translation. The gray oval represents an amino acid.
Incorporation of non-natural amino acids into proteins Hohsaka and Sisido
811
Figure 2
COOH
COOH
H 2N
COOH
H2N
COOH
H2N
COOH
COOH
H2N
H2N
H2N
N
N+
Cl
1
2
NO2
COOH
3
OH
COOH
H2N
4
N−
COOH
COOH
H2N
H2N
H2N
6
O
COOH
COOH
H2N
5
OCH3
H2N
OH
S
NH
N
O
N3
7
8
9
NH2
N
10
11
O
COOH
H2N
NO2
COOH
H2N
H2N
H 2N
N
O
COOH
COOH
COOH
12
O
COOH
H2N
H2N
NH
O2N
O
N
O
18
O
S
13
HN
COOH
COOH
O2N
H2N
H2N
H2N
H2N
S
O
NHAc
N
COOH
COOH
COOH
H2N
H2N
17
NH
O
COOH
N
16
15
14
O
O2N
NH
23
24
I
O
N
O
O
OH
n (n=2–5)
N
OH
HO
19
20
21
22
HO
H2N
COOH
HN
COOH
H2N
COOH
COOH
CH2
HN
H2N
N
O
29
O
N
O
O
27
OCH3
F
F
HN
N
26
NH
O
H2N
H2N
25
N(CH3)3+
COOH
S
B
N
H2N
28
Current Opinion in Chemical Biology
812
Biopolymers
transcription/translation systems. Hirao et al. [11••,12–14]
designed non-natural base pairs incorporating an orthogonal
hydrogen bond and steric hindrance. On the other hand,
Romesberg and co-workers [15–17] have attempted to
generate non-natural base pairs by introducing hydrophobic
and stacking interactions into base pairs.
Aminoacylation of tRNAs with non-natural
amino acids
Because the chemical aminoacylation (Figure 3a) cannot
be made in living cells, the non-natural mutagenesis had
been carried out only in cell-free translation systems or in
Xenopus oocytes through microinjection of the aa-tRNA.
Exceptionally, amino acids that are analogous to natural
ones are accepted as substrates by one of the aminoacyltRNA synthetases (aaRSs). For example, Kiick et al. [18•]
found that a methionine analog, azidohomoalanine (33), was
accepted by MetRS and incorporated into proteins in place
of methionine in E. coli. The incorporated azido group was
selectively modified by the Staudinger ligation with a
phosphine-containing peptide.
Recently, non-natural aminoacylation has been achieved
by mutated aaRS (Figure 3b) by Schultz’s group. A pair of
an amber suppressor tRNATyr and a TyrRS from
Methanococcus jannaschii has been mutated to become
orthogonal to any aaRS/tRNA pair from E. coli [19,20]. The
latter TyrRS was further mutated not to accept tyrosine or
any other amino acids, but to accept O-methyl-tyrosine (44)
exclusively as the substrate [21••]. Five residues located in
the active site of the TyrRS were randomly mutated. The
library of the mutant TyrRSs was screened by two rounds
of positive selection to accept O-methyl-tyrosine and
subsequent negative selection to reject any natural amino
acids. The final aaRS was found to accept O-methyl-tyrosine exclusively. Co-expression of the mutant TyrRS and
amber suppressor tRNA in the presence of a dihydrofolate
reductase (DHFR) gene containing an amber codon
produced a mutant DHFR protein containing an O-methyltyrosine at the amber codon position in E. coli [21••]. An
ion trap mass analysis demonstrated that O-methyl-tyrosine
was incorporated at more than 95% fidelity.
Similar approaches were carried out for 2-naphthylalanine
(55) [22], p-azidophenylalanine (66) [23], and p-benzoylphenylalanine (77) [24••]. In addition, mutant aaRSs for
p-aminophenylalanine (88) and p-isopropylphenylalanine
(99) were also selected [25•]. Mutant aaRSs for non-natural
amino acids with spin (110), fluorescent (111,12), and biotin
labels (113), an aldehyde (114) or allyl (115) functionality, a
metal binder (116), photocaged groups (117,18), a photoisomerizable group (119), and a sugar moiety (220) are being
investigated [25•].
Hirao, Yokoyama and co-workers [26•] reported a mutated
E. coli TyrRS that recognizes m-iodotyrosine (221) more
efficiently than tyrosine. Three positions of the TyrRS
were systematically mutated, and the aminoacylation activities
of each mutant with iodotyrosine and with tyrosine
were compared by use of an amber suppressor E. coli
tRNATyr. Then, two effective point mutations were combined
together. One of these double mutants, V37C195, recognized
iodotyrosine 10-fold more efficiently than it recognized
tyrosine. Addition of the mutated enzyme and the amber
suppressor tRNA to a wheat germ cell-free translation system
expressing a c-Ha-Ras gene with an amber codon gave the
mutant Ras protein containing an iodotyrosine at the
directed position. LC-MS (liquid chromatography coupled
with mass spectrometry) analysis demonstrated that
iodotyrosine was incorporated in more than 95% fidelity.
The wheat germ cell-free translation system has the
advantage that eukaryotic proteins are liable to fold correctly.
In addition, the E. coli TyrRS-tRNATyr pair was orthogonal
to wheat germ tRNA-aaRS pairs without any mutagenesis.
RajBhandary and co-workers [27,28] have developed a
method for importing aminoacylated amber and ochre
suppressor tRNAs into mammalian cells. They showed
that both suppressor tRNAs aminoacylated with tyrosine
were imported by using a transfecting reagent Effectene,
and an active chloramphenicol acetyl transferase was
expressed from co-transfected chloramphenicol acetyl
transferase genes containing the amber or ochre codon at
an internal position.
Suga and co-workers [29–31] have reported aminoacylation
of tRNAs with ribozymes. They screened a ribozyme from
random combinatorial pools and aminoacylated tRNA with
specific N-biotinylated amino acids including ε-biotinylated
lysine (222, Figure 3c) [32••]. The aminoacylating ribozyme
interacts with both the 3′ terminal and the anticodon loop
of the target tRNA. The ribozyme strategy is not only useful
for labeling of proteins with biotin, but is potentially
applicable to customized ribozymes that aminoacylate
desired non-natural amino acids to specific tRNAs.
Extension of protein functions with
non-natural amino acids
By introducing non-natural amino acids with specialty
functions, protein functions will be widely expanded
as described below. Dougherty’s group [33] have been
investigating ion channels by using the amber suppression
method in Xenopus oocytes. Caged amino acids,
S-o-nitrobenzyl-cystein (223) and O-o-nitrobenzyl-tyrosine
(117) have been introduced into nicotinic acetylcholine
receptor and Kir2.1 channel [34,35]. The resulting
receptors recovered their activity when the o-nitrobenzyl
group was removed by UV irradiation. The receptor activity
could be controlled also by introducing agonist-tethered
amino acids (224) [36•]. Four kinds of O-(trimethylammoniumalkyl)tyrosine have been synthesized and incorporated
at three different positions of the acetylcholine receptor.
Because the trimethylammoniumalkyl group works as an
agonist for the receptor, the resulting receptor–agonist
conjugates showed constitutive activity in the absence
of acetylcholine.
Incorporation of non-natural amino acids into proteins Hohsaka and Sisido
813
Figure 3
Methods for aminoacylation of tRNAs with
non-natural amino acids. (a) Chemical
aminoacylation. A chemically synthesized
aminoacyl-dinucleotide is enzymatically ligated
by T4 RNA ligase with a tRNA lacking the
terminal dinucleotide unit. (b) Aminoacylation
by aaRSs generated by directed evolution to
recognize both a non-natural amino acid and
an anticodon of tRNA. (c) Aminoacylation by
ribozymes that were screened by directed
evolution. The ribozyme consists of acyltransfease and tRNA-binding domains. The
5′-OH of the ribozyme is aminoacylated by an
aminoacylated pentaribonucleotide before the
aminoacylation of 3′-OH of the tRNA. The
gray oval represents an amino acid.
(a) Chemical aminoacylaion
A
dC
C
A
dC + C
T4 RNA Ligase
(b) Enzymatic aminoacylation
A
C
C
A
C
C
A
U
C
A
UC
Mutated
aminoacyl-tRNA
synthetase
(c) Aminoacylation by ribozyme
O=C
A A GG
UUUU
:
5' O
OH
A
C
C
A
A
U C
G C
G A
U
U
3'
Acyl-transfer ribozyme
with tRNA binding domain
A
U
A
U C
U
A
C
G
Current Opinion in Chemical Biology
Hecht and co-workers reported position-specific proteolysis
by introducing allylglycine (225 ). Proteins containing
allylglycine have been cleaved by treatment with
iodine through a presumed iodolactone intermediate.
Introduction of allylglycine to a trypsinogen [37•] or a
trypsin inhibitor (ecotin) [38] successfully controlled the
trypsin activity. Non-natural amino acids with sugar
moieties were also investigated by their group [39].
814
Biopolymers
Schultz and co-workers have been using their in vivo
method to investigate protein–protein interactions in living
cells. They incorporated p-benzoylphenylalanine (77) into a
Phe52 position of dimeric glutathione S-transferase.
Subsequent UV irradiation of the mutant resulted in a
formation of a covalently linked homodimer [24••]. This
photocrosslinking method will be useful for mapping
protein–protein interactions in living cells.
Acknowledgements
Reversible photoregulation of proteins has been achieved
by incorporating a photoisomerizable group such as
azobenzene into specific positions. We have introduced
p-phenylazophenylalanine (226) into various positions of a
horseradish peroxidase [40]. The F179-mutant and F162mutant showed reversible photoregulation of the enzyme
activity. Incorporation of phenylazophenylalanine into a
camel anti-lysozyme antibody or into a DNA-binding
protein also induced reversible photoresponse of ligand
binding (Hohsaka T, Sisido M, unpublished data).
• of special interest
•• of outstanding interest
Position-specific incorporation of fluorescent groups is an
important step toward comprehensive proteome analysis.
Because of the size limitation of non-natural amino acids
that are accepted by the translation machinery, relatively
small but long-wavelength emitting fluorescent amino acids
have been searched for. Amino acids carrying coumarin (227)
[41] and anthraniloyl (228) [42] group are in such class of
amino acids. They can be incorporated into proteins in
relatively high yields and their fluorescence intensity and
wavelength are sensitive to the ligand binding. Larger
fluorescent groups such as fluorescein had never been
introduced into proteins. Very recently, however, a greenfluorescent BODIPY FL-labeled p-aminophenylalanine
(229) was designed, synthesized and successfully incorporated
into proteins through an E. coli cell-free translation system
(Hohsaka T, Sisido M, unpublished data). This finding
will open a way for practical application of the non-natural
mutagenesis for comprehensive analysis of protein–protein
and protein–nucleic acid interactions.
Conclusions
Introduction of non-natural amino acids into proteins is
a potential tool that is widely applicable to genomic,
proteomic and cellular biological researches. Positionspecific incorporation of probes such as biotin and fluorescent groups will be useful for high-throughput analyses of
protein–protein and protein–nucleic-acid interactions. The
possibility is further expanded when multiple non-natural
amino acids are incorporated by using the four-base codon
strategy. For these purposes, cell-free protein synthesis
is advantageous for its rapid and simple processing.
In vivo synthesis of non-natural mutants using artificial
aaRS is essential for large-scale expression and for
analyses of protein–protein interactions in living cells.
Large-scale synthesis is particularly required for utilization
of the non-natural mutants as nano-sized chemical devices,
such as biosensors, biophotoelectronic devices and
protein microarrays.
Works on four-base codons was supported by the Grant-in-Aid for Specially
Promoted Research from the Ministry of Education, Science, Sports and
Culture, Japan (No. 11 102 003) and by Industrial Technology Research
Grant Program in ’00 from the New Energy and Industrial Technology
Development Organization (NEDO) of Japan.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
1.
Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG: A general
method for site-specific incorporation of unnatural amino acids
into proteins. Science 1989, 244:182-188.
2.
Bain JD, Glabe CG, Dix TA, Chamberlin AR, Diala ES: Biosynthetic
site-specific incorporation of a non-natural amino acid into a
polypeptide. J Am Chem Soc 1989, 111:8013-8014.
3.
Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM:
T4 RNA ligase mediated preparation of novel “chemically
misacylated” tRNAPhes. Biochemistry 1984, 23:1468-1473.
4.
Cload ST, Liu DR, Froland AF, Schultz PG: Development of
improved tRNAs for in vitro biosynthesis of proteins containing
unnatural amino acids. Chem Biol 1996, 3:1033-1038.
5.
•
Hohsaka T, Ashizuka Y, Taira H, Murakami H, Sisido M: Incorporation
of nonnatural amino acids into proteins by using various fourbase codons in an Escherichia coli in vitro translation system.
Biochemistry 2001, 40:11060-11064.
Various kinds of four-base codons are developed. A highly efficient four-base
codon pair for incorporation of two different non-natural amino acids into a
single protein is also described.
6.
Sisido M, Hohsaka T: Introduction of specialty functions by the
position-specific incorporation of nonnatural amino acids into
proteins through four-base codon/anticodon pairs. Appl Microbiol
Biotechnol 2001, 57:274-281.
7.
•
Hohsaka T, Ashizuka Y, Murakami H, Sisido M: Five-base codons for
incorporation of nonnatural amino acids into proteins. Nucleic
Acids Res 2001, 29:3646-3651.
This is the first report that five-base codons are decoded by aa-tRNAs
containing five-base anticodons in a translation system. Sixteen five-base
codons are developed for incorporation of non-natural amino acids into proteins.
8.
Magliery TJ, Anderson JC, Schultz PG: Expanding the genetic code:
selection of efficient suppressors of four-base codons and
identification of ‘shifty’ four-base codons with a library approach
in Escherichia coli. J Mol Biol 2001, 307:755-769.
9.
Anderson JC, Magliery TJ, Schultz PG: Exploring the limits of codon
and anticodon size. Chem Biol 2002, 9:237-244.
10. Bain JD, Switzer C, Chamberlin AR, Benner SA: Ribosome-mediated
incorporation of a non-standard amino acid into a peptide through
expansion of the genetic code. Nature 1992, 356:537-539.
11. Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T,
•• Nakayama H, Takio K, Yabuki T, Kigawa T et al.: An unnatural base
pair for incorporating amino acid analogs into proteins. Nat
Biotechnol 2002, 20:177-182.
An artificial codon containing a non-natural base on DNA is transcribed to a
mRNA, then the artificial codon on mRNA is translated to a non-natural
amino acid in an E. coli extract.
12. Fujiwara T, Kimoto M, Sugiyama H, Hirao I, Yokoyama S: Synthesis of
6-(2-thienyl)purine nucleoside derivatives that form unnatural
base pairs with pyridin-2-one nucleosides. Bioorg Med Chem Lett
2001, 11:2221-2223.
13. Ohtsuki T, Kimoto M, Ishikawa M, Mitsui T, Hirao I, Yokoyama S:
Unnatural base pairs for specific transcription. Proc Natl Acad Sci
USA 2001, 98:4922-4925.
14. Hirao I, Kimoto M, Yamakage S, Ishikawa M, Kikuchi J, Yokoyama S:
A unique unnatural base pair between a C analogue,
pseudoisocytosine, and an A analogue, 6-methoxypurine, in
replication. Bioorg Med Chem Lett 2002, 12:1391-1393.
15. Tae EL, Wu Y, Xia G, Schultz PG, Romesberg FE: Efforts toward
expansion of the genetic alphabet: replication of DNA with three
base pairs. J Am Chem Soc 2001, 123:7439-7440.
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16. Berger M, Wu Y, Ogawa AK, McMinn DL, Schultz PG, Romesberg FE:
Universal bases for hybridization, replication and chain
termination. Nucleic Acids Res 2000, 28:2911-2914.
17.
Berger M, Ogawa AK, McMinn DL, Wu Y, Schultz PG, Romesberg FE:
Stable and selective hybridization of oligonucleotides with
unnatural hydrophobic bases. Angew Chem Int Ed Engl 2000,
39:2940-2942.
18. Kiick KL, Saxon E, Tirrell DA, Bertozzi CR: Incorporation of azides
•
into recombinant proteins for chemoselective modification by the
Staudinger ligation. Proc Natl Acad Sci USA 2002, 99:19-24.
An azidohomoalanine is incorporated into a protein as a methionine analogue
in E. coli. The resulting protein is ligated with phosphine-labeled peptides.
19. Wang L, Magliery TJ, Liu DR, Schultz PG: A new functional
suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo
incorporation of unnatural amino acids into proteins. J Am Chem
Soc 2000, 122:5010-5011.
20. Wang L, Schultz PG: A general approach for the generation of
orthogonal tRNAs. Chem Biol 2001, 8:883-890.
21. Wang L, Brock A, Herberich B, Schultz PG: Expanding the genetic
•• code of Escherichia coli. Science 2001, 292:498-500.
A breakthrough report describing the incorporation of a 21st amino acid into
a protein in a growing E. coli. A mutated TyrRS that exclusively recognizes an
amber suppressor tRNA and O-methyl-tyrosine is developed, and expressed
in an E. coli together with a DHFR gene containing an amber codon. The
produced DHFR contains O-methyl-tyrosine at the directed position.
22. Wang L, Brock A, Schultz PG: Adding L-3-(2-naphthyl)alanine to
the genetic code of E. coli. J Am Chem Soc 2002, 124:1836-1837.
23. Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG:
Addition of p-azido-L-phenylalanine to the genetic code of
Escherichia coli. J Am Chem Soc 2002, 124:9026-9027.
24. Chin JW, Martin AB, King DS, Wang L, Schultz PG: Addition of a
•• photocrosslinking amino acid to the genetic code of Escherichia
coli. Proc Natl Acad Sci USA 2002, 99:11020-11024.
This report describes a novel mutated aaRS that catalyzes an aminoacylation
of tRNA with p-benzoylphenylalanine. The result raises the possibility that
non-natural amino acids with large side chains may be accepted by ARSs
after appropriate mutations.
25. Wang L, Schultz PG: Expanding the genetic code. Chem Commun
•
2002, 7:1-11.
This is a review of expanded translation systems for introducing non-natural
amino acids into proteins. Recent work of author’s group are described.
26. Kiga D, Sakamoto K, Kodama K, Kigawa T, Matsuda T, Yabuki T,
•
Shirouzu M, Harada Y, Nakayama H, Takio K et al.: An engineered
Escherichia coli tyrosyl-tRNA synthetase for site-specific
incorporation of an unnatural amino acid into proteins in
eukaryotic translation and its application in a wheat germ cellfree system. Proc Natl Acad Sci USA 2002, 99:9715-9720.
A mutated TyrRS catalyzes an aminoacylation of a nonsense suppressor
tRNA with a non-natural amino acid, m-iodotyrosine. The resulting aminoacyltRNA introduces iodotyrosine into a desired position of proteins in a wheat
germ translation system. This system, enabling the introduction of iodine into
specific positions of proteins, will be useful for X-ray structure analyses.
27.
Kohrer C, Xie L, Kellerer S, Varshney U, RajBhandary UL: Import of
amber and ochre suppressor tRNAs into mammalian cells:
a general approach to site-specific insertion of amino acid
analogues into proteins. Proc Natl Acad Sci USA 2001,
98:14310-14315.
28. Kowal AK, Kohrer C, RajBhandary UL: Twenty-first aminoacyl-tRNA
synthetase-suppressor tRNA pairs for possible use in sitespecific incorporation of amino acid analogues into proteins in
815
eukaryotes and in eubacteria. Proc Natl Acad Sci USA 2001,
98:2268-2273.
29. Saito H, Watanabe K, Suga H: Concurrent molecular recognition of
the amino acid and tRNA by a ribozyme. RNA 2001, 7:1867-1878.
30. Saito H, Suga H: A ribozyme exclusively aminoacylates the
3′′-hydroxyl group of the tRNA terminal adenosine. J Am Chem
Soc 2001, 123:7178-7179.
31. Murakami H, Bonzagni NJ, Suga H: Aminoacyl-tRNA synthesis by a
resin-immobilized ribozyme. J Am Chem Soc 2002,
124:6834-6835.
32. Bessho Y, Hodgson DR, Suga H: A tRNA aminoacylation system
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