U.S. patent application number 11/975351 was filed with the patent office on 2008-07-10 for genetic incorporation of unnatural amino acids into proteins in mammalian cells.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Wenshe Liu, Peter G. Schultz.
Application Number | 20080166766 11/975351 |
Document ID | / |
Family ID | 39512252 |
Filed Date | 2008-07-10 |
United States Patent
Application |
20080166766 |
Kind Code |
A1 |
Liu; Wenshe ; et
al. |
July 10, 2008 |
Genetic incorporation of unnatural amino acids into proteins in
mammalian cells
Abstract
The invention relates to orthogonal pairs of tRNAs and
aminoacyl-tRNA synthetases that can incorporate unnatural amino
acids into proteins in mammalian host cells, for example, primate
host cells and rodent host cells. The invention provides, for
example but not limited to, translation systems that include host
cells (e.g., primate or rodent cells), orthogonal aminoacyl-tRNA
synthetases derived from eubacterial synthetases, orthogonal tRNAs,
and the unnatural amino acid. The invention also relates to methods
for producing proteins of interest comprising at least one
unnatural amino acid in mammalian host cell systems.
Inventors: |
Liu; Wenshe; (College
Station, TX) ; Schultz; Peter G.; (La Jolla,
CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
The Scripps Research
Institute
La Jolla
CA
|
Family ID: |
39512252 |
Appl. No.: |
11/975351 |
Filed: |
October 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60853008 |
Oct 18, 2006 |
|
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60923458 |
Apr 12, 2007 |
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Current U.S.
Class: |
435/69.1 ;
435/352; 435/353; 435/354; 435/358; 435/363; 435/366 |
Current CPC
Class: |
C12P 21/02 20130101;
C12N 9/93 20130101 |
Class at
Publication: |
435/69.1 ;
435/363; 435/352; 435/353; 435/354; 435/366; 435/358 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C12N 5/10 20060101 C12N005/10; C12N 5/16 20060101
C12N005/16; C12N 5/18 20060101 C12N005/18 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support from the
National Institutes of Health under Grant No. GM62159. The
government may have certain rights to this invention.
Claims
1. A translation system comprising: (a) a first unnatural amino
acid selected from the group consisting of: i)
p-methoxyphenylalanine (pMpa); ii) p-acetylphenylalanine (pApa);
iii) p-benzoylphenylalanine (pBpa); iv) p-iodophenylalanine (pIpa);
v) p-azidophenylalanine (pAzpa); vi) p-propargyloxyphenylalanine
(pPpa); vii) .alpha.-aminocaprylic acid; viii)
o-nitrobenzylcysteine (o-NBC); ix) 1,5-dansylalanine; and x)
o-nitrobenzylserine (o-NBS); (b) a first orthogonal tRNA (O-tRNA);
(c) a first orthogonal aminoacyl-tRNA synthetase (O-RS) derived
from a eubacterial aminoacyl-tRNA synthetase, wherein said O-RS
preferentially aminoacylates said O-tRNA with said unnatural amino
acid; and (d) a host cell comprising (a), (b) and (c), said cell
selected from a rodent cell and a primate cell.
2. The translation system of claim 1, wherein said first O-RS
preferentially aminoacylates said first O-tRNA with said first
unnatural amino acid with an efficiency that is at least 50% of the
efficiency observed for a translation system comprising said
O-tRNA, said unnatural amino acid, and an aminoacyl-tRNA synthetase
comprising an amino acid sequence selected from SEQ ID NOS:
57-101.
3. The translation system of claim 1, wherein said first O-tRNA
comprises or is encoded by the polynucleotide sequence set forth in
SEQ ID NO: 3.
4. The translation system of claim 1, wherein said first O-RS is
derived from an E. coli aminoacyl-tRNA synthetase.
5. The translation system of claim 1, wherein said first O-RS
comprises an amino acid sequence selected from the group of amino
acid sequences set forth in SEQ ID NOS: 57-101, and conservative
variants thereof.
6. The translation system of claim 1, wherein said host cell
further comprises a nucleic acid encoding a protein of interest,
said nucleic acid comprising at least one selector codon, wherein
said selector codon is recognized by said first O-tRNA.
7. The translation system of claim 6, wherein said host cell
further comprises a second unnatural amino acid that is different
from the first unnatural amino acid, a second O-RS and a second
O-tRNA, wherein the second O-RS preferentially aminoacylates the
second O-tRNA with the second unnatural amino acid, and wherein the
second O-tRNA recognizes a selector codon encoded by the nucleic
acid that is different from the selector codon recognized by the
first O-tRNA.
8. The translation system of claim 1, wherein said host cell
comprises a polynucleotide encoding said first O-RS.
9. The translation system of claim 8, wherein said polynucleotide
comprises a nucleotide sequence selected from the nucleotide
sequences set forth in SEQ ID NOS: 8-56.
10. The translation system of claim 1, wherein said host cell
comprises a polynucleotide encoding said first O-tRNA.
11. The translation system of claim 1, wherein said rodent host
cell is selected from a rat host cell and a mouse host cell.
12. The translation system of claim 1, wherein said primate host
cell is selected from the group consisting of human host cell,
chimpanzee host cell, bonobo host cell, gorilla host cell,
orangutan host cell, gibbon host cell, macaque host cell, tamarin
host cell and marmoset host cell.
13. The translation system of claim 1, wherein said rodent host
cell is a CHO host cell.
14. The translation system of claim 1, wherein said primate host
cell is a 293T host cell.
15. A method for producing a protein comprising an unnatural amino
acid at a selected position, the method comprising: (a) providing:
(i) a first unnatural amino acid selected from the group consisting
of: A) p-methoxyphenylalanine (pMpa); B) p-acetylphenylalanine
(pApa); C) p-benzoylphenylalanine (pBpa); D) p-iodophenylalanine
(pIpa); E) p-azidophenylalanine (pAzpa); F)
p-propargyloxyphenylalanine (pPpa); G) .alpha.-aminocaprylic acid;
H) o-nitrobenzylcysteine (o-NBC); I) 1,5-dansylalanine; and J)
o-nitrobenzylserine (o-NBS); (ii) a first orthogonal tRNA (O-tRNA);
(iii) a first orthogonal aminoacyl-tRNA synthetase (O-RS), wherein
said first O-RS preferentially aminoacylates said first O-tRNA with
said unnatural amino acid; (iv) a nucleic acid encoding said
protein, wherein said nucleic acid comprises at least one selector
codon that is recognized by said first O-tRNA, and wherein position
of selector codon in said nucleic acid corresponds to selected
position in said protein; and (v) a host cell comprising (i), (ii),
(iii) and (iv), said host cell selected from a rodent cell and a
primate cell; and (b) incorporating said unnatural amino acid at
said selected position in said protein during translation of said
protein in response to said selector codon in the presence of said
unnatural amino acid, O-tRNA and O-RS, thereby producing said
protein comprising said unnatural amino acid at the selected
position.
16. The method of claim 15, wherein said first O-RS preferentially
aminoacylates said first O-tRNA with said first unnatural amino
acid with an efficiency that is at least 50% of the efficiency
observed for a translation system comprising said O-tRNA, said
unnatural amino acid, and an aminoacyl-tRNA synthetase comprising
an amino acid sequence selected from the group of amino acid
sequences of SEQ ID NOS: 57-101.
17. The method of claim 15, wherein said first O-tRNA comprises or
is encoded by the polynucleotide sequence set forth in SEQ ID NO:
3.
18. The method of claim 15, wherein providing a first O-RS
comprises providing a polynucleotide encoding said first O-RS.
19. The method of claim 15, wherein said first O-RS is derived from
an E. coli aminoacyl-tRNA synthetase.
20. The method of claim 15, wherein said first O-RS comprises an
amino acid sequence selected from the group of amino acid sequences
set forth in SEQ ID NOS: 57-101, and conservative variants
thereof.
21. The method of claim 15, wherein said providing a first O-RS
comprises providing a polynucleotide encoding said O-RS.
22. The method of claim 21, wherein said polynucleotide comprises a
nucleotide sequence set selected from the nucleotide sequences set
forth in SEQ ID NOS: 8-56.
23. The method of claim 15, wherein said providing a first O-RS
comprises mutating an amino acid binding pocket of a wild-type
aminoacyl-tRNA synthetase by site-directed mutagenesis, and
selecting a resulting O-RS that preferentially aminoacylates said
first O-tRNA with said first unnatural amino acid.
24. The method of claim 23, wherein said selecting step comprises
positively selecting and negatively selecting for said first O-RS
from a pool comprising a plurality of resulting aminoacyl-tRNA
synthetase molecules following site-directed mutagenesis.
25. The method of claim 15, wherein said providing a first O-tRNA
comprises providing a polynucleotide encoding said first
O-tRNA.
26. The method of claim 15, wherein said protein comprises a second
unnatural amino acid that is different from said first unnatural
amino acid, the method further providing said second unnatural
amino acid, a second O-RS and a second O-tRNA, wherein the second
O--RS preferentially aminoacylates the second O-tRNA with the
second unnatural amino acid, and wherein the second O-tRNA
recognizes a selector codon in the nucleic acid that is different
from the selector codon recognized by the first O-tRNA.
27. The method of claim 15, wherein said incorporating step
comprises culturing said host cell.
28. The method of claim 15, wherein the first unnatural amino acid
comprises a photocaged amino acid, and wherein the method further
comprises: exposing the host cell to UV light.
29. The method of claim 29, wherein exposing the host cell to UV
light provides spatial control of protein activity in the host
cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/853,008, filed Oct. 18, 2006, and Ser. No.
60/923,458, filed Apr. 12, 2007, the disclosures of which is are
incorporated herein by reference in its their entirety for all
purposes.
FIELD OF THE INVENTION
[0003] The invention is in the field of translation biochemistry.
The invention relates to compositions and methods for making and
using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and
pairs thereof, that incorporate unnatural amino acids into
proteins. The invention also relates to methods of producing
proteins in cells using such pairs and the proteins produced by the
methods.
BACKGROUND OF THE INVENTION
[0004] Methods have been previously described to incorporate
unnatural amino acids site-specifically into proteins in mammalian
cells. Chemically aminoacylated suppressor tRNAs have been
microinjected or electroporated into CHO cells and neurons,
respectively, and used to suppress nonsense amber mutations with a
series of unnatural amino acids (Monahan et al. (2003),
"Site-specific incorporation of unnatural amino acids into
receptors expressed in Mammalian cells," Chem Biol 10:573-580).
However, the use of the aminoacylated tRNA as a stoichiometric
reagent severely limits the amount of protein that can be
produced.
[0005] Alternatively, heterologous suppressor tRNA/aaRS pairs that
do not cross react with host tRNAs, aaRSs or amino acids
(orthogonal tRNA/aaRSs) have been engineered to incorporate
unnatural amino acids selectively into proteins. For example,
Yokoyama and coworkers modified a Bacillus stearothermophilus amber
suppressor tRNA.sub.CUA.sup.Tyr (BstRNA.sub.CUA.sup.Tyr) and E.
coli tyrosyl-tRNA synthetase (EcTyrRS) to incorporate
3-iodo-L-tyrosine into proteins in CHO cells (Sakamoto et al.
(2002), "Site-specific incorporation of an unnatural amino acid
into proteins in mammalian cells," Nucleic Acids Res 30:4692-4699).
Similarly, Zhang and coworkers engineered an orthogonal Bacillus
subtilis suppressor tRNA/tryptophanyl-tRNA synthetase pair to
incorporate 5-hydroxytryptophan into proteins in mammalian cells
with high fidelity (Zhang et al. (2004), "Selective incorporation
of 5-hydroxytryptophan into proteins in mammalian cells," Proc Natl
Acad Sci USA 101:8882-8887).
[0006] However, the use of structure-based mutagenesis to generate
aaRS variants that aminoacylate an amino acid whose side chain
differs significantly from that of the wild type substrate requires
mutations of multiple active site residues which are difficult to
predict a priori (Zhang et al. (2002), "Structure-based design of
mutant Methanococcus jannaschii tyrosyl-tRNA synthetase for
incorporation of O-methyl-L-tyrosine," Proc Natl Acad Sci USA
99:6579-6584; Turner et al. (2005), "Structural characterization of
a p-acetylphenylalanyl aminoacyl-tRNA synthetase," J Am Chem Soc
127:14976-14977; Turner et al. (2006), "Structural plasticity of an
aminoacyl-tRNA synthetase active site," Proc Natl Acad Sci USA
103:6483-6488). Moreover, the engineered mutant may still recognize
host amino acids, as is the case with a mutant aaRS that charges
its cognate tRNA.sub.CUA.sup.Tyr with 3-iodo-L-tyrosine (Sakamoto
et al. (2002), "Site-specific incorporation of an unnatural amino
acid into proteins in mammalian cells," Nucleic Acids Res
30:4692-4699; and Kiga et al. (2002), "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 cell-free system," Proc Natl Acad
Sci USA 99:9715-9720).
[0007] Alternatively, one can attempt to evolve aaRSs with altered
specificities directly in mammalian cells. For example, Wang and
coworkers recently used somatic hypermutation in a human B cell
line to directly evolve a monomeric red fluorescent protein with
enhanced photostability and far-red emissions (Wang et al. (2004),
"Evolution of new nonantibody proteins via iterative somatic
hypermutation," Proc Natl Acad Sci USA 101:16745-16749). However,
somatic hypermutation introduces random mutations in the whole
protein, which may be less effective than genetic diversity created
by targeted mutagenesis of the active site when evolving variants
with altered substrate specificity. The latter, however, is limited
by difficulties in generating large stable libraries in mammalian
cells.
Orthogonal Translation Technology
[0008] A general methodology has been developed for the in vivo
site-specific incorporation of structurally diverse unnatural amino
acids with non-native physical, chemical and biological properties
into proteins in prokaryotic organisms and yeast. These methods
rely on orthogonal protein translation components that recognize a
suitable selector codon to insert a desired unnatural amino acid at
a defined position in a gene of interest during polypeptide
translation in vivo. These methods utilize an orthogonal tRNA
(O-tRNA) that recognizes a selector codon (e.g., a nonsense amber
codon), and where a corresponding specific orthogonal
aminoacyl-tRNA synthetase (an O-RS) specifically charges the O-tRNA
with the unnatural amino acid. These components do not cross-react
with any of the endogenous tRNAs or RSs in the host organism (i.e.,
the engineered tRNA and RS are orthogonal).
[0009] Using this technique in E. coli host systems, functional
amber and frameshift suppressor tRNA/aaRS pairs have been derived
from a Methanococcus jannaschii tRNA.sup.Tyr/TyrRS pair, an
archaeal tRNA.sup.Glu/Pyrococcus horikoshii glutamyl-tRNA
synthetase pair, and an archaeal tRNA.sup.Lys/Pyrococcus horikoshii
lysyl-tRNA synthetase pair. In Saccharomyces cerevisiae (S.
cerevisiae), functional tRNA.sub.CUA/aaRS pairs have been derived
from the corresponding E. coli tRNA.sup.Tyr/TyrRS and
tRNA.sup.Leu/leucyl-tRNA synthetase pairs. Directed evolution of
these suppressor tRNA/aaRS pairs using a combination of positive
and negative selections has allowed the efficient, highly selective
in vivo incorporation of a large number of diverse unnatural amino
acids in E. coli and S. cerevisiae. These include fluorescent,
glycosylated, sulfated, metal-ion-binding, and redox-active amino
acids, as well as amino acids with novel chemical and photochemical
reactivity. This methodology provides a powerful tool for exploring
protein structure and function in vitro and in vivo, and for
generating proteins, e.g., therapeutic proteins, with new or
enhanced properties. The extension of this methodology to mammalian
cells, for example primate and rodent cell lines, would
significantly enhance the utility of this technology.
[0010] The practice of using orthogonal translation systems that
are suitable for in vivo production of proteins that comprise one
or more unnatural amino acid is generally known in the art. For
example, see International Publication Numbers WO 2002/086075,
entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL
tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN
VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593,
entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415,
filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO
2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct. 27,
2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS." Each of these applications
is incorporated herein by reference in its entirety.
[0011] Additional discussion of orthogonal translation systems is
also found in, for example, Wang et al. (2001), "Expanding the
genetic code of Escherichia coli," Science 292:498-500; Wang and
Schultz (2002), "Expanding the Genetic Code," Chem. Commun. (Camb.)
1:1-11; Alfonta et al. (2003), "Site-Specific Incorporation of a
Redox-Active Amino Acid into Proteins," J Am Chem Soc
125:14662-14663; Santoro et al. (2003), "An archaebacteria-derived
glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid
mutagenesis of proteins in Escherichia coli," Nucleic Acids Res
31:6700-6709; Chin et al. (2003), "An expanded eukaryotic genetic
code," Science 301, 964-967; Chin et al. (2003), "Progress toward
an expanded eukaryotic genetic code," Chem Biol 10, 511-519; and Wu
et al. (2004), "A genetically encoded photocaged amino acid," J Am
Chem Soc 126, 14306-14307; Summerer et al. (2006), "A Genetically
Encoded Fluorescent Amino Acid," PNAS 103(26):9785-9789; Anderson
et al. (2004), "An expanded genetic code with a functional
quadruplet codon," Proc Natl Acad Sci USA 101:7566-7571; Zhang et
al. (2004), "A new strategy for the synthesis of glycoproteins,"
Science 303, 371-373; Wang and Schultz "Expanding the Genetic
Code," Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and
Schultz, "An Expanding Genetic Code," Methods 36(3):227-238 (2005);
Xie and Schultz, "Adding Amino Acids to the Genetic Repertoire,"
Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al.,
"Expanding the Genetic Code," Annu. Rev. Biophys. Biomol. Struct.,
35:225-249 (2006); Xie and Schultz (2006), "A Chemical Toolkit for
Proteins--an Expanded Genetic Code," Nat. Rev. Mol. Cell. Biol.,
7(10):775-782; Summerer et al. (2006), "A Genetically Encoded
Fluorescent Amino Acid," Proc Natl Acad Sci USA 103, 9785-9789;
Wang et al. (2006), "A Genetically Encoded Fluorescent Amino Acid,"
J Am Chem Soc 128, 8738-8739; and Liu and Schultz (2006),
"Recombinant Expression of Selectively Sulfated Proteins in E.
coli," Nat. Biotechnol., 24(11): 1436-1440. The content of each of
these publications above is hereby incorporated by reference.
[0012] There is a need in the art for the development of improved
orthogonal translation components that incorporate unnatural amino
acids into proteins in mammalian cell host systems, for example in
primate and rodent host cell systems, where a desired unnatural
amino acid is incorporated at defined positions. There is a need in
the art for improved methods for screening and identifying
orthogonal translation components (e.g., mutant aminoacyl-tRNA
synthetase enzymes) that can function in mammalian cells, such as
rodent cells and human cells. The invention described herein
fulfills these and other needs, as will be apparent upon review of
the following disclosure.
SUMMARY OF THE INVENTION
[0013] The present invention provides a general approach that
allows unnatural amino acids with diverse physicochemical and
biological properties to be genetically encoded in mammalian cells,
e.g., rodent cells and primate cells. Mutant Escherichia coli (E.
coli) aminoacyl-tRNA synthetases (RS) are first evolved in yeast to
selectively utilize the unnatural amino acid of interest. The
mutant RS together with an amber suppressor tRNA from a eubacteria,
e.g., Bacillus stearothermophilus (B. stearothermophilus), are then
used to site-specifically incorporate the unnatural amino acid into
proteins in the mammalian cells in response to an amber nonsense
codon. Ten unnatural amino acids (the unnatural amino acids
provided in FIG. 1) were independently incorporated into model
proteins expressed in Chinese Hamster Ovary (CHO) cells or human
293T cells with efficiencies up to 1 .mu.g protein per
2.times.10.sup.7 cells. Mass spectrometry confirmed a high
translational fidelity for the unnatural amino acid. This
methodology can facilitate the introduction of biological probes
into proteins for cellular studies and may ultimately facilitate
the synthesis of therapeutic proteins containing unnatural amino
acids in mammalian cells such as rodent cells and primate
cells.
[0014] The invention provides compositions and methods for
incorporating unnatural amino acids into a growing polypeptide
chain in response to a selector codon, e.g., an amber stop codon,
in vivo in a host cell such as a rodent host cell or a primate host
cell. These compositions include the host cells, as well as pairs
of orthogonal-tRNAs (O-tRNAs) and orthogonal aminoacyl-tRNA
synthetases (O-RSs) that do not interact with the host cell
translation machinery. That is to say, the O-tRNA is not charged
(or not charged to a significant level) with an amino acid (natural
or unnatural) by an endogenous host cell aminoacyl-tRNA synthetase.
Similarly, the O-RSs provided by the invention do not charge any
endogenous tRNA with an amino acid (natural or unnatural) to a
significant or detectable level. These novel compositions permit
the production of large quantities of proteins having
translationally incorporated unnatural amino acids in mammalian
host cell systems.
[0015] In some aspects, the invention provides translation systems.
These systems comprise (a) a mammalian host cell such as a rodent
or primate host cell, comprising (b) a first orthogonal
aminoacyl-tRNA synthetase (O-RS), (c) a first orthogonal tRNA
(O-tRNA), and (d) a first unnatural amino acid, where the first
O-RS preferentially aminoacylates the first O-tRNA with the first
unnatural amino acid. The unnatural amino acid can be, but not
limited by: [0016] p-methoxyphenylalanine (pMpa); [0017]
p-acetylphenylalanine (pApa); [0018] p-benzoylphenylalanine (pBpa);
[0019] p-iodophenylalanine (pIpa); [0020] p-azidophenylalanine
(pAzpa); [0021] p-propargyloxyphenylalanine (pPpa); [0022]
.alpha.-aminocaprylic acid; [0023] o-nitrobenzylcysteine (o-NBC);
[0024] 1,5-dansylalanine; and [0025] o-nitrobenzylserine
(o-NBS).
[0026] In some aspects, the O-RS preferentially aminoacylates the
O-tRNA with said unnatural amino acid with an efficiency that is at
least 50% of the efficiency observed for a translation system
comprising that same O-tRNA, the unnatural amino acid, and an
aminoacyl-tRNA synthetase comprising an amino acid sequence
selected from SEQ ID NO: 57-101.
[0027] The translation systems can use components derived from a
variety of sources. In one embodiment, the first O-RS is derived
from an E. coli aminoacyl-tRNA synthetase, e.g., a wild-type E.
coli tyrosyl or leucyl-tRNA synthetase. In some embodiments, the
O-tRNA comprises or is encoded by SEQ ID NO: 3. The O-RS used in
the system can comprise the amino acid sequence of SEQ ID NOS:
57-101, and conservative variants of that sequence. In some
aspects, the host cell can comprise one or more polynucleotides
that encode components of the translation system, including the
O-RS or O-tRNA. In some embodiments, the polynucleotide encoding
the O-RS comprises a nucleotide sequence selected from SEQ ID NO:
8-56.
[0028] In some aspects, the translation system further comprises a
nucleic acid encoding a protein of interest, where the nucleic acid
has at least one selector codon that is recognized by the
O-tRNA.
[0029] In some aspects, the translation system (i.e., the host
cell) incorporates a second orthogonal pair (that is, a second O-RS
and a second O-tRNA) that utilizes a second unnatural amino acid,
so that the system is now able to incorporate at least two
different unnatural amino acids at different selected sites in a
polypeptide. In this dual system, the second O-RS preferentially
aminoacylates the second O-tRNA with a second unnatural amino acid
that is different from the first unnatural amino acid, and the
second O-tRNA recognizes a selector codon that is different from
the selector codon recognized by the first O-tRNA.
[0030] The mammalian host cell used in the translation system is
not particularly limited, as long as the O-RS and O-tRNA retain
their orthogonality in their host cell environment. The host cell
can be a rodent host cell such as a mouse cell or a rat cell. When
the host cell is a primate cell, the cell can be, e.g., a human
host cell, chimpanzee host cell, bonobo host cell, gorilla host
cell, orangutan host cell, gibbon host cell, macaque host cell,
tamarin host cell or a marmoset host cell. The host cell can be,
for example, a CHO cell or a human 293T.
[0031] In other aspects, the invention provides methods for
producing proteins of interest having one or more unnatural amino
acids at selected positions. These methods utilize the translation
systems described above. Generally, these methods start with the
step of providing a translation system comprising: (i) a first
unnatural amino acid (e.g., an unnatural amino acid of FIG. 1);
(ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a
first orthogonal tRNA (O-tRNA), wherein the O-RS preferentially
aminoacylates the O-tRNA with the unnatural amino acid; and, (iv) a
nucleic acid encoding the protein of interest, where the nucleic
acid comprises at least one selector codon that is recognized by
the first O-tRNA, and where the position of selector codon in the
nucleic acid corresponds to the selected position in the protein of
interest. All of these system components are contained in a
suitable host cell, e.g., a rodent cell or a primate cell. The
methods for producing a protein with an unnatural amino acid entail
generally culturing the host cell in the presence of all the
components of the translation system described above. In these
methods, the unnatural amino acid can be selected from (but not
limited to): [0032] p-methoxyphenylalanine (pMpa); [0033]
p-acetylphenylalanine (pApa); [0034] p-benzoylphenylalanine (pBpa);
[0035] p-iodophenylalanine (pIpa); [0036] p-azidophenylalanine
(pAzpa); [0037] p-propargyloxyphenylalanine (pPpa); [0038]
.alpha.-aminocaprylic acid; [0039] o-nitrobenzylcysteine (o-NBC);
[0040] 1,5-dansylalanine; and [0041] o-nitrobenzylserine
(o-NBS).
[0042] In some aspects of these methods, the O-RS preferentially
aminoacylates the O-tRNA with the unnatural amino acid with an
efficiency that is at least 50% of the efficiency observed for a
translation system comprising that same O-tRNA, the unnatural amino
acid, and an aminoacyl-tRNA synthetase (O-RS) comprising an amino
acid sequence selected from SEQ ID NOs: 57-101.
[0043] This methods can be widely applied using a variety of
reagents. In some embodiments of the methods, the O-tRNA comprises
or is encoded by SEQ ID NO: 3. In some embodiments, a
polynucleotide encoding the O-RS and/or the O-tRNA is provided. In
some embodiments, the O-RS is derived from an E. coli
aminoacyl-tRNA synthetase, such as a wild-type E. coli tyrosyl or
leucyl-tRNA synthetase. In some embodiments, the providing step
includes providing an O-RS comprising an amino acid sequence
selected from SEQ ID NOS: 57-101, and conservative variants
thereof. In some aspects of these methods, a polynucleotide that
encodes the O-RS is provided. For example, the polynucleotide
encoding the O-RS can comprise a nucleotide sequence selected from
SEQ ID NO: 8-56.
[0044] In some embodiments of these methods, the providing the O-RS
entails mutating an amino acid binding pocket of a wild-type
aminoacyl-tRNA synthetase by site-directed mutagenesis, and
selecting a resulting O-RS that preferentially aminoacylates the
O-tRNA with the unnatural amino acid. The selecting step can
comprises positively selecting and negatively selecting for the
O-RS from a pool of resulting aminoacyl-tRNA synthetase molecules
following site-directed mutagenesis.
[0045] These methods can also be modified to incorporate more than
one unnatural amino acid into a protein. In those methods, a second
orthogonal translation system is employed in conjunction with the
first translation system, where the second system has different
amino acid and selector codon specificities. For example, the
providing step can include providing a second O-RS and a second
O-tRNA, where the second O-RS preferentially aminoacylates the
second O-tRNA with a second unnatural amino acid that is different
from the first unnatural amino acid, and where the second O-tRNA
recognizes a selector codon in the nucleic acid that is different
from the selector codon recognized by the first O-tRNA.
DEFINITIONS
[0046] Before describing the invention in detail, it is to be
understood that this invention is not limited to particular
biological systems, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting. As used in this specification and the appended claims,
the singular forms "a", "an" and "the" include plural referents
unless the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes combinations of two or more cells;
reference to "a polynucleotide" includes, as a practical matter,
many copies of that polynucleotide.
[0047] Unless defined herein and below in the reminder of the
specification, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in
the art to which the invention pertains.
[0048] Orthogonal: As used herein, the term "orthogonal" refers to
a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal
aminoacyl-tRNA synthetase (O-RS)) that functions with endogenous
components of a cell with reduced efficiency as compared to a
corresponding molecule that is endogenous to the cell or
translation system, or that fails to function with endogenous
components of the cell. In the context of tRNAs and aminoacyl-tRNA
synthetases, orthogonal refers to an inability or reduced
efficiency, e.g., less than 20% efficiency, less than 10%
efficiency, less than 5% efficiency, or less than 1% efficiency, of
an orthogonal tRNA to function with an endogenous tRNA synthetase
compared to an endogenous tRNA to function with the endogenous tRNA
synthetase, or of an orthogonal aminoacyl-tRNA synthetase to
function with an endogenous tRNA compared to an endogenous tRNA
synthetase to function with the endogenous tRNA. The orthogonal
molecule lacks a functionally normal endogenous complementary
molecule in the cell. For example, an orthogonal tRNA in a cell is
aminoacylated by any endogenous RS of the cell with reduced or even
zero efficiency, when compared to aminoacylation of an endogenous
tRNA by the endogenous RS. In another example, an orthogonal RS
aminoacylates any endogenous tRNA a cell of interest with reduced
or even zero efficiency, as compared to aminoacylation of the
endogenous tRNA by an endogenous RS. A second orthogonal molecule
can be introduced into the cell that functions with the first
orthogonal molecule. For example, an orthogonal tRNA/RS pair
includes introduced complementary components that function together
in the cell with an efficiency (e.g., 45% efficiency, 50%
efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80%
efficiency, 90% efficiency, 95% efficiency, or 99% or more
efficiency) as compared to that of a control, e.g., a corresponding
tRNA/RS endogenous pair, or an active orthogonal pair (e.g., a
tyrosyl or leucyl derived orthogonal tRNA/RS pair).
[0049] Orthogonal tyrosyl-tRNA: As used herein, an orthogonal
tyrosyl-tRNA (tyrosyl-O-tRNA) is a tRNA that is orthogonal to a
translation system of interest, where the tRNA is: (1) identical or
substantially similar to a naturally occurring tyrosyl-tRNA, (2)
derived from a naturally occurring tyrosyl-tRNA by natural or
artificial mutagenesis, (3) derived by any process that takes a
sequence of a wild-type or mutant tyrosyl-tRNA sequence of (1) or
(2) into account, (4) homologous to a wild-type or mutant
tyrosyl-tRNA; (5) homologous to any tRNA that is designated as a
substrate for a tyrosyl-tRNA synthetase, or (6) a conservative
variant of any example tRNA that is designated as a substrate for a
tyrosyl-tRNA synthetase. The tyrosyl-tRNA can exist charged with an
amino acid, or in an uncharged state. It is also to be understood
that a "tyrosyl-O-tRNA" optionally is charged (aminoacylated) by a
cognate synthetase with an amino acid other than tyrosine,
respectively, e.g., with an unnatural amino acid. Indeed, it will
be appreciated that a tyrosyl-O-tRNA of the invention is
advantageously used to insert essentially any amino acid, whether
natural or unnatural, into a growing polypeptide, during
translation, in response to a selector codon.
[0050] Orthogonal tyrosyl amino acid synthetase: As used herein, an
orthogonal tyrosyl amino acid synthetase (tyrosyl-O-RS) is an
enzyme that preferentially aminoacylates the tyrosyl-O-tRNA with an
amino acid in a translation system of interest. The amino acid that
the tyrosyl-O-RS loads onto the tyrosyl-O-tRNA can be any amino
acid, whether natural, unnatural or artificial, and is not limited
herein. The synthetase is optionally the same as or homologous to a
naturally occurring tyrosyl amino acid synthetase, or the same as
or homologous to a synthetase designated as an O-RS provided
herein. For example, the O-RS can be a conservative variant of a
tyrosyl-O-RS of FIG. 16, and/or can be at least 50%, 60%, 70%, 80%,
90%, 95%, 98%, 99% or more identical in sequence to an O-RS of FIG.
16 (SEQ ID NOS: 57-101).
[0051] Orthogonal leucyl-tRNA: As used herein, an orthogonal
leucyl-tRNA (leucyl-O-tRNA) is a tRNA that is orthogonal to a
translation system of interest, where the tRNA is: (1) identical or
substantially similar to a naturally occurring leucyl-tRNA, (2)
derived from a naturally occurring leucyl-tRNA by natural or
artificial mutagenesis, (3) derived by any process that takes a
sequence of a wild-type or mutant leucyl-tRNA sequence of (1) or
(2) into account, (4) homologous to a wild-type or mutant
tyrosyl-tRNA; (5) homologous to any tRNA that is designated as a
substrate for a leucyl-tRNA synthetase, or (6) a conservative
variant of any example tRNA that is designated as a substrate for a
leucyl-tRNA synthetase. The leucyl-tRNA can exist charged with an
amino acid, or in an uncharged state. It is also to be understood
that a "leucyl-O-tRNA" optionally is charged (aminoacylated) by a
cognate synthetase with an amino acid other than leucine,
respectively, e.g., with an unnatural amino acid. Indeed, it will
be appreciated that a leucyl-O-tRNA of the invention is
advantageously used to insert essentially any amino acid, whether
natural or unnatural, into a growing polypeptide, during
translation, in response to a selector codon.
[0052] Orthogonal leucyl-amino acid synthetase: As used herein, an
orthogonal leucyl amino acid synthetase (leucyl-O-RS) is an enzyme
that preferentially aminoacylates the leucyl-O-tRNA with an amino
acid in a translation system of interest. The amino acid that the
leucyl-O-RS loads onto the leucyl-O-tRNA can be any amino acid,
whether natural, unnatural or artificial, and is not limited
herein. The synthetase is optionally the same as or homologous to a
naturally occurring leucyl amino acid synthetase, or the same as or
homologous to a synthetase designated as an O-RS provided herein.
For example, the O-RS can be a conservative variant of a
leucyl-O-RS of FIG. 16, and/or can be at least 50%, 60%, 70%, 80%,
90%, 95%, 98%, 99% or more identical in sequence to an O-RS of FIG.
16.
[0053] Cognate: The term "cognate" refers to components that
function together, or have some aspect of specificity for each
other, e.g., an orthogonal tRNA and an orthogonal aminoacyl-tRNA
synthetase. The components can also be referred to as being
complementary.
[0054] Preferentially aminoacylates: As used herein in reference to
orthogonal translation systems, an O-RS "preferentially
aminoacylates" a cognate O-tRNA when the O-RS charges the O-tRNA
with an amino acid more efficiently than it charges any endogenous
tRNA in an expression system. That is, when the O-tRNA and any
given endogenous tRNA are present in a translation system in
approximately equal molar ratios, the O-RS will charge the O-tRNA
more frequently than it will charge the endogenous tRNA.
Preferably, the relative ratio of O-tRNA charged by the O-RS to
endogenous tRNA charged by the O-RS is high, preferably resulting
in the O-RS charging the O-tRNA exclusively, or nearly exclusively,
when the O-tRNA and endogenous tRNA are present in equal molar
concentrations in the translation system. The relative ratio
between O-tRNA and endogenous tRNA that is charged by the O-RS,
when the O-tRNA and O-RS are present at equal molar concentrations,
is greater than 1:1, preferably at least about 2:1, more preferably
5:1, still more preferably 10:1, yet more preferably 20:1, still
more preferably 50:1, yet more preferably 75:1, still more
preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or
higher.
[0055] The O-RS "preferentially aminoacylates an O-tRNA with an
unnatural amino acid" when (a) the O-RS preferentially
aminoacylates the O-tRNA compared to an endogenous tRNA, and (b)
where that aminoacylation is specific for the unnatural amino acid,
as compared to aminoacylation of the O-tRNA by the O-RS with any
natural amino acid. That is, when the unnatural and natural amino
acids are present in equal molar amounts in a translation system
comprising the O-RS and O-tRNA, the O-RS will load the O-tRNA with
the unnatural amino acid more frequently than with the natural
amino acid. Preferably, the relative ratio of O-tRNA charged with
the unnatural amino acid to O-tRNA charged with the natural amino
acid is high. More preferably, O-RS charges the O-tRNA exclusively,
or nearly exclusively, with the unnatural amino acid. The relative
ratio between charging of the O-tRNA with the unnatural amino acid
and charging of the O-tRNA with the natural amino acid, when both
the natural and unnatural amino acids are present in the
translation system in equal molar concentrations, is greater than
1:1, preferably at least about 2:1, more preferably 5:1, still more
preferably 10:1, yet more preferably 20:1, still more preferably
50:1, yet more preferably 75:1, still more preferably 95:1, 98:1,
99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.
[0056] Selector codon: The term "selector codon" refers to codons
recognized by the O-tRNA in the translation process and not
recognized by an endogenous tRNA. The O-tRNA anticodon loop
recognizes the selector codon on the mRNA and incorporates its
amino acid, e.g., an unnatural amino acid, at this site in the
polypeptide. Selector codons can include, e.g., nonsense codons,
such as, stop codons, e.g., amber, ochre, and opal codons; four or
more base codons; rare codons; codons derived from natural or
unnatural base pairs and/or the like.
[0057] Suppressor tRNA: A suppressor tRNA is a tRNA that alters the
reading of a messenger RNA (mRNA) in a given translation system,
typically by allowing the incorporation of an amino acid in
response to a stop codon (i.e., "read-through") during the
translation of a polypeptide. In some aspects, a selector codon of
the invention is a suppressor codon, e.g., a stop codon (e.g., an
amber, ocher or opal codon), a four base codon, a rare codon,
etc.
[0058] Suppression activity: As used herein, the term "suppression
activity" refers, in general, to the ability of a tRNA (e.g., a
suppressor tRNA) to allow translational read-through of a codon
(e.g., a selector codon that is an amber codon or a 4-or-more base
codon) that would otherwise result in the termination of
translation or mistranslation (e.g., frame-shifting). Suppression
activity of a suppressor tRNA can be expressed as a percentage of
translational read-through activity observed compared to a second
suppressor tRNA, or as compared to a control system, e.g., a
control system lacking an O-RS.
[0059] The present invention provides various methods by which
suppression activity can be quantitated. Percent suppression of a
particular O-tRNA and O-RS against a selector codon (e.g., an amber
codon) of interest refers to the percentage of activity of a given
expressed test marker (e.g., LacZ), that includes a selector codon,
in a nucleic acid encoding the expressed test marker, in a
translation system of interest, where the translation system of
interest includes an O-RS and an O-tRNA, as compared to a positive
control construct, where the positive control lacks the O-tRNA, the
O-RS and the selector codon. Thus, for example, if an active
positive control marker construct that lacks a selector codon has
an observed activity of X in a given translation system, in units
relevant to the marker assay at issue, then percent suppression of
a test construct comprising the selector codon is the percentage of
X that the test marker construct displays under essentially the
same environmental conditions as the positive control marker was
expressed under, except that the test marker construct is expressed
in a translation system that also includes the O-tRNA and the O-RS.
Typically, the translation system expressing the test marker also
includes an amino acid that is recognized by the O-RS and O-tRNA.
Optionally, the percent suppression measurement can be refined by
comparison of the test marker to a "background" or "negative"
control marker construct, which includes the same selector codon as
the test marker, but in a system that does not include the O-tRNA,
O-RS and/or relevant amino acid recognized by the O-tRNA and/or
O-RS. This negative control is useful in normalizing percent
suppression measurements to account for background signal effects
from the marker in the translation system of interest.
[0060] Suppression efficiency can be determined by any of a number
of assays known in the art. For example, a .beta.-galactosidase
reporter assay can be used, e.g., a derivatived lacZ plasmid (where
the construct has a selector codon n the lacZ nucleic acid
sequence) is introduced into cells from an appropriate organism
(e.g., an organism where the orthogonal components can be used)
along with plasmid comprising an O-tRNA of the invention. A cognate
synthetase can also be introduced (either as a polypeptide or a
polynucleotide that encodes the cognate synthetase when expressed).
The cells are grown in media to a desired density, e.g., to an
OD.sub.600 of about 0.5, and .beta.-galactosidase assays are
performed, e.g., using the BetaFluor.TM. .beta.-Galactosidase Assay
Kit (Novagen). Percent suppression can be calculated as the
percentage of activity for a sample relative to a comparable
control, e.g., the value observed from the derivatized lacZ
construct, where the construct has a corresponding sense codon at
desired position rather than a selector codon.
[0061] Translation system: The term "translation system" refers to
the components that incorporate an amino acid into a growing
polypeptide chain (protein). Components of a translation system can
include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like.
The O-tRNA and/or the O-RSs of the invention can be added to or be
part of an in vitro or in vivo translation system, e.g., in a
mammalian cell, e.g., a rodent cell or a primate cell.
[0062] Unnatural amino acid: As used herein, the term "unnatural
amino acid" refers to any amino acid, modified amino acid, and/or
amino acid analogue, that is not one of the 20 common naturally
occurring amino acids or seleno cysteine or pyrrolysine. For
example, see the unnatural amino acids provided in FIG. 1.
[0063] Derived from: As used herein, the term "derived from" refers
to a component that is isolated from or made using a specified
molecule or organism, or information from the specified molecule or
organism. For example, a polypeptide that is derived from a second
polypeptide can include an amino acid sequence that is identical or
substantially similar to the amino acid sequence of the second
polypeptide. In the case of polypeptides, the derived species can
be obtained by, for example, naturally occurring mutagenesis,
artificial directed mutagenesis or artificial random mutagenesis.
The mutagenesis used to derive polypeptides can be intentionally
directed or intentionally random, or a mixture of each. The
mutagenesis of a polypeptide to create a different polypeptide
derived from the first can be a random event (e.g., caused by
polymerase infidelity) and the identification of the derived
polypeptide can be made by appropriate screening methods, e.g., as
discussed herein. Mutagenesis of a polypeptide typically entails
manipulation of the polynucleotide that encodes the
polypeptide.
[0064] Positive selection or screening marker: As used herein, the
term "positive selection or screening marker" refers to a marker
that, when present, e.g., expressed, activated or the like, results
in identification of a cell, which comprises the trait, e.g., a
cell with the positive selection marker, from those without the
trait.
[0065] Negative selection or screening marker: As used herein, the
term "negative selection or screening marker" refers to a marker
that, when present, e.g., expressed, activated, or the like, allows
identification of a cell that does not comprise a selected property
or trait (e.g., as compared to a cell that does possess the
property or trait).
[0066] Reporter: As used herein, the term "reporter" refers to a
component that can be used to identify and/or select target
components of a system of interest. For example, a reporter can
include a protein, e.g., an enzyme, that confers antibiotic
resistance or sensitivity (e.g., .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), and the like), a fluorescent screening
marker (e.g., green fluorescent protein (e.g., (GFP), YFP, EGFP,
RFP, etc.), a luminescent marker (e.g., a firefly luciferase
protein), an affinity based screening marker, or positive or
negative selectable marker genes such as lacZ, .beta.-gal/lacZ
(.beta.-galactosidase), ADH (alcohol dehydrogenase), his3, ura3,
leu2, lys2, or the like.
[0067] Eukaryote: As used herein, the term "eukaryote" refers to
organisms belonging to the Kingdom Eucarya. Eukaryotes are
generally distinguishable from prokaryotes by their typically
multicellular organization (but not exclusively multicellular, for
example, yeast), the presence of a membrane-bound nucleus and other
membrane-bound organelles, linear genetic material (i.e., linear
chromosomes), the absence of operons, the presence of introns,
message capping and poly-A mRNA, and other biochemical
characteristics, such as a distinguishing ribosomal structure.
Eukaryotic organisms include, for example, animals (e.g., mammals,
insects, reptiles, birds, etc.), ciliates, plants (e.g., monocots,
dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia,
protists, etc.
[0068] Prokaryote: As used herein, the term "prokaryote" refers to
organisms belonging to the Kingdom Monera (also termed Procarya).
Prokaryotic organisms are generally distinguishable from eukaryotes
by their unicellular organization, asexual reproduction by budding
or fission, the lack of a membrane-bound nucleus or other
membrane-bound organelles, a circular chromosome, the presence of
operons, the absence of introns, message capping and poly-A mRNA,
and other biochemical characteristics, such as a distinguishing
ribosomal structure. The Prokarya include subkingdoms Eubacteria
and Archaea (sometimes termed "Archaebacteria"). Cyanobacteria (the
blue green algae) and mycoplasma, although historically considered
"bacteria," are sometimes given separate classifications under the
Kingdom Monera.
[0069] Eubacteria: As used herein, the terms "eubacteria" and
"bacteria" refer to prokaryotic organisms that are distinguishable
from Archaea. Similarly, Archaea refers to prokaryotes that are
distinguishable from eubacteria. Eubacteria and Archaea can be
distinguished by a number morphological and biochemical criteria.
For example, differences in ribosomal RNA sequences, RNA polymerase
structure, the presence or absence of introns, antibiotic
sensitivity, the presence or absence of cell wall peptidoglycans
and other cell wall components, the branched versus unbranched
structures of membrane lipids, and the presence/absence of histones
and histone-like proteins are used to assign an organism to
Eubacteria or Archaea.
[0070] Examples of Eubacteria include Escherichia coli, Thermus
thermophilus, Bacillus subtilis and Bacillus stearothermophilus.
Example of Archaea include Methanococcus jannaschii (Mj),
Methanosarcina mazei (Mm), Methanobacterium thermoautotrophicum
(Mt), Methanococcus maripaludis, Methanopyrus kandleri,
Halobacterium such as Haloferax volcanii and Halobacterium species
NRC-1, Archaeoglobus fulgidus (Af), Pyrococcus furiosus (Pf),
Pyrococcus horikoshii (Ph), Pyrobaculum aerophilum, Pyrococcus
abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii,
Aeuropyrum pernix (Ap), Thermoplasma acidophilum and Thermoplasma
volcanium.
[0071] Conservative variant: As used herein, the term "conservative
variant," in the context of a translation component, refers to a
translation component, e.g., a conservative variant O-tRNA or a
conservative variant O-RS, that functionally performs similar to a
base component that the conservative variant is similar to, e.g.,
an O-tRNA or O-RS, having variations in the sequence as compared to
a reference O-tRNA or O-RS. For example, an O-RS, or a conservative
variant of that O-RS, will aminoacylate a cognate O-tRNA with an
unnatural amino acid, e.g., an unnatural amino acid of FIG. 1. In
this example, the O-RS and the conservative variant O-RS do not
have the same amino acid sequences. The conservative variant can
have, e.g., one variation, two variations, three variations, four
variations, or five or more variations in sequence, as long as the
conservative variant is still complementary to (e.g., functions
with) the cognate corresponding O-tRNA or O-RS.
[0072] In some embodiments, a conservative variant O-RS comprises
one or more conservative amino acid substitutions compared to the
O-RS from which it was derived. In some embodiments, a conservative
variant O-RS comprises one or more conservative amino acid
substitutions compared to the O-RS from which it was derived, and
furthermore, retains O-RS biological activity; for example, a
conservative variant O-RS that retains at least 10% of the
biological activity of the parent O-RS molecule from which it was
derived, or alternatively, at least 20%, at least 30%, or at least
40%. In some preferred embodiments, the conservative variant O-RS
retains at least 50% of the biological activity of the parent O-RS
molecule from which it was derived. The conservative amino acid
substitutions of a conservative variant O-RS can occur in any
domain of the O-RS, including the amino acid binding pocket.
[0073] Selection or screening agent: As used herein, the term
"selection or screening agent" refers to an agent that, when
present, allows for selection/screening of certain components from
a population. For example, a selection or screening agent can be,
but is not limited to, e.g., a nutrient, an antibiotic, a
wavelength of light, an antibody, an expressed polynucleotide, or
the like. The selection agent can be varied, e.g., by
concentration, intensity, etc.
[0074] In response to: As used herein, the term "in response to"
refers to the process in which an O-tRNA of the invention
recognizes a selector codon and mediates the incorporation of the
unnatural amino acid, which is coupled to the tRNA, into the
growing polypeptide chain.
[0075] Encode: As used herein, the term "encode" refers to any
process whereby the information in a polymeric macromolecule or
sequence string is used to direct the production of a second
molecule or sequence string that is different from the first
molecule or sequence string. As used herein, the term is used
broadly, and can have a variety of applications. In some aspects,
the term "encode" describes the process of semi-conservative DNA
replication, where one strand of a double-stranded DNA molecule is
used as a template to encode a newly synthesized complementary
sister strand by a DNA-dependent DNA polymerase.
[0076] In another aspect, the term "encode" refers to any process
whereby the information in one molecule is used to direct the
production of a second molecule that has a different chemical
nature from the first molecule. For example, a DNA molecule can
encode an RNA molecule (e.g., by the process of transcription
incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA
molecule can encode a polypeptide, as in the process of
translation. When used to describe the process of translation, the
term "encode" also extends to the triplet codon that encodes an
amino acid. In some aspects, an RNA molecule can encode a DNA
molecule, e.g., by the process of reverse transcription
incorporating an RNA-dependent DNA polymerase. In another aspect, a
DNA molecule can encode a polypeptide, where it is understood that
"encode" as used in that case incorporates both the processes of
transcription and translation.
BRIEF DESCRIPTION OF THE FIGURES
[0077] FIG. 1 provides the chemical structures and nomenclature of
the unnatural amino acids p-methoxyphenylalanine (pMpa),
p-acetylphenylalanine (pApa), p-benzoylphenylalanine (pBpa),
p-iodophenylalanine (pIpa), p-azidophenylalanine (pAzpa),
p-propargyloxyphenylalanine (pPpa), .alpha.-aminocaprylic acid,
o-nitrobenzylcysteine (o-NBC), 1,5-dansylalanine and
o-nitrobenzylserine (o-NBS). Most of these unnatural amino acids
have variably been referred to using various alternative
nomenclatures, which are also indicated in the figure.
[0078] FIG. 2 shows a Western blot analysis of full-length GFP
expression in Invitrogen.TM. T-REx.TM. CHO cells using orthogonal
BstRNA.sub.CUA.sup.Tyr and EcTyrRS pairs. The cells were
cotransfected with plasmids pcDNA4-GFP37TAG, pcDNA4-EcTyrRS variant
and pUC18-3BstRNA.sub.CUA.sup.Tyr and grown in the presence or
absence of unnatural amino acids. The first lane is wild type GFP
expression in T-rex CHO cells that were transiently transfected
with pcDNA4-GFP. The second lane is full-length expression of
GFP37TAG suppressed by wild type EcTyrRS together with
BstRNA.sub.CUA.sup.Tyr. The following lanes are full-length
expression of GFP37TAG suppressed by EcTyrRS variants together with
BstRNA.sub.CUA.sup.Tyr in the presence or absence of unnatural
amino acids. pMpaRS represents pMpa-tRNA synthetase, etc. A 40
.mu.g aliquot of the cell lysate for each reaction (10 .mu.g cell
lysate was loaded into the first lane) was analyzed with anti-c-myc
antibody. The concentrations of unnatural amino acids for protein
expression were 10 mM for pMpa, 10 mM for pApa, 1 mM for pBpa, 8 mM
for plpa, 5 mM for pAzpa and 1 mM for pPpa.
[0079] FIG. 3 shows a Western blot analysis of full-length GFP
expression in Invitrogen.TM. T-REx.TM. 293 cells using orthogonal
BstRNA.sub.CUA.sup.Tyr and EcTyrRS pairs as described in FIG. 2. A
20 .mu.g aliquot of the cell lysate for each reaction (5 .mu.g for
the control reaction) was analyzed with anti-His-HRP antibody. The
reagents, concentrations of unnatural amino acids and lane
designations were the same as described in FIG. 2.
[0080] FIG. 4A provides a plasmid map of pSWAN-pMpaRS.
[0081] FIG. 4B provides a plasmid map of pSWAN-GFP37TAG.
[0082] FIG. 5 provides the amino acid sequence of the wild type GFP
and GFP-pMpa peptides. Y* denotes tyrosine in wild type GFP or pMpa
in GFP-pMpa. The y- and b-type ions generated during fragmentation
of the peptide FSVSGEGEGDATY*GK (SEQ ID NO: 103) are shown.
[0083] FIG. 6 provides an annotated tandem MS spectra of the wild
type GFP peptide FSVSGEGEGDATY*GK (SEQ ID NO: 103) provided in FIG.
5. For comparison and clarity, only the abundant Y*-ion series are
annotated as well as the b.sub.13 ion that locates pMpa
unambiguously at position 37. Proteins were purified by anti-myc
affinity column and analyzed by SDS-PAGE. The GFP bands were
excised for the MS analysis.
[0084] FIG. 7 provides an annotated tandem MS spectra of the
FSVSGEGEGDATY*GK (SEQ ID NO: 103) peptide from GFP-pMpa. Tandem MS
spectra of the Y*-ion series exhibit the expected mass shift of 14
Da.
[0085] FIG. 8 provides an intact protein ESI TOF-MS spectrum of
affinity purified GFP-pMpa. The deconvoluted charge state envelope
(shown in the figure insert) shows one major component of 29696 Da,
which matches the expected mass of the modified protein minus the
N-terminal methionine and plus acetylation (theoretical mass
29696.52) within experimental error. The smaller feature at 29654
Da is assigned to GFP-pMpa minus the N-terminal methionine. The two
side bands are likely due to nonspecific modification.
[0086] FIG. 9 provides a tandem MS spectra of mutant GFP containing
pApa. The Y*-ions exhibit a 26 Da mass shift with respect to the
same ions in the spectrum of wild type GFP.
[0087] FIG. 10 provides a tandem MS spectrum of mutant GFP
containing pBpa. The characteristic mass shift of 86 Da between
mutant GFP and wild type GFP is clearly observed.
[0088] FIG. 11 provides a tandem MS spectrum of mutant GFP
containing pAzpa. Most of pAzpa decays to pAmpa
(p-aminophenylalanine; see FIG. 15 for tandem MS spectra of tryptic
pAmpa containing fragment). However, the characteristic mass shift
of 25 Da of Y*-ions in comparison to signals of wild type GFP is
still clearly observed.
[0089] FIG. 12 provides a tandem MS spectrum of mutant GFP
containing pPpa. The signals of the pPpa fragment are largely
swamped by background. Despite this, it is still identified with
good discrimination by database searching of the spectrum against
MSDB. The stronger Y*-ions are observed with a characteristic mass
shift of 38 Da.
[0090] FIGS. 13A and 13B illustrate that amber suppression is
dependent upon both the EcTyrRS and BstRNA.sub.CUA.sup.Tyr genes in
both Invitrogen.TM. T-REx.TM. CHO and Invitrogen.TM. T-REx.TM. 293
cells. FIG. 13A provides panels with green fluorescence images of
Invitrogen.TM. T-REx.TM. CHO cells transiently transfected with
different constructs. In the first panel, cells were transfected
with pcDNA4-GFP. In the second panel, cells were transfected with
pcDNA4-EcTyrRS and pcDNA4-GFP37TAG. In the third panel, cells were
transfected with pUC18-3BstRNA.sub.CUA.sup.Tyr and pcDNA4-GFP37TAG.
In the fourth panel, cells were transfected with
pUC18-3BstRNA.sub.CUA.sup.Tyr, pcDNA4-EcTyrRS and pcDNA4-GFP37TAG.
FIG. 13B provides panels with green fluorescence images of
Invitrogen.TM. T-REx.TM. 293 cells transiently transfected with
different constructs. The constructs used in the various panels
were identical to that shown in FIG. 13A.
[0091] FIG. 14 provides a Western blot analysis of expression of
six EcTyrRS variants in Invitrogen.TM. T-REx.TM. CHO and 293 cells.
A 40 .mu.g aliquot of the cell lysate for each reaction was
analyzed with anti-c-myc antibody. The expression of EcTyrRS
variants is not affected by the active site mutations. The variants
express equally in both T-REx.TM. CHO and T-REx.TM. 293 cell
lines.
[0092] FIG. 15 provides an annotated tandem MS spectra of the
peptide FSVSGEGEGDATY*GK (SEQ ID NO: 103) from mutant GFP
containing pAzpa. Y* denotes pAmpa (p-aminophenylalanine). For
clarity only the Y*-ion series is annotated. Most of the pAzpa
decayed to pAmpa as the signal strength for the pAzpa peptide is
quite weak (see FIG. 11) and the detectable mass 1 Da mass
difference between fragments of wild type GFP and those of mutant
GFP containing pAzpa.
[0093] FIG. 16 provides nucleotide and amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0094] The present invention provides a general methodology that
allows for the rapid screening and isolation of mutant
aminoacyl-tRNA synthetase enzymes (RS) that have the ability to
charge a suitable orthogonal suppressor tRNA with unnatural amino
acids with diverse physicochemical and biological properties in
mammalian cell host systems, and further, where the screening
method does away with the need for slow and technically challenging
screening in mammalian cells. In these methods, mutant Escherichia
coli (E. coli) aminoacyl-tRNA synthetases (RS) are evolved in yeast
to selectively utilize the unnatural amino acid of interest. The
same mutant RS together with an amber suppressor tRNA from Bacillus
stearothermophilus (B. stearothermophilus) are then used to
site-specifically incorporate the unnatural amino acid into protein
of interest in mammalian cells in response to an selector codon
(e.g., an amber nonsense codon). The incorporation of the unnatural
amino acid into the protein is programmed to occur at any desired
position by engineering the polynucleotide encoding the protein of
interest to contain the selector codon that signals the
incorporation of the unnatural amino acid.
[0095] The present invention also provides novel translation
systems, where the systems comprise host cells that have hereto not
been contemplated. For example, a translation system of the
invention includes (i) an unnatural amino acid, (ii) an orthogonal
tRNA (O-tRNA), (iii) an orthogonal aminoacyl-tRNA synthetase (O-RS)
derived from a eubacterial aminoacyl-tRNA synthetase, typically an
E. coli RS, and (iv) a mammalian host cell that contains these
components, where the mammalian host cell can be a rodent cell
(e.g., a mouse cell or a rat cell), or a primate cell, i.e., any
cell belonging to any of the groups commonly termed simians
(including apes, humans and Old World monkeys), New World monkeys
and pro-simians (e.g., lemurs). Human cells find particular use
with the invention. However, it is not intended that the invention
be limited to the host cells listed above, as other host cells also
find use with the invention.
[0096] The invention also provides methods for producing a protein
having at least one unnatural amino acid at a selected position,
where the methods use the translation systems described above. The
methods result in the incorporation of the unnatural amino acid by
programming the incorporation of the unnatural amino acid at the
site of a selector codon during protein translation.
[0097] The invention provides a general approach in which mutant
RSs (originally evolved in S. cerevisiae) together with an amber
suppressor from a suitable eubacteria, e.g., B. stearothermophilus,
are used to incorporate a wide variety of unnatural amino acids
into proteins in mammalian host cells, such as rodent cells and
primate cells. As a proof of principle, this methodology was
successfully employed to site specifically incorporate a total of
ten different unnatural amino acids into model proteins in response
to amber nonsense codons with excellent fidelity and good
efficiencies in Chinese hamster ovary (CHO) and human 293T host
cells. For example, unnatural amino acids were independently
incorporated into green fluorescent protein (GFP) expressed in CHO
cells with efficiencies up to 1 .mu.g protein per 2.times.10.sup.7
cells. Mass spectrometry confirmed a high translational fidelity
for the unnatural amino acid. This methodology should facilitate
the introduction of biological probes into proteins for cellular
studies and may ultimately facilitate the synthesis of therapeutic
proteins containing unnatural amino acids in mammalian cells.
[0098] In some aspects, to demonstrate (but not to limit) the
present invention, the disclosure herein demonstrates that the
unnatural amino acid moiety can be incorporated into various model
proteins in mammalian host cells. It is not intended that the
incorporation of the unnatural amino acid be limited to any
particular protein of interest. From the present disclosure, it
will be clear that the incorporation of the various unnatural amino
acids into particular proteins of interest is advantageous for a
wide variety of proteins and for a wide variety of purposes.
[0099] It is noted that the genes that control the expression of
the mutant E. coli aminoacyl-tRNA synthetases (O-RSs) that are
evolved in yeast and the O-tRNA can not be used immediately in
mammalian host cells (e.g., rodent or human cells). These genes
must be reengineered with suitable regulatory elements to drive
their expression in the mammalian cell background. The yeast
transcription regulatory elements originally associated with the
O-tRNA and O-RS genes would be ineffective (or at best not optimal)
for use in the mammalian cells. One of skill in the art recognizes
this point. Mammalian cell gene expression elements are well
characterized and readily available for use in recombinant
constructs for the purpose of optimizing (e.g., maximizing) the
gene sequences with which they are associated.
Unnatural Amino Acids Finding Use with the Invention
[0100] The invention provides orthogonal translation systems that
have the ability to produce polypeptides comprising unnatural amino
acids in mammalian host cell systems, for example, primate and
rodent host cell systems. The unnatural amino acids that are
employed in these translation systems is not particularly limited.
Generally, the unnatural amino acids that find use with the
invention are those that can be utilized by an E. coli derived
mutant aminoacyl-tRNA synthetase to charge a corresponding
orthogonal tRNA in a Saccharomyces cerevisiae (S. cerevisiae) yeast
host cell system. For example, as utilized herein, the following
unnatural amino acids find use with the invention: [0101]
p-methoxyphenylalanine (pMpa) [0102] p-acetylphenylalanine (pApa)
[0103] p-benzoylphenylalanine (pBpa) [0104] p-iodophenylalanine
(pIpa) [0105] p-azidophenylalanine (pAzpa) [0106]
p-propargyloxyphenylalanine (pPpa) The structures of each of these
six unnatural amino acids is shown in FIG. 1. These amino acids can
also be known by alternative nomenclatures and various
abbreviations, which are also shown in FIG. 1. As shown in the
Examples, these six unnatural amino acids were independently
incorporated into green fluorescent protein (GFP) in human and
rodent host cells using E. coli derived O-RS species that were
originally screened and isolated using yeast host cell systems.
[0107] In addition to those listed above, still other unnatural
amino acids also find use with the invention. The unnatural amino
acids listed below have previously been used successfully by E.
coli derived orthogonal RS's to charge an O-tRNA in yeast host cell
systems. These unnatural amino acids include: [0108]
.alpha.-aminocaprylic acid [0109] o-nitrobenzylcysteine (o-NBC)
[0110] 1,5-dansylalanine [0111] o-nitrobenzylserine The structures
of these unnatural amino acids and their various alternative
nomenclatures and abbreviations are shown in FIG. 1.
[0112] The successful use of all these unnatural amino acids in
yeast host cells is described, for example, in Chin et al. (2003),
"An expanded eukaryotic genetic code," Science 301:964-967; Deiters
et al. (2003), "Adding amino acids with novel reactivity to the
genetic code of Saccharomyces cerevisiae," J Am Chem Soc
125:11782-11783; Summerer et al. (2006), "A Genetically Encoded
Fluorescent Amino Acid," PNAS 103(26):9785-9789; WO 2005/003294 to
Deiters et al., "UNNATURAL REACTIVE AMINO ACID GENETIC CODE
ADDITIONS," filed Apr. 16, 2004; WO 2006/034410 to Deiters et al.,
"ADDING PHOTOREGULATED AMINO ACIDS TO THE GENETIC CODE," filed Sep.
21, 2005; and WO 2006/110182, filed Oct. 27, 2005, entitled
"ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF
UNNATURAL AMINO ACIDS."
Host Cells Finding Use with the Invention
[0113] The present invention provides novel translation systems,
where the systems utilize host cells that have hereto not been
contemplated. For example, a translation system of the invention
includes (i) an unnatural amino acid, (ii) an orthogonal tRNA
(O-tRNA), (iii) an orthogonal aminoacyl-tRNA synthetase (O-RS)
derived from an E. coli aminoacyl-tRNA synthetase, and (iv) a
mammalian host cell that contains these components, where the
mammalian host cell can be a rodent cell (e.g., a mouse cell or a
rat cell), or a primate cell, i.e., any cell belonging to any of
the groups commonly termed simians (including apes, humans and Old
World monkeys), New World monkeys and pro-simians (e.g., lemurs).
Human cells find particular use with the invention. The animal cell
lines finding use with the invention can be derived from any tissue
from the animal. It is not intended that the invention be limited
to the host cells listed above, as other host cells also find use
with the invention.
[0114] As used herein, the term "host cell" also includes a
plurality of individual cells, commonly referred to as a "cell
line," and in this case, a "host cell line."
[0115] The host cells used for the production of polypeptides that
contain at least one unnatural amino acid can be any type of host
cell, but some host cells find particular use. For example, some
host cell types can be used to produce polypeptides that find use
in the production of recombinant therapeutic polypeptides.
[0116] When the host cell is a rodent cell, the type of rodent cell
is not particularly limited. As described in the Examples, Chinese
hamster ovary (CHO) cells can be used as host cells. CHO cells are
widely adapted and are a well established system for the expression
of recombinant proteins. However, it is not intended that the
invention be limited to this rodent cell type. Indeed, one of skill
in the art knows a vast array of rodent cell lines that can be used
as host cells for recombinant protein production.
[0117] Rodent cell lines include any cell line derived from the
mammalian order Rodentia, and includes, but not limited to, true
mice, rats, hamsters, chipmunks, beavers and squirrels. Rodent cell
lines include cell lines derived from the common house mouse, Mus
musculus, and any and all strains thereof. Similarly, rodent cell
lines include cell lines derived from the rat, e.g., any species of
the genus Rattus, such as Rattus norvegicus (Norwegian rat) and
Rattus rattus (the black rat), and any and all strains thereof.
[0118] The host cell of the invention can be a primate cell. The
type of primate cell is not particularly limited. As described in
the Examples, human embryonic kidney cells (also known as HEK
cells, HEK 293 or just 293) can be used as host cells. 293 cells
are widely adapted and are a well established system for the
expression of recombinant proteins. However, it is not intended
that the invention be limited to this human cell type. Indeed, one
of skill in the art knows a vast array of human and other primate
cell lines that can be used as host cells for recombinant protein
production.
[0119] Primate cell lines include any cell line derived from the
mammalian order Primate, and includes, but not limited to (i)
simians, which include Old World monkeys apes, including humans),
(ii) New World monkeys and (iii) prosimians (e.g., lemurs). The
primate cell line can be derived from any primate species. For
example, but not limited to, the primate cell lines can be derived
from chimpanzees, baboons, marmosets, macaques, and African green
monkeys (e.g., the COS-1 cell line).
[0120] The table below provides a non-exhaustive list of primate
species from which derived cells lines find use with the
invention.
TABLE-US-00001 Species Common Name Pan troglodytes schweinfurthii
Eastern chimpanzee Pan troglodytes Chimpanzee Pan paniscus Bonobo
Pan troglodytes verus Western chimpanzee Gorilla gorilla Western
lowland gorilla Pongo pygmaeus abelii Sumatran orangutan Pongo
pygmaeus pygmaeus Bornean orangutan Hylobates sp. gibbon sp. Macaca
arctoides stump-tailed macaque Macaca fascicularis Long-tailed
macaque Cercopithecus mitis albogularis blue monkey Macaca
fascicularis Long-tailed macaque Macaca mulatta Rhesus macaque
Macaca sylvanus Barbary macaque Pan troglodytes verus Western
chimpanzee Papio hamadryas anubis Olive baboon Papio sp. baboon sp.
Saguinus oedipus Cotton top tamarin Callithrix jacchus Common
marmoset Pan paniscus Bonobo Homo sapiens human
[0121] Cell lines derived from these primate species can be found,
for example, on the websites for the Research Infrastructure to
Promote Primate Molecular Biology (INPRIMAT; Barcelona, Spain) and
the Biomedical Primate Research Centre (BPRC; Rijswijk, The
Netherlands).
[0122] The use of human cell lines for the production of
recombinant polypeptides is well established and within the scope
of the present invention. The human host cell lines can be from any
source, and can be derived from any tissue. Human cell lines
suitable for the production of recombinant proteins are widely
known, and are available from a large number of commercial and
private sources well know to one of skill in the art.
[0123] Furthermore, host cell lines derived from other diverse
mammalian species are also within the scope of the invention. These
include bovine (cattle), porcine (swine), ovine (sheep), canine
(dog), equine (horse) and caprine (goat) cell lines. Cell lines
derived from these species are known in the art, and are readily
available to one of average skill in the art.
[0124] Rodent cell lines, primate cell lines, and numerous other
cell lines from other mammalian groups, and materials and methods
for the culture of these animal cell lines, are well established in
the art, and are widely available from a number of sources. For
example, see the American Type Culture Collection (ATCC; Manassas,
Va.) for extensive listings of available cell lines. Cell culture
media is available from a variety of sources, for example,
GIBCO.RTM. brand (Invitrogen.TM.) cell culture media and Mediatech,
Inc. culture media by Cellgro.RTM.. Furthermore, the de novo
establishment of new cell lines (primary cell lines or established
cell lines) also finds use with the invention as host cells for the
production of recombinant proteins having at least one unnatural
amino acid.
O-RS Species Finding Use with the Invention
[0125] The present invention provides novel orthogonal translation
systems, where the systems have the ability to produce polypeptides
comprising unnatural amino acids in mammalian host cells that have
not been previously contemplated, for example, primate and rodent
host cell systems. A translation system of the invention includes
(i) an unnatural amino acid, (ii) an orthogonal tRNA (O-tRNA),
(iii) an orthogonal aminoacyl-tRNA synthetase (O-RS) derived from
an E. coli aminoacyl-tRNA synthetase, and (iv) a mammalian host
cell that contains these components, where the mammalian host cell
can be, for example, a rodent cell (e.g., a mouse cell or a rat
cell), or a primate cell, (e.g., a human cell or a mouse cell). The
invention also provides methods for producing a protein having at
least one unnatural amino acid at a selected position, where the
methods use the translation systems described above. The methods
result in the incorporation of the unnatural amino acid in the
mammalian host cell by programming the incorporation of the
unnatural amino acid at a selector codon during protein
translation.
[0126] The O-RS species finding use with the invention are not
particularly limited, but generally has the following properties.
An O-RS finding use with the invention is generally derived from an
E. coli aminoacyl-tRNA synthetase, such as a wild-type E. coli
leucyl-tRNA synthetase (Ec LeuRS; amino acid sequence provided at
SEQ ID NO: 4, and polynucleotide sequence provided at SEQ ID NO: 5)
or a wild-type E. coli tyrosyl-tRNA synthetase (Ec TyrRS; amino
acid sequence provided at SEQ ID NO: 6, and polynucleotide sequence
provided at SEQ ID NO: 7). The mutant O-RS species finding use with
the invention (e.g., synthetases derived from the SEQ ID NOS: 4 or
6) are originally evolved and selected in a Saccharomyces
cerevisiae (S. cerevisiae) yeast host cell system.
[0127] A large number of such O-RS species that find use with the
invention are known in the art. In S. cerevisiae, functional
tRNA.sub.CUA/RS pairs have been successfully derived from the
corresponding E. coli tRNA.sup.Tyr/TyrRS and
tRNA.sup.Leu/leucyl-tRNA synthetase pairs. See, for example, Chin
et al., "An Expanded Eukaryotic Genetic Code," Science 301, 964-967
(2003); Chin et al., "Progress Toward an Expanded Eukaryotic
Genetic Code," Chem Biol 10, 511-519 (2003); Deiters et al. (2003),
"Adding amino acids with novel reactivity to the genetic code of
Saccharomyces cerevisiae," J Am Chem Soc 125:11782-11783; Summerer
et al. (2006), "A Genetically Encoded Fluorescent Amino Acid," PNAS
103(26):9785-9789; and Wu et al., "A Genetically Encoded Photocaged
Amino Acid," J Am Chem Soc 126, 14306-14307 (2004)). See also WO
2005/003294 to Deiters et al., "UNNATURAL REACTIVE AMINO ACID
GENETIC CODE ADDITIONS," filed Apr. 16, 2004; WO 2006/034410 to
Deiters et al., "ADDING PHOTOREGULATED AMINO ACIDS TO THE GENETIC
CODE," filed Sep. 21, 2005; and WO 2006/110182, filed Oct. 27,
2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS." Each of these references
is incorporated by reference in their entirety. This teaching in
the art also provides sufficient guidance for the construction of
additional O-RS species that find use with the invention.
[0128] Examples of O-RS species that find use with the invention
are provided in FIG. 16. The polynucleotide sequences that encode
the O-RSs are provided in SEQ ID NOS: 8-56. The corresponding O-RS
polypeptide sequences are provided in SEQ ID NOS: 57-101. It is
noted that a full length O-RS is not necessarily required for the
RS to be operative in the invention. Expression of only an RS
active site fragment can be sufficient for use with the invention,
as shown in some of the sequences in FIG. 16. Each of the O-RS
sequences in FIG. 16 is known in the art, as described in the
references cited herein.
[0129] It is not intended that the invention be limited to the use
of the O-RS provided in FIG. 16. Any O-RS species identified herein
that finds use with the invention can serve as a template for the
derivation is still other O-RS species that can be used with the
invention, for example, by making conservative amino acid
substitutions within the existing O-RS species. The number of
substitutions that are allowed is not limited, with the condition
that the newly derived O-RS retains biological activity to
preferentially aminoacylate the corresponding O-tRNA with the
unnatural amino acid.
[0130] The biological activity of an O-RS can be expressed as a
percentage of the biological activity of another "reference" O-RS
that is shown to be of use with the invention. For example, an O-RS
finding use with the invention can include an O-RS that
preferentially aminoacylates an O-tRNA with an unnatural amino acid
with an efficiency that is at least 50% of the efficiency observed
for a "reference" translation system comprising the O-tRNA, the
unnatural amino acid, and an aminoacyl-tRNA synthetase (RS) of
known sequence. The selection of the threshold activity is
arbitrary, and the value of "50%" serves only as an example.
Indeed, an O-RS finding use with the invention can include an O-RS
that preferentially aminoacylates an O-tRNA with an unnatural amino
acid with an efficiency that is at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 98% or 99% of the efficiency observed for a
"reference" translation system.
O-tRNA Species Finding Use with the Invention
[0131] The mutant O-RS species finding use with the invention are
originally evolved and selected in a yeast host cell system. The
O-RS species that are evolved in the yeast host cell system
function in conjunction with a suitable O-tRNA that is
preferentially aminoacylated by the O-RS with the unnatural amino
acid. For example, suppressor O-tRNA species that function in the
yeast host system can be, e.g., the tRNA of SEQ ID NOS: 1 or 2.
[0132] In contrast, the translation systems of the present
invention use a different O-tRNA that is orthogonal in a mammalian
host cell (such as a rodent cell or a primate cell). For example,
an O-tRNA finding use with the invention can be the Bacillus
stearothermophilus amber suppressor tyrosyl-tRNA.sub.CUA
(Bs-tRNATyrCUA), as provided in SEQ ID NO: 3. It is not intended
that the invention be limited to this one tRNA species, as variants
of this tRNA sequence, other tRNAs derived from Bacillus
stearothermophilus, as well as tRNA species derived from other
organisms (e.g., other eubacterial species) are also
contemplated.
Orthogonal tRNA/Aminoacyl-tRNA Synthetase Technology
[0133] An understanding of the novel compositions and methods of
the present invention requires an understanding of the activities
associated with orthogonal tRNA and orthogonal aminoacyl-tRNA
synthetase pairs. In order to add additional unnatural amino acids
to the genetic code, new orthogonal pairs comprising an
aminoacyl-tRNA synthetase and a suitable tRNA are needed that can
function efficiently in the host translational machinery, but that
are "orthogonal" to the translation system at issue, meaning that
it functions independently of the synthetases and tRNAs endogenous
to the translation system. Desired characteristics of the
orthogonal pair include tRNA that decode or recognize only a
specific codon, e.g., a selector codon, that is not decoded by any
endogenous tRNA, and aminoacyl-tRNA synthetases that preferentially
aminoacylate (or "charge") its cognate tRNA with only one specific
unnatural amino acid. The O-tRNA is also not typically
aminoacylated (or is poorly aminoacylated, i.e., charged) by
endogenous synthetases. For example, in an E. coli host system, an
orthogonal pair will include an aminoacyl-tRNA synthetase that does
not cross-react with any of the endogenous tRNA, e.g., which there
are 40 in E. coli, and an orthogonal tRNA that is not aminoacylated
by any of the endogenous synthetases, e.g., of which there are 21
in E. coli.
[0134] The general principles of orthogonal translation systems
that are suitable for making proteins that comprise one or more
unnatural amino acid are known in the art, as are the general
methods for producing orthogonal translation systems. For example,
see International Publication Numbers WO 2002/086075, entitled
"METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL
tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN
VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593,
entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415,
filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO
2005/007624, filed Jul. 7, 2004; International Application No.
PCT/US2005/039210, filed on Oct. 27, 2005, entitled "ORTHOGONAL
TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL
AMINO ACIDS;" and U.S. Provisional Application Ser. No. 60/783,497,
entitled "SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATION
COMPONENTS IN EUBACTERIAL HOST CELLS," filed Mar. 17, 2006. Each of
these applications is incorporated herein by reference in its
entirety. For discussion of orthogonal translation systems that
incorporate unnatural amino acids, and methods for their production
and use, see also, Wang and Schultz "Expanding the Genetic Code,"
Angewandte Chemie Int. Ed., 44(1):34-66 (2005), Xie and Schultz,
"An Expanding Genetic Code," Methods 36(3):227-238 (2005); Xie and
Schultz, "Adding Amino Acids to the Genetic Repertoire," Curr.
Opinion in Chemical Biology 9(6):548-554 (2005); and Wang et al.,
"Expanding the Genetic Code," Annu. Rev. Biophys. Biomol. Struct.,
35:225-249 (2006); the contents of which are each incorporated by
reference in their entirety.
[0135] Orthogonal Translation Systems
[0136] Orthogonal translation systems generally comprise cells
(which can be mammalian cells such as rodent cells or primate
cells) that include an orthogonal tRNA (O-tRNA), an orthogonal
aminoacyl tRNA synthetase (O-RS), and an unnatural amino acid,
where the O-RS aminoacylates the O-tRNA with the unnatural amino
acid. An orthogonal pair of the invention can include an O-tRNA,
e.g., a suppressor tRNA, a frameshift tRNA, or the like, and a
cognate O-RS. The orthogonal systems of the invention can typically
comprise O-tRNA/O-RS pairs, either in the context of a host cell or
without the host cell. In addition to multi-component systems, the
invention also provides individual components, for example,
orthogonal aminoacyl-tRNA synthetase polypeptides (e.g., SEQ ID
NOs: 57-101), and the polynucleotides that encodes those
polypeptides (e.g., SEQ ID NOs: 8-56).
[0137] In general, when an orthogonal pair recognizes a selector
codon and loads an amino acid in response to the selector codon,
the orthogonal pair is said to "suppress" the selector codon. That
is, a selector codon that is not recognized by the translation
system's (e.g., the cell's) endogenous machinery is not ordinarily
charged, which results in blocking production of a polypeptide that
would otherwise be translated from the nucleic acid. In an
orthogonal pair system, the O-RS aminoacylates the O-tRNA with a
specific unnatural amino acid. The charged O-tRNA recognizes the
selector codon and suppresses the translational block caused by the
selector codon.
[0138] In some aspects, an O-tRNA of the invention recognizes a
selector codon and includes at least about, e.g., a 45%, a 50%, a
60%, a 75%, a 80%, or a 90% or more suppression efficiency in the
presence of a cognate synthetase in response to a selector codon as
compared to the suppression efficiency of an O-tRNA comprising or
encoded by a polynucleotide sequence as set forth in the sequence
listing herein.
[0139] In some embodiments, the suppression efficiency of the O-RS
and the O-tRNA together is about, e.g., 5 fold, 10 fold, 15 fold,
20 fold, or 25 fold or more greater than the suppression efficiency
of the O-tRNA lacking the O-RS. In some aspect, the suppression
efficiency of the O-RS and the O-tRNA together is at least about,
e.g., 35%, 40%, 45%, 50%, 60%, 75%, 80%, or 90% or more of the
suppression efficiency of an orthogonal synthetase pair as set
forth in the sequence listings herein.
[0140] The host cell uses the O-tRNA/O-RS pair to incorporate the
unnatural amino acid into a growing polypeptide chain, e.g., via a
nucleic acid that comprises a polynucleotide that encodes a
polypeptide of interest, where the polynucleotide comprises a
selector codon that is recognized by the O-tRNA. In certain
preferred aspects, the cell can include one or more additional
O-tRNA/O-RS pairs, where the additional O-tRNA is loaded by the
additional O-RS with a different unnatural amino acid. For example,
one of the O-tRNAs can recognize a four base codon and the other
O-tRNA can recognize a stop codon. Alternately, multiple different
stop codons or multiple different four base codons can be used in
the same coding nucleic acid.
[0141] As noted, in some embodiments, there exists multiple
O-tRNA/O-RS pairs in a cell or other translation system, which
allows incorporation of more than one unnatural amino acid into a
polypeptide. For example, the cell can further include an
additional different O-tRNA/O-RS pair and a second unnatural amino
acid, where this additional O-tRNA recognizes a second selector
codon and this additional O-RS preferentially aminoacylates the
O-tRNA with the second unnatural amino acid. For example, a cell
that includes an O-tRNA/O-RS pair (where the O-tRNA recognizes,
e.g., an amber selector codon), can further comprise a second
orthogonal pair, where the second O-tRNA recognizes a different
selector codon, e.g., an opal codon, a four-base codon, or the
like. Desirably, the different orthogonal pairs are derived from
different sources, which can facilitate recognition of different
selector codons.
[0142] In certain embodiments, systems comprise a cell such as a
mammalian cell, including but not limited to rodent and primate
cells, that includes an orthogonal tRNA (O-tRNA), an orthogonal
aminoacyl-tRNA synthetase (O-RS), an unnatural amino acid and a
nucleic acid that comprises a polynucleotide that encodes a
polypeptide of interest, where the polynucleotide comprises the
selector codon that is recognized by the O-tRNA. The translation
system can also be a cell-free system, e.g., any of a variety of
commercially available "in vitro" transcription/translation systems
in combination with an O-tRNA/O-RS pair and an unnatural amino acid
as described herein.
[0143] The O-tRNA and/or the O-RS can be naturally occurring or can
be, e.g., derived by mutation of a naturally occurring tRNA and/or
RS, e.g., by generating libraries of tRNAs and/or libraries of RSs,
from any of a variety of organisms and/or by using any of a variety
of available mutation strategies. For example, one strategy for
producing an orthogonal tRNA/aminoacyl-tRNA synthetase pair
involves importing a heterologous (to the host cell)
tRNA/synthetase pair from, e.g., a source other than the host cell,
or multiple sources, into the host cell. The properties of the
heterologous synthetase candidate include, e.g., that it does not
charge any host cell tRNA, and the properties of the heterologous
tRNA candidate include, e.g., that it is not aminoacylated by any
host cell synthetase. In addition, the heterologous tRNA is
orthogonal to all host cell synthetases. A second strategy for
generating an orthogonal pair involves generating mutant libraries
from which to screen and/or select an O-tRNA or O-RS. These
strategies can also be combined.
[0144] Orthogonal tRNA (O-tRNA)
[0145] An orthogonal tRNA (O-tRNA) of the invention desirably
mediates incorporation of an unnatural amino acid into a protein
that is encoded by a polynucleotide that comprises a selector codon
that is recognized by the O-tRNA, e.g., in vivo or in vitro. In
certain embodiments, an O-tRNA of the invention includes at least
about, e.g., a 45%, a 50%, a 60%, a 75%, a 80%, or a 90% or more
suppression efficiency in the presence of a cognate synthetase in
response to a selector codon as compared to an O-tRNA comprising or
encoded by a polynucleotide sequence as set forth in the O-tRNA
sequences in the sequence listing herein.
[0146] Suppression efficiency can be determined by any of a number
of assays known in the art. For example, a .beta.-galactosidase
reporter assay can be used, e.g., a derivatized lacZ plasmid (where
the construct has a selector codon in the lacZ nucleic acid
sequence) is introduced into cells from an appropriate organism
(e.g., an organism where the orthogonal components can be used)
along with plasmid comprising an O-tRNA of the invention. A cognate
synthetase can also be introduced (either as a polypeptide or a
polynucleotide that encodes the cognate synthetase when expressed).
The cells are grown in media to a desired density, e.g., to an
OD.sub.600 of about 0.5, and .beta.-galactosidase assays are
performed, e.g., using the BetaFluor.TM. .beta.-Galactosidase Assay
Kit (Novagen). Percent suppression can be calculated as the
percentage of activity for a sample relative to a comparable
control, e.g., the value observed from the derivatized lacZ
construct, where the construct has a corresponding sense codon at
desired position rather than a selector codon.
[0147] Examples of O-tRNAs finding use with the invention are set
forth in the sequence listing herein, for example, see FIG. 16 and
SEQ ID NO: 3. The disclosure herein also provides guidance for the
design of additional equivalent O-tRNA species. In an RNA molecule,
such as an O-RS mRNA, or O-tRNA molecule, Thymine (T) is replace
with Uracil (U) relative to a given sequence (or vice versa for a
coding DNA), or complement thereof. Additional modifications to the
bases can also be present to generate largely functionally
equivalent molecules.
[0148] The invention also encompasses conservative variations of
O-tRNAs corresponding to particular O-tRNAs herein. For example,
conservative variations of O-tRNA include those molecules that
function like the particular O-tRNAs, e.g., as in the sequence
listing herein and that maintain the tRNA L-shaped structure by
virtue of appropriate self-complementarity, but that do not have a
sequence identical to those, e.g., in the sequence listing or FIG.
16, and desirably, are other than wild type tRNA molecules.
[0149] The composition comprising an O-tRNA can further include an
orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS
preferentially aminoacylates the O-tRNA with an unnatural amino
acid. In certain embodiments, a composition including an O-tRNA can
further include a translation system (e.g., in vitro or in vivo). A
nucleic acid that comprises a polynucleotide that encodes a
polypeptide of interest, where the polynucleotide comprises a
selector codon that is recognized by the O-tRNA, or a combination
of one or more of these can also be present in the cell.
[0150] Methods of producing an orthogonal tRNA (O-tRNA) are also a
feature of the invention. An O-tRNA produced by the method is also
a feature of the invention. In certain embodiments of the
invention, the O-tRNAs can be produced by generating a library of
mutants. The library of mutant tRNAs can be generated using various
mutagenesis techniques known in the art. For example, the mutant
tRNAs can be generated by site-specific mutations, random point
mutations, homologous recombination, DNA shuffling or other
recursive mutagenesis methods, chimeric construction or any
combination thereof, e.g., of the O-tRNA of SEQ ID NO: 3.
[0151] Additional mutations can be introduced at a specific
position(s), e.g., at a nonconservative position(s), or at a
conservative position, at a randomized position(s), or a
combination of both in a desired loop or region of a tRNA, e.g., an
anticodon loop, the acceptor stem, D arm or loop, variable loop,
TPC arm or loop, other regions of the tRNA molecule, or a
combination thereof. Typically, mutations in a tRNA include
mutating the anticodon loop of each member of the library of mutant
tRNAs to allow recognition of a selector codon. The method can
further include adding additional sequences to the O-tRNA.
Typically, an O-tRNA possesses an improvement of orthogonality for
a desired organism compared to the starting material, e.g., the
plurality of tRNA sequences, while preserving its affinity towards
a desired RS.
[0152] The methods optionally include analyzing the similarity
(and/or inferred homology) of sequences of tRNAs and/or
aminoacyl-tRNA synthetases to determine potential candidates for an
O-tRNA, O-RS and/or pairs thereof, that appear to be orthogonal for
a specific organism. Computer programs known in the art and
described herein can be used for the analysis, e.g., BLAST and
pileup programs can be used. In one example, to choose potential
orthogonal translational components for use in mammalian cells such
as rodent cells and primate cells, a synthetase and/or a tRNA is
chosen that does not display close sequence similarity to the host
organisms.
[0153] Typically, an O-tRNA is obtained by subjecting to, e.g.,
negative selection, a population of cells of a first species, where
the cells comprise a member of the plurality of potential O-tRNAs.
The negative selection eliminates cells that comprise a member of
the library of potential O-tRNAs that is aminoacylated by an
aminoacyl-tRNA synthetase (RS) that is endogenous to the cell. This
provides a pool of tRNAs that are orthogonal to the cell of the
first species.
[0154] In certain embodiments, in the negative selection, a
selector codon(s) is introduced into a polynucleotide that encodes
a negative selection marker, e.g., an enzyme that confers
antibiotic resistance, e.g., .beta.-lactamase, an enzyme that
confers a detectable product, e.g., .beta.-galactosidase,
chloramphenicol acetyltransferase (CAT), e.g., a toxic product,
such as barnase, at a nonessential position (e.g., still producing
a functional barnase), etc. Screening/selection is optionally done
by growing the population of cells in the presence of a selective
agent (e.g., an antibiotic, such as ampicillin). In one embodiment,
the concentration of the selection agent is varied.
[0155] For example, to measure the activity of suppressor tRNAs, a
selection system is used that is based on the in vivo suppression
of selector codon, e.g., nonsense (e.g., stop) or frameshift
mutations introduced into a polynucleotide that encodes a negative
selection marker, e.g., a gene for .beta.-lactamase (bla). For
example, polynucleotide variants, e.g., bla variants, with a
selector codon at a certain position (e.g., A184), are constructed.
Cells, e.g., bacteria, are transformed with these polynucleotides.
In the case of an orthogonal tRNA, which cannot be efficiently
charged by endogenous E. coli synthetases, antibiotic resistance,
e.g., ampicillin resistance, should be about or less than that for
a bacteria transformed with no plasmid. If the tRNA is not
orthogonal, or if a heterologous synthetase capable of charging the
tRNA is co-expressed in the system, a higher level of antibiotic,
e.g., ampicillin, resistance is be observed. Cells, e.g., bacteria,
are chosen that are unable to grow on LB agar plates with
antibiotic concentrations about equal to cells transformed with no
plasmids.
[0156] In the case of a toxic product (e.g., ribonuclease or
barnase), when a member of the plurality of potential tRNAs is
aminoacylated by endogenous host, e.g., Escherichia coli
synthetases (i.e., it is not orthogonal to the host, e.g.,
Escherichia coli synthetases), the selector codon is suppressed and
the toxic polynucleotide product produced leads to cell death.
Cells harboring orthogonal tRNAs or non-functional tRNAs
survive.
[0157] In one embodiment, the pool of tRNAs that are orthogonal to
a desired organism are then subjected to a positive selection in
which a selector codon is placed in a positive selection marker,
e.g., encoded by a drug resistance gene, such a .beta.-lactamase
gene. The positive selection is performed on a cell comprising a
polynucleotide encoding or comprising a member of the pool of tRNAs
that are orthogonal to the cell, a polynucleotide encoding a
positive selection marker, and a polynucleotide encoding a cognate
RS. In certain embodiments, the second population of cells
comprises cells that were not eliminated by the negative selection.
The polynucleotides are expressed in the cell and the cell is grown
in the presence of a selection agent, e.g., ampicillin. tRNAs are
then selected for their ability to be aminoacylated by the
coexpressed cognate synthetase and to insert an amino acid in
response to this selector codon. Typically, these cells show an
enhancement in suppression efficiency compared to cells harboring
non-functional tRNA(s), or tRNAs that cannot efficiently be
recognized by the synthetase of interest. The cell harboring the
non-functional tRNAs or tRNAs that are not efficiently recognized
by the synthetase of interest, are sensitive to the antibiotic.
Therefore, tRNAs that: (i) are not substrates for endogenous host,
e.g., Escherichia coli, synthetases; (ii) can be aminoacylated by
the synthetase of interest; and (iii) are functional in
translation, survive both selections.
[0158] Accordingly, the same marker can be either a positive or
negative marker, depending on the context in which it is screened.
That is, the marker is a positive marker if it is screened for, but
a negative marker if screened against.
[0159] The stringency of the selection, e.g., the positive
selection, the negative selection or both the positive and negative
selection, in the above described-methods, optionally includes
varying the selection stringency. For example, because barnase is
an extremely toxic protein, the stringency of the negative
selection can be controlled by introducing different numbers of
selector codons into the barnase gene and/or by using an inducible
promoter. In another example, the concentration of the selection or
screening agent is varied (e.g., ampicillin concentration). In some
aspects of the invention, the stringency is varied because the
desired activity can be low during early rounds. Thus, less
stringent selection criteria are applied in early rounds and more
stringent criteria are applied in later rounds of selection. In
certain embodiments, the negative selection, the positive selection
or both the negative and positive selection can be repeated
multiple times. Multiple different negative selection markers,
positive selection markers or both negative and positive selection
markers can be used. In certain embodiments, the positive and
negative selection marker can be the same.
[0160] Other types of selections/screening can be used in the
invention for producing orthogonal translational components, e.g.,
an O-tRNA, an O-RS, and an O-tRNA/O--RS pair that loads an
unnatural amino acid in response to a selector codon. For example,
the negative selection marker, the positive selection marker or
both the positive and negative selection markers can include a
marker that fluoresces or catalyzes a luminescent reaction in the
presence of a suitable reactant. In another embodiment, a product
of the marker is detected by fluorescence-activated cell sorting
(FACS) or by luminescence. Optionally, the marker includes an
affinity based screening marker. See also, Francisco, J. A., et
al., (1993) Production and fluorescence-activated cell sorting of
Escherichia coli expressing a functional antibody fragment on the
external surface. Proc Natl Acad Sci USA. 90:10444-8.
[0161] Additional methods for producing a recombinant orthogonal
tRNA can be found, e.g., in International Application Publications
WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE
PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;" WO
2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" and
WO 2005/019415, filed Jul. 7, 2004. See also Forster et al., (2003)
Programming peptidomimetic synthetases by translating genetic codes
designed de novo PNAS 100(11):6353-6357; and, Feng et al., (2003),
Expanding tRNA recognition of a tRNA synthetase by a single amino
acid change, PNAS 100(10): 5676-5681.
[0162] Orthogonal Aminoacyl-tRNA Synthetase (O-RS)
[0163] An O-RS of the invention preferentially aminoacylates an
O-tRNA with an unnatural amino acid, in vitro or in vivo. An O-RS
of the invention can be provided to the translation system, e.g., a
cell, by a polypeptide that includes an O-RS and/or by a
polynucleotide that encodes an O-RS or a portion thereof. For
example, an example O-RS comprises an amino acid sequence selected
from SEQ ID NOs: 57-101, or a conservative variation thereof. In
another example, an O-RS, or a portion thereof, is encoded by a
polynucleotide sequence that encodes an amino acid sequence in the
sequence listing or examples herein, or a complementary
polynucleotide sequence thereof. See, e.g., the polynucleotides of
SEQ ID NOs: 8-56.
[0164] Methods for identifying an orthogonal aminoacyl-tRNA
synthetase (O-RS), e.g., an O-RS, for use with an O-tRNA, are also
a feature of the invention. For example, a method includes
subjecting to selection, e.g., positive selection, a population of
cells of a first species, where the cells individually comprise: 1)
a member of a plurality of aminoacyl-tRNA synthetases (RSs), (e.g.,
the plurality of RSs can include mutant RSs, RSs derived from a
species other than the first species or both mutant RSs and RSs
derived from a species other than the first species); 2) the
orthogonal tRNA (O-tRNA) (e.g., from one or more species); and 3) a
polynucleotide that encodes an (e.g., positive) selection marker
and comprises at least one selector codon. Cells are selected or
screened for those that show an enhancement in suppression
efficiency compared to cells lacking or with a reduced amount of
the member of the plurality of RSs. Suppression efficiency can be
measured by techniques known in the art and as described herein.
Cells having an enhancement in suppression efficiency comprise an
active RS that aminoacylates the O-tRNA. A level of aminoacylation
(in vitro or in vivo) by the active RS of a first set of tRNAs from
the first species is compared to the level of aminoacylation (in
vitro or in vivo) by the active RS of a second set of tRNAs from
the second species. The level of aminoacylation can be determined
by a detectable substance (e.g., a labeled unnatural amino acid).
The active RS that more efficiently aminoacylates the second set of
tRNAs compared to the first set of tRNAs is typically selected,
thereby providing an efficient (e.g., optimized) orthogonal
aminoacyl-tRNA synthetase for use with the O-tRNA. An O-RS,
identified by the method, is also a feature of the invention.
[0165] Any of a number of assays can be used to determine
aminoacylation. These assays can be performed in vitro or in vivo.
For example, in vitro aminoacylation assays are described in, e.g.,
Hoben and Soil (1985) Methods Enzymol. 113:55-59. Aminoacylation
can also be determined by using a reporter along with orthogonal
translation components and detecting the reporter in a cell
expressing a polynucleotide comprising at least one selector codon
that encodes a protein. See also, WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" and WO 2004/094593,
entitiled "EXPANDING THE EUKARYOTIC GENETIC CODE."
[0166] Identified O-RS can be further manipulated to alter
substrate specificity of the synthetase, so that only a desired
unnatural amino acid, but not any of the common 20 amino acids, are
charged to the O-tRNA. Methods to generate an orthogonal
aminoacyl-tRNA synthetase with a substrate specificity for an
unnatural amino acid include mutating the synthetase, e.g., at the
active site in the synthetase, at the editing mechanism site in the
synthetase, at different sites by combining different domains of
synthetases, or the like, and applying a selection process. A
strategy is used, which is based on the combination of a positive
selection followed by a negative selection. In the positive
selection, suppression of the selector codon introduced at a
nonessential position(s) of a positive marker allows cells to
survive under positive selection pressure. In the presence of both
natural and unnatural amino acids, survivors thus encode active
synthetases charging the orthogonal suppressor tRNA with either a
natural or unnatural amino acid. In the negative selection,
suppression of a selector codon introduced at a nonessential
position(s) of a negative marker removes synthetases with natural
amino acid specificities. Survivors of the negative and positive
selection encode synthetases that aminoacylate (charge) the
orthogonal suppressor tRNA with unnatural amino acids only. These
synthetases can then be subjected to further mutagenesis, e.g., DNA
shuffling or other recursive mutagenesis methods.
[0167] A library of mutant O-RSs can be generated using various
mutagenesis techniques known in the art. For example, the mutant
RSs can be generated by site-specific mutations, random point
mutations, homologous recombination, DNA shuffling or other
recursive mutagenesis methods, chimeric construction or any
combination thereof. For example, a library of mutant RSs can be
produced from two or more other, e.g., smaller, less diverse
"sub-libraries." Chimeric libraries of RSs are also included in the
invention. It should be noted that libraries of tRNA synthetases
from various organism (e.g., microorganisms such as eubacteria or
archaebacteria) such as libraries that comprise natural diversity
(see, e.g., U.S. Pat. No. 6,238,884 to Short et al; U.S. Pat. No.
5,756,316 to Schallenberger et al; U.S. Pat. No. 5,783,431 to
Petersen et al; U.S. Pat. No. 5,824,485 to Thompson et al; U.S.
Pat. No. 5,958,672 to Short et al), are optionally constructed and
screened for orthogonal pairs.
[0168] Once the synthetases are subject to the positive and
negative selection/screening strategy, these synthetases can then
be subjected to further mutagenesis. For example, a nucleic acid
that encodes the O-RS can be isolated; a set of polynucleotides
that encode mutated O-RSs (e.g., by random mutagenesis,
site-specific mutagenesis, recombination or any combination
thereof) can be generated from the nucleic acid; and, these
individual steps or a combination of these steps can be repeated
until a mutated O-RS is obtained that preferentially aminoacylates
the O-tRNA with the unnatural amino acid. In some aspects of the
invention, the steps are performed multiple times, e.g., at least
two times.
[0169] Additional levels of selection/screening stringency can also
be used in the methods of the invention, for producing O-tRNA,
O-RS, or pairs thereof. The selection or screening stringency can
be varied on one or both steps of the method to produce an O-RS.
This could include, e.g., varying the amount of selection/screening
agent that is used, etc. Additional rounds of positive and/or
negative selections can also be performed. Selecting or screening
can also comprise one or more of a change in amino acid
permeability, a change in translation efficiency, a change in
translational fidelity, etc. Typically, the one or more change is
based upon a mutation in one or more gene in an organism in which
an orthogonal tRNA-tRNA synthetase pair is used to produce
protein.
[0170] Additional general details for producing O-RS, and altering
the substrate specificity of the synthetase can be found in
Internal Publication Number WO 2002/086075, entitled "METHODS AND
COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA
SYNTHETASE PAIRS; " and WO 2004/094593, entitled "EXPANDING THE
EUKARYOTIC GENETIC CODE." See also, Wang and Schultz "Expanding the
Genetic Code," Angewandte Chemie Int. Ed., 44(1):34-66 (2005), the
content of which is incorporated by reference in its entirety.
Source and Host Organisms
[0171] The orthogonal translational components (O-tRNA and O-RS) of
the invention can be derived from any organism (or a combination of
organisms) for use in a host translation system from any other
species, with the caveat that the O-tRNA/O-RS components and the
host system work in an orthogonal manner. It is not a requirement
that the O-tRNA and the O-RS from an orthogonal pair be derived
from the same organism. In some aspects, the orthogonal components
are derived from E. coli genes (i.e., eubacteria) for use in
mammalian host systems (e.g., rodent or primate host systems).
[0172] For example, the orthogonal O-tRNA can be derived from an
Archae organism, e.g., an archaebacterium, such as Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Halobacterium
such as Haloferax volcanii and Halobacterium species NRC-1,
Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii,
Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus
kandleri, Methanosarcina mazei (Mm), Pyrobaculum aerophilum,
Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus
tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the
like, or a eubacterium, such as Escherichia coli, Thermus
thermophilus, Bacillus subtilis, Bacillus stearothermphilus, or the
like, while the orthogonal O-RS can be derived from an organism or
combination of organisms, e.g., an archaebacterium, such as
Methanococcus jannaschii, Methanobacterium thermoautotrophicum,
Halobacterium such as Haloferax volcanii and Halobacterium species
NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus
horikoshii, Aeuropyrum pernix, Methanococcus maripaludis,
Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum
aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus
tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the
like, or a eubacterium, such as Escherichia coli, Thermus
thermophilus, Bacillus subtilis, Bacillus stearothermphilus, or the
like. In one embodiment, eukaryotic sources, e.g., plants, algae,
protists, fungi, yeasts, animals (e.g., mammals, insects,
arthropods, etc.), or the like, can also be used as sources of
O-tRNAs and O-RSs.
[0173] The individual components of an O-tRNA/O-RS pair can be
derived from the same organism or different organisms. In one
embodiment, the O-tRNA/O-RS pair is from the same organism.
Alternatively, the O-tRNA and the O-RS of the O-tRNA/O-RS pair are
from different organisms.
[0174] The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or
screened in vivo or in vitro and/or used in a cell, e.g., a
mammalian cell such as a rodent cell or primate cell, to produce a
polypeptide with an unnatural amino acid. The mammalian host cell
used is not particularly limited. Compositions of mammalian cells
comprising translational components of the invention are also a
feature of the invention.
[0175] See also, International Application Publication Number WO
2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE,"
filed Apr. 16, 2004, for screening O-tRNA and/or O-RS in one
species for use in another species.
[0176] Although orthogonal translation systems (e.g., comprising an
O-RS, an O-tRNA and an unnatural amino acid) can utilize cultured
host cells to produce proteins having unnatural amino acids, it is
not intended that an orthogonal translation system of the invention
require an intact, viable host cell. For example, a orthogonal
translation system can utilize a cell-free system in the presence
of a cell extract. Indeed, the use of cell free, in vitro
transcription/translation systems for protein production is a well
established technique. Adaptation of these in vitro systems to
produce proteins having unnatural amino acids using orthogonal
translation system components described herein is well within the
scope of the invention.
Selector Codons
[0177] Selector codons of the invention expand the genetic codon
framework of protein biosynthetic machinery. For example, a
selector codon includes, e.g., a unique three base codon, a
nonsense codon, such as a stop codon, e.g., an amber codon (UAG),
or an opal codon (UGA), an unnatural codon, at least a four base
codon, a rare codon, or the like. A number of selector codons can
be introduced into a desired gene, e.g., one or more, two or more,
more than three, etc. By using different selector codons, multiple
orthogonal tRNA/synthetase pairs can be used that allow the
simultaneous site-specific incorporation of multiple unnatural
amino acids e.g., including at least one unnatural amino acid,
using these different selector codons.
[0178] In one embodiment, the methods involve the use of a selector
codon that is a stop codon for the incorporation of an unnatural
amino acid in vivo in a cell. For example, an O-tRNA is produced
that recognizes the stop codon and is aminoacylated by an O-RS with
an unnatural amino acid. This O-tRNA is not recognized by the
naturally occurring host's aminoacyl-tRNA synthetases. Conventional
site-directed mutagenesis can be used to introduce the stop codon
at the site of interest in a polynucleotide encoding a polypeptide
of interest. See, e.g., Sayers, J. R., et al. (1988), 5',3'
Exonuclease in phosphorothioate-based oligonucleotide-directed
mutagenesis. Nucleic Acids Res, 791-802. When the O-RS, O-tRNA and
the nucleic acid that encodes a polypeptide of interest are
combined, e.g., in vivo, the unnatural amino acid is incorporated
in response to the stop codon to give a polypeptide containing the
unnatural amino acid at the specified position. In one embodiment
of the invention, the stop codon used as a selector codon is an
amber codon, UAG, and/or an opal codon, UGA. In one example, a
genetic code in which UAG and UGA are both used as a selector codon
can encode 22 amino acids while preserving the ochre nonsense
codon, UAA, which is the most abundant termination signal.
[0179] The incorporation of unnatural amino acids in vivo can be
done without significant perturbation of the host cell. For example
in non-eukaryotic cells, such as Escherichia coli, because the
suppression efficiency for the UAG codon depends upon the
competition between the O-tRNA, e.g., the amber suppressor tRNA,
and the release factor 1 (RF1) (which binds to the UAG codon and
initiates release of the growing peptide from the ribosome), the
suppression efficiency can be modulated by, e.g., either increasing
the expression level of O-tRNA, e.g., the suppressor tRNA, or using
an RF1 deficient strain. In eukaryotic cells, because the
suppression efficiency for the UAG codon depends upon the
competition between the O-tRNA, e.g., the amber suppressor tRNA,
and a eukaryotic release factor (e.g., eRF) (which binds to a stop
codon and initiates release of the growing peptide from the
ribosome), the suppression efficiency can be modulated by, e.g.,
increasing the expression level of O-tRNA, e.g., the suppressor
tRNA. In addition, additional compounds can also be present, e.g.,
reducing agents such as dithiothretiol (DTT).
[0180] Unnatural amino acids can also be encoded with rare codons.
For example, when the arginine concentration in an in vitro protein
synthesis reaction is reduced, the rare arginine codon, AGG, has
proven to be efficient for insertion of Ala by a synthetic tRNA
acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939
(1993). In this case, the synthetic tRNA competes with the
naturally occurring tRNA.sup.Arg, which exists as a minor species
in Escherichia coli. In addition, some organisms do not use all
triplet codons. An unassigned codon AGA in Micrococcus luteus has
been utilized for insertion of amino acids in an in vitro
transcription/translation extract. See, e.g., Kowal and Oliver,
Nucl. Acid. Res. 25:4685 (1997). Components of the invention can be
generated to use these rare codons in vivo.
[0181] Selector codons can also comprise extended codons, e.g.,
four or more base codons, such as, four, five, six or more base
codons. Examples of four base codons include, e.g., AGGA, CUAG,
UAGA, CCCU, and the like. Examples of five base codons include,
e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.
Methods of the invention include using extended codons based on
frameshift suppression. Four or more base codons can insert, e.g.,
one or multiple unnatural amino acids, into the same protein. In
other embodiments, the anticodon loops can decode, e.g., at least a
four-base codon, at least a five-base codon, or at least a six-base
codon or more. Since there are 256 possible four-base codons,
multiple unnatural amino acids can be encoded in the same cell
using a four or more base codon. See also, Anderson et al., (2002)
Exploring the Limits of Codon and Anticodon Size, Chemistry and
Biology, 9:237-244; and, Magliery, (2001) 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. 307: 755-769.
[0182] For example, four-base codons have been used to incorporate
unnatural amino acids into proteins using in vitro biosynthetic
methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and
Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU
were used to simultaneously incorporate 2-naphthylalanine and an
NBD derivative of lysine into streptavidin in vitro with two
chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka
et al., (1999) J. Am. Chem. Soc., 121:12194. In an in vivo study,
Moore et al. examined the ability of tRNA.sup.Leu derivatives with
NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C),
and found that the quadruplet UAGA can be decoded by a tRNA.sup.Leu
with a UCUA anticodon with an efficiency of 13 to 26% with little
decoding in the 0 or -1 frame. See Moore et al., (2000) J. Mol.
Biol., 298:195. In one embodiment, extended codons based on rare
codons or nonsense codons can be used in invention, which can
reduce missense readthrough and frameshift suppression at other
unwanted sites. Four base codons have been used as selector codons
in a variety of orthogonal systems. See, e.g., WO 2005/019415; WO
2005/007870 and WO 2005/07624. See also, Wang and Schultz
"Expanding the Genetic Code," Angewandte Chemie Int. Ed.,
44(1):34-66 (2005), the content of which is incorporated by
reference in its entirety. While the examples below utilize an
amber selector codon, four or more base codons can be used as well,
by modifying the examples herein to include four-base O-tRNAs and
synthetases modified to include mutations similar to those
previously described for various unnatural amino acid O-RSs.
[0183] For a given system, a selector codon can also include one of
the natural three base codons, where the endogenous system does not
use (or rarely uses) the natural base codon. For example, this
includes a system that is lacking a tRNA that recognizes the
natural three base codon, and/or a system where the three base
codon is a rare codon.
[0184] Selector codons optionally include unnatural base pairs.
These unnatural base pairs further expand the existing genetic
alphabet. One extra base pair increases the number of triplet
codons from 64 to 125. Properties of third base pairs include
stable and selective base pairing, efficient enzymatic
incorporation into DNA with high fidelity by a polymerase, and the
efficient continued primer extension after synthesis of the nascent
unnatural base pair. Descriptions of unnatural base pairs which can
be adapted for methods and compositions include, e.g., Hirao, et
al., (2002) An unnatural base pair for incorporating amino acid
analogues into protein, Nature Biotechnology, 20:177-182. See also
Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other
relevant publications are listed below.
[0185] For in vivo usage, the unnatural nucleoside is membrane
permeable and is phosphorylated to form the corresponding
triphosphate. In addition, the increased genetic information is
stable and not destroyed by cellular enzymes. Previous efforts by
Benner and others took advantage of hydrogen bonding patterns that
are different from those in canonical Watson-Crick pairs, the most
noteworthy example of which is the iso-C:iso-G pair. See, e.g.,
Switzer et al., (1989) J. Am. Chem. Soc., 111:8322; and Piccirilli
et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem.
Biol., 4:602. These bases in general mispair to some degree with
natural bases and cannot be enzymatically replicated. Kool and
co-workers demonstrated that hydrophobic packing interactions
between bases can replace hydrogen bonding to drive the formation
of base pair. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and
Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In
an effort to develop an unnatural base pair satisfying all the
above requirements, Schultz, Romesberg and co-workers have
systematically synthesized and studied a series of unnatural
hydrophobic bases. A PICS:PICS self-pair is found to be more stable
than natural base pairs, and can be efficiently incorporated into
DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF).
See, e.g., McMinn et al., (1999) J. Am. Chem. Soc., 121:11586; and
Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A 3MN:3MN
self-pair can be synthesized by KF with efficiency and selectivity
sufficient for biological function. See, e.g., Ogawa et al., (2000)
J. Am. Chem. Soc., 122:8803. However, both bases act as a chain
terminator for further replication. A mutant DNA polymerase has
been recently evolved that can be used to replicate the PICS self
pair. In addition, a 7AI self pair can be replicated. See, e.g.,
Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase
pair, Dipic:Py, has also been developed, which forms a stable pair
upon binding Cu(II). See Meggers et al., (2000) J. Am. Chem. Soc.,
122:10714. Because extended codons and unnatural codons are
intrinsically orthogonal to natural codons, the methods of the
invention can take advantage of this property to generate
orthogonal tRNAs for them.
[0186] A translational bypassing system can also be used to
incorporate an unnatural amino acid in a desired polypeptide. In a
translational bypassing system, a large sequence is inserted into a
gene but is not translated into protein. The sequence contains a
structure that serves as a cue to induce the ribosome to hop over
the sequence and resume translation downstream of the
insertion.
Unnatural Amino Acids
[0187] As used herein, an unnatural amino acid refers to any amino
acid, modified amino acid, or amino acid analogue other than
selenocysteine and/or pyrrolysine and the following twenty
genetically encoded alpha-amino acids: alanine, arginine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
valine. The generic structure of an alpha-amino acid is illustrated
by Formula I:
##STR00001##
[0188] An unnatural amino acid is typically any structure having
Formula I wherein the R group is any substituent other than one
used in the twenty natural amino acids. See e.g., Biochemistry by
L. Stryer, 3.sup.rd ed. 1988, Freeman and Company, New York, for
structures of the twenty natural amino acids. Note that, the
unnatural amino acids of the invention can be naturally occurring
compounds other than the twenty alpha-amino acids above.
[0189] Because the unnatural amino acids of the invention typically
differ from the natural amino acids in side chain, the unnatural
amino acids form amide bonds with other amino acids, e.g., natural
or unnatural, in the same manner in which they are formed in
naturally occurring proteins. However, the unnatural amino acids
have side chain groups that distinguish them from the natural amino
acids.
[0190] Although the unnatural amino acids shown in FIG. 1 are of
primary interest in the Examples described herein, it is not
intended that the invention be strictly limited to these structure.
Indeed, a variety of easily-derived, structurally related analogs
can be readily produced that retain the principle characteristics
of the structures from which they were derived (i.e., the
structures shown in FIG. 1), and also are specifically recognized
by the aminoacyl-tRNA synthetases references herein (e.g., the
O-RSs of SEQ ID NOS: 57-101). It is intended that these related
amino acid analogues are within the scope of the invention.
[0191] In other unnatural amino acids, for example, R in Formula I
optionally comprises an alkyl-, aryl-, acyl-, hydrazine, cyano-,
halo-, hydrazide, alkenyl, ether, borate, boronate, phospho,
phosphono, phosphine, enone, imine, ester, hydroxylamine, amine,
and the like, or any combination thereof. Other unnatural amino
acids of interest include, but are not limited to, amino acids
comprising a photoactivatable cross-linker, spin-labeled amino
acids, fluorescent amino acids, metal binding amino acids,
metal-containing amino acids, radioactive amino acids, amino acids
with novel functional groups, amino acids that covalently or
noncovalently interact with other molecules, photocaged and/or
photoisomerizable amino acids, biotin or biotin-analogue containing
amino acids, keto containing amino acids, glycosylated amino acids,
a saccharide moiety attached to the amino acid side chain, amino
acids comprising polyethylene glycol or polyether, heavy atom
substituted amino acids, chemically cleavable or photocleavable
amino acids, amino acids with an elongated side chain as compared
to natural amino acids (e.g., polyethers or long chain
hydrocarbons, e.g., greater than about 5, greater than about 10
carbons, etc.), carbon-linked sugar-containing amino acids, amino
thioacid containing amino acids, and amino acids containing one or
more toxic moiety.
[0192] In another aspect, the invention provides unnatural amino
acids having the general structure illustrated by Formula IV
below:
##STR00002##
[0193] An unnatural amino acid having this structure is typically
any structure where R.sub.1 is a substituent used in one of the
twenty natural amino acids (e.g., tyrosine or phenylalanine) and
R.sub.2 is a substituent. Thus, this type of unnatural amino acid
can be viewed as a natural amino acid derivative.
[0194] In addition to the unnatural amino acid structures shown in
FIG. 1, unnatural amino acids can also optionally comprise modified
backbone structures, e.g., as illustrated by the structures of
Formula II and III:
##STR00003##
wherein Z typically comprises OH, NH.sub.2, SH, NH--R', or S--R'; X
and Y, which can be the same or different, typically comprise S or
O, and R and R', which are optionally the same or different, are
typically selected from the same list of constituents for the R
group described above for the unnatural amino acids having Formula
I as well as hydrogen. For example, unnatural amino acids of the
invention optionally comprise substitutions in the amino or
carboxyl group as illustrated by Formulas II and III. Unnatural
amino acids of this type include, but are not limited to,
.alpha.-hydroxy acids, .alpha.-thioacids
.alpha.-aminothiocarboxylates, e.g., with side chains corresponding
to the common twenty natural amino acids or unnatural side chains.
In addition, substitutions at the .alpha.-carbon optionally include
L, D, or .alpha.-.alpha.-disubstituted amino acids such as
D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and
the like. Other structural alternatives include cyclic amino acids,
such as proline analogues as well as 3, 4, 6, 7, 8, and 9 membered
ring proline analogues, .beta. and .gamma. amino acids such as
substituted .beta.-alanine and .gamma.-amino butyric acid.
[0195] In some aspects, the invention utilizes unnatural amino
acids in the L-configuration. However, it is not intended that the
invention be limited to the use of L-configuration unnatural amino
acids. It is contemplated that the D-enantiomers of these unnatural
amino acids also find use with the invention.
[0196] The unnatural amino acids finding use with the invention are
not strictly limited to the unnatural amino acids shown in FIG. 1.
One of skill in the art will recognize that a wide variety of
unnatural analogs of naturally occurring amino acids are easily
derived. For example, but not limited to, unnatural amino acids
derived from phenylalanine or tyrosine are readily produced.
Tyrosine analogs include, e.g., para-substituted tyrosines,
ortho-substituted tyrosines, and meta substituted tyrosines,
wherein the substituted tyrosine comprises an alkynyl group, acetyl
group, a benzoyl group, an amino group, a hydrazine, an
hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a
methyl group, a C.sub.6-C.sub.20 straight chain or branched
hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl
group, a polyether group, a nitro group, or the like. In addition,
multiply substituted aryl rings are also contemplated. Glutamine
analogs of the invention include, but are not limited to,
.alpha.-hydroxy derivatives, .gamma.-substituted derivatives,
cyclic derivatives, and amide substituted glutamine derivatives.
Example phenylalanine analogs include, but are not limited to,
para-substituted phenylalanines, ortho-substituted phenylalanines,
and meta-substituted phenylalanines, wherein the substituent
comprises an alkynyl group, a hydroxy group, a methoxy group, a
methyl group, an allyl group, an aldehyde, a nitro, a thiol group,
or keto group, or the like. Specific examples of unnatural amino
acids include, but are not limited to, sulfotyrosine,
p-ethylthiocarbonyl-L-phenylalanine,
p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine,
7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid,
nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,
p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine,
m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine,
bipyridyl alanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine,
p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine and
p-nitro-L-phenylalanine. Also, a p-propargyloxyphenylalanine, a
3,4-dihydroxy-L-phenyalanine (DHP), a
3,4,6-trihydroxy-L-phenylalanine, a
3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, a
p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, an
L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an
O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a 3-nitro-tyrosine, a
3-thiol-tyrosine, a tri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, a
fluorinated phenylalanine, an isopropyl-L-phenylalanine, a
p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a
p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a
phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine,
a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, and
the like. The structures of a variety of unnatural amino acids are
disclosed in the references cited herein. See also, Published
International Applications WO 2004/094593, entitled "EXPANDING THE
EUKARYOTIC GENETIC CODE;" and International Application
PCT/US2005/039210, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR
THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS," filed Oct. 27,
2005.
[0197] Chemical Synthesis of Unnatural Amino Acids
[0198] Many of the unnatural amino acids provided above are
commercially available, e.g., from Sigma (USA) or Aldrich
(Milwaukee, Wis., USA). Those that are not commercially available
are optionally synthesized as provided in various publications or
using standard methods known to those of skill in the art. For
organic synthesis techniques, see, e.g., Organic Chemistry by
Fessendon and Fessendon, (1982, Second Edition, Willard Grant
Press, Boston Mass.); Advanced Organic Chemistry by March (Third
Edition, 1985, Wiley and Sons, New York); and Advanced Organic
Chemistry by Carey and Sundberg (Third Edition, Parts A and B,
1990, Plenum Press, New York). Additional publications describing
the synthesis of unnatural amino acids include, e.g., WO
2002/085923 entitled "In Vivo Incorporation of Unnatural Amino
Acids;" Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King
and Kidd (1949) "A New Synthesis of Glutamine and of
.gamma.-Dipeptides of Glutamic Acid from Phthylated Intermediates,"
J. Chem. Soc., 3315-3319; Friedman and Chatterrji, (1959)
"Synthesis of Derivatives of Glutamine as Model Substrates for
Anti-Tumor Agents," J. Am. Chem. Soc. 81, 3750-3752; Craig et al.
(1988) "Absolute Configuration of the Enantiomers of 7-Chloro-4
[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine)," J.
Org. Chem. 53, 1167-1170; Azoulay et al., (1991) "Glutamine
analogues as Potential Antimalarials," Eur. J. Med. Chem. 26,
201-5; Koskinen and Rapoport, (1989) "Synthesis of 4-Substituted
Prolines as Conformationally Constrained Amino Acid Analogues,". J.
Org. Chem. 54, 1859-1866; Christie and Rapoport, (1985) "Synthesis
of Optically Pure Pipecolates from L-Asparagine. Application to the
Total Synthesis of (+)-Apovincamine through Amino Acid
Decarbonylation and Iminium Ion Cyclization," J. Org. Chem.
1989:1859-1866; Barton et al., (1987) "Synthesis of Novel
a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of
L- and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and
Appropriate Unsaturated Derivatives," Tetrahedron Lett.
43:4297-4308; and, Subasinghe et al., (1992) "Quisqualic acid
analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid
derivatives and their activity at a novel quisqualate-sensitized
site," J. Med. Chem. 35:4602-7. See also, International Publication
WO 2004/058946, entitled "PROTEIN ARRAYS," filed on Dec. 22,
2003.
[0199] Cellular Uptake of Unnatural Amino Acids
[0200] Unnatural amino acid uptake by a cell is one issue that is
typically considered when designing and selecting unnatural amino
acids, e.g., for incorporation into a protein. For example, the
high charge density of .alpha.-amino acids suggests that these
compounds are unlikely to be cell permeable. Natural amino acids
are taken up into the cell via a collection of protein-based
transport systems often displaying varying degrees of amino acid
specificity. A rapid screen can be done which assesses which
unnatural amino acids, if any, are taken up by cells. See, e.g.,
the toxicity assays in, e.g., International Publication WO
2004/058946, entitled "PROTEIN ARRAYS," filed on Dec. 22, 2003; and
Liu and Schultz (1999) Progress toward the evolution of an organism
with an expanded genetic code. PNAS 96:4780-4785. Although uptake
is easily analyzed with various assays, an alternative to designing
unnatural amino acids that are amenable to cellular uptake pathways
is to provide biosynthetic pathways to create amino acids in
vivo.
[0201] Biosynthesis of Unnatural Amino Acids
[0202] Many biosynthetic pathways already exist in cells for the
production of amino acids and other compounds. While a biosynthetic
method for a particular unnatural amino acid may not exist in
nature, e.g., in a cell, the invention provides such methods. For
example, biosynthetic pathways for unnatural amino acids are
optionally generated in host cell by adding new enzymes or
modifying existing host cell pathways. Additional new enzymes are
optionally naturally occurring enzymes or artificially evolved
enzymes. For example, the biosynthesis of p-aminophenylalanine (as
presented in an example in WO 2002/085923, supra) relies on the
addition of a combination of known enzymes from other organisms.
The genes for these enzymes can be introduced into a cell by
transforming the cell with a plasmid comprising the genes. The
genes, when expressed in the cell, provide an enzymatic pathway to
synthesize the desired compound. Examples of the types of enzymes
that are optionally added are provided in the examples below.
Additional enzymes sequences are found, e.g., in Genbank.
Artificially evolved enzymes are also optionally added into a cell
in the same manner. In this manner, the cellular machinery and
resources of a cell are manipulated to produce unnatural amino
acids.
[0203] Indeed, any of a variety of methods can be used for
producing novel enzymes for use in biosynthetic pathways, or for
evolution of existing pathways, for the production of unnatural
amino acids, in vitro or in vivo. Many available methods of
evolving enzymes and other biosynthetic pathway components can be
applied to the present invention to produce unnatural amino acids
(or, indeed, to evolve synthetases to have new substrate
specificities or other activities of interest). For example, DNA
shuffling is optionally used to develop novel enzymes and/or
pathways of such enzymes for the production of unnatural amino
acids (or production of new synthetases), in vitro or in vivo. See,
e.g., Stemmer (1994), "Rapid evolution of a protein in vitro by DNA
shuffling," Nature 370(4):389-391; and, Stemmer, (1994), "DNA
shuffling by random fragmentation and reassembly: In vitro
recombination for molecular evolution," Proc. Natl. Acad. Sci.
USA., 91:10747-10751. A related approach shuffles families of
related (e.g., homologous) genes to quickly evolve enzymes with
desired characteristics. An example of such "family gene shuffling"
methods is found in Crameri et al. (1998) "DNA shuffling of a
family of genes from diverse species accelerates directed
evolution" Nature, 391(6664): 288-291. New enzymes (whether
biosynthetic pathway components or synthetases) can also be
generated using a DNA recombination procedure known as "incremental
truncation for the creation of hybrid enzymes" ("ITCHY"), e.g., as
described in Ostermeier et al. (1999) "A combinatorial approach to
hybrid enzymes independent of DNA homology" Nature Biotech 17:1205.
This approach can also be used to generate a library of enzyme or
other pathway variants which can serve as substrates for one or
more in vitro or in vivo recombination methods. See, also,
Ostermeier et al. (1999) "Combinatorial Protein Engineering by
Incremental Truncation," Proc. Natl. Acad. Sci. USA, 96: 3562-67,
and Ostermeier et al. (1999), "Incremental Truncation as a Strategy
in the Engineering of Novel Biocatalysts," Biological and Medicinal
Chemistry, 7: 2139-44. Another approach uses exponential ensemble
mutagenesis to produce libraries of enzyme or other pathway
variants that are, e.g., selected for an ability to catalyze a
biosynthetic reaction relevant to producing an unnatural amino acid
(or a new synthetase). In this approach, small groups of residues
in a sequence of interest are randomized in parallel to identify,
at each altered position, amino acids which lead to functional
proteins. Examples of such procedures, which can be adapted to the
present invention to produce new enzymes for the production of
unnatural amino acids (or new synthetases) are found in Delegrave
and Youvan (1993) Biotechnology Research 11:1548-1552. In yet
another approach, random or semi-random mutagenesis using doped or
degenerate oligonucleotides for enzyme and/or pathway component
engineering can be used, e.g., by using the general mutagenesis
methods of e.g., Arkin and Youvan (1992) "Optimizing nucleotide
mixtures to encode specific subsets of amino acids for semi-random
mutagenesis" Biotechnology 10:297-300; or Reidhaar-Olson et al.
(1991) "Random mutagenesis of protein sequences using
oligonucleotide cassettes" Methods Enzymol. 208:564-86. Yet another
approach, often termed a "non-stochastic" mutagenesis, which uses
polynucleotide reassembly and site-saturation mutagenesis can be
used to produce enzymes and/or pathway components, which can then
be screened for an ability to perform one or more synthetase or
biosynthetic pathway function (e.g., for the production of
unnatural amino acids in vivo). See, e.g., Short "NON-STOCHASTIC
GENERATION OF GENETIC VACCINES AND ENZYMES" WO 00/46344.
[0204] An alternative to such mutational methods involves
recombining entire genomes of organisms and selecting resulting
progeny for particular pathway functions (often referred to as
"whole genome shuffling"). This approach can be applied to the
present invention, e.g., by genomic recombination and selection of
an organism (e.g., an E. coli or other cell) for an ability to
produce an unnatural amino acid (or intermediate thereof). For
example, methods taught in the following publications can be
applied to pathway design for the evolution of existing and/or new
pathways in cells to produce unnatural amino acids in vivo: Patnaik
et al. (2002) "Genome shuffling of lactobacillus for improved acid
tolerance" Nature Biotechnology, 20(7): 707-712; and Zhang et al.
(2002) "Genome shuffling leads to rapid phenotypic improvement in
bacteria" Nature, February 7, 415(6872): 644-646.
[0205] Other techniques for organism and metabolic pathway
engineering, e.g., for the production of desired compounds are also
available and can also be applied to the production of unnatural
amino acids. Examples of publications teaching useful pathway
engineering approaches include: Nakamura and White (2003)
"Metabolic engineering for the microbial production of 1,3
propanediol" Curr. Opin. Biotechnol. 14(5):454-9; Berry et al.
(2002) "Application of Metabolic Engineering to improve both the
production and use of Biotech Indigo" J. Industrial Microbiology
and Biotechnology 28:127-133; Banta et al. (2002) "Optimizing an
artificial metabolic pathway: Engineering the cofactor specificity
of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in
vitamin C biosynthesis" Biochemistry, 41(20), 6226-36; Selivonova
et al. (2001) "Rapid Evolution of Novel Traits in Microorganisms"
Applied and Environmental Microbiology, 67:3645, and many
others.
[0206] Regardless of the method used, typically, the unnatural
amino acid produced with an engineered biosynthetic pathway of the
invention is produced in a concentration sufficient for efficient
protein biosynthesis, e.g., a natural cellular amount, but not to
such a degree as to significantly affect the concentration of other
cellular amino acids or to exhaust cellular resources. Typical
concentrations produced in vivo in this manner are about 10 mM to
about 0.05 mM. Once a cell is engineered to produce enzymes desired
for a specific pathway and an unnatural amino acid is generated, in
vivo selections are optionally used to further optimize the
production of the unnatural amino acid for both ribosomal protein
synthesis and cell growth.
[0207] Orthogonal Components for Incorporating Unnatural Amino
Acids
[0208] The invention provides compositions and methods for
producing orthogonal components for incorporating the unnatural
amino acids shown in FIG. 1 into a growing polypeptide chain in
response to a selector codon, e.g., an amber stop codon, a nonsense
codon, a four or more base codon, etc., e.g., in vivo. For example,
the invention provides orthogonal-tRNAs (O-tRNAs), orthogonal
aminoacyl-tRNA synthetases (O-RSs) and pairs thereof. These pairs
can be used to incorporate an unnatural amino acid into growing
polypeptide chains.
[0209] A composition of the invention includes an orthogonal
aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially
aminoacylates an O-tRNA with an unnatural amino acid of FIG. 1. In
certain embodiments, the O-RS comprises an amino acid sequence
selected from SEQ ID NO: 57-101, and conservative variations
thereof. In certain embodiments of the invention, the O-RS
preferentially aminoacylates the O-tRNA over any endogenous tRNA
with an the particular unnatural amino acid, where the O-RS has a
bias for the O-tRNA, and where the ratio of O-tRNA charged with an
unnatural amino acid to the endogenous tRNA charged with the same
unnatural amino acid is greater than 1:1, and more preferably where
the O-RS charges the O-tRNA exclusively or nearly exclusively.
[0210] A composition that includes an O-RS can optionally further
include an orthogonal tRNA (O-tRNA), where the O-tRNA recognizes a
selector codon. Typically, an O-tRNA of the invention includes at
least about, e.g., a 45%, a 50%, a 60%, a 75%, an 80%, or a 90% or
more suppression efficiency in the presence of a cognate synthetase
in response to a selector codon as compared to the suppression
efficiency of an O-tRNA comprising or encoded by a polynucleotide
sequence as set forth in the sequence listings (e.g., SEQ ID NO: 3)
and examples herein. In one embodiment, the suppression efficiency
of the O-RS and the O-tRNA together is, e.g., 5 fold, 10 fold, 15
fold, 20 fold, 25 fold or more greater than the suppression
efficiency of the O-tRNA in the absence of an O-RS. In some
aspects, the suppression efficiency of the O-RS and the O-tRNA
together is at least 45% of the suppression efficiency of an
orthogonal leucyl or tyrosyl-tRNA synthetase pair derived from E.
coli.
[0211] A composition that includes an O-tRNA can optionally include
a host cell (e.g., a mammalian cell such as a rodent cell or a
primate cell), and/or a complete translation system.
[0212] A cell (e.g., a rodent cell or a primate cell) comprising a
translation system is also provided by the invention, where the
translation system includes an orthogonal-tRNA (O-tRNA); an
orthogonal aminoacyl-tRNA synthetase (O-RS); and, an unnatural
amino acid provided in FIG. 1. Typically, the O-RS preferentially
aminoacylates the O-tRNA over any endogenous tRNA with the
unnatural amino acid, where the O-RS has a bias for the O-tRNA, and
where the ratio of O-tRNA charged with the unnatural amino acid to
the endogenous tRNA charged with the unnatural amino acid is
greater than 1:1, and more preferably where the O-RS charges the
O-tRNA exclusively or nearly exclusively. The O-tRNA recognizes the
first selector codon, and the O-RS preferentially aminoacylates the
O-tRNA with an unnatural amino acid. In one embodiment, the O-tRNA
comprises or is encoded by a polynucleotide sequence as set forth
in SEQ ID NO: 3, or a complementary polynucleotide sequence
thereof. In one embodiment, the O-RS comprises an amino acid
sequence selected from SEQ ID NOs: 57-101, and conservative
variations thereof.
[0213] A cell of the invention can optionally further comprise an
additional different O-tRNA/O-RS pair and a second unnatural amino
acid, e.g., where this O-tRNA recognizes a second selector codon
and this O-RS preferentially aminoacylates the corresponding O-tRNA
with the second unnatural amino acid, where the second amino acid
is different from the first unnatural amino acid. Optionally, a
cell of the invention includes a nucleic acid that comprises a
polynucleotide that encodes a polypeptide of interest, where the
polynucleotide comprises a selector codon that is recognized by the
O-tRNA.
[0214] In certain embodiments, a host cell of the invention is a
mammalian cell (such as a rodent cell or a primate cell) that
includes an orthogonal-tRNA (O-tRNA), an orthogonal aminoacyl-tRNA
synthetase (O-RS), an unnatural amino acid, and a nucleic acid that
comprises a polynucleotide that encodes a polypeptide of interest,
where the polynucleotide comprises the selector codon that is
recognized by the O-tRNA. In certain embodiments of the invention,
the O-RS preferentially aminoacylates the O-tRNA with the unnatural
amino acid with an efficiency that is greater than the efficiency
with which the O--RS aminoacylates any endogenous tRNA.
[0215] In certain embodiments of the invention, an O-tRNA of the
invention comprises or is encoded by a polynucleotide sequence as
set forth in the sequence listings (e.g., SEQ ID NO: 3) or examples
herein, or a complementary polynucleotide sequence thereof. In
certain embodiments of the invention, an O-RS comprises an amino
acid sequence as set forth in the sequence listings, or a
conservative variation thereof. In one embodiment, the O-RS or a
portion thereof is encoded by a polynucleotide sequence encoding an
amino acid as set forth in the sequence listings or examples
herein, or a complementary polynucleotide sequence thereof.
[0216] The O-tRNA and/or the O-RS of the invention can be derived
from any of a variety of organisms (e.g., eukaryotic and/or
non-eukaryotic organisms).
[0217] Polynucleotides are also a feature of the invention. A
polynucleotide of the invention (e.g., SEQ ID NOs: 8-56) includes
an artificial (e.g., man-made, and not naturally occurring)
polynucleotide comprising a nucleotide sequence encoding a
polypeptide as set forth in the sequence listings herein, and/or is
complementary to or that polynucleotide sequence. A polynucleotide
of the invention can also include a nucleic acid that hybridizes to
a polynucleotide described above, under highly stringent
conditions, over substantially the entire length of the nucleic
acid. A polynucleotide of the invention also includes a
polynucleotide that is, e.g., at least 75%, at least 80%, at least
90%, at least 95%, at least 98% or more identical to that of a
naturally occurring tRNA or corresponding coding nucleic acid (but
a polynucleotide of the invention is other than a naturally
occurring tRNA or corresponding coding nucleic acid), where the
tRNA recognizes a selector codon, e.g., a four base codon.
Artificial polynucleotides that are, e.g., at least 80%, at least
90%, at least 95%, at least 98% or more identical to any of the
above and/or a polynucleotide comprising a conservative variation
of any the above, are also included in polynucleotides of the
invention.
[0218] Vectors comprising a polynucleotide of the invention are
also a feature of the invention. For example, a vector of the
invention can include a plasmid, a cosmid, a phage, a virus, an
expression vector, and/or the like. A cell comprising a vector of
the invention is also a feature of the invention.
[0219] Methods of producing components of an O-tRNA/O-RS pair are
also features of the invention. Components produced by these
methods are also a feature of the invention. For example, methods
of producing at least one tRNA that is orthogonal to a cell
(O-tRNA) include generating a library of mutant tRNAs; mutating an
anticodon loop of each member of the library of mutant tRNAs to
allow recognition of a selector codon, thereby providing a library
of potential O-tRNAs, and subjecting to negative selection a first
population of cells of a first species, where the cells comprise a
member of the library of potential O-tRNAs. The negative selection
eliminates cells that comprise a member of the library of potential
O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase (RS)
that is endogenous to the cell. This provides a pool of tRNAs that
are orthogonal to the cell of the first species, thereby providing
at least one O-tRNA. An O-tRNA produced by the methods of the
invention is also provided.
[0220] In certain embodiments, the methods further comprise
subjecting to positive selection a second population of cells of
the first species, where the cells comprise a member of the pool of
tRNAs that are orthogonal to the cell of the first species, a
cognate aminoacyl-tRNA synthetase, and a positive selection marker.
Using the positive selection, cells are selected or screened for
those cells that comprise a member of the pool of tRNAs that is
aminoacylated by the cognate aminoacyl-tRNA synthetase and that
shows a desired response in the presence of the positive selection
marker, thereby providing an O-tRNA. In certain embodiments, the
second population of cells comprise cells that were not eliminated
by the negative selection.
[0221] Methods for identifying an orthogonal-aminoacyl-tRNA
synthetase that charges an O-tRNA with an unnatural amino acid are
also provided. For example, methods include subjecting a population
of cells of a first species to a selection, where the cells each
comprise: 1) a member of a plurality of aminoacyl-tRNA synthetases
(RSs), (e.g., the plurality of RSs can include mutant RSs, RSs
derived from a species other than a first species or both mutant
RSs and RSs derived from a species other than a first species); 2)
the orthogonal-tRNA (O-tRNA) (e.g., from one or more species); and
3) a polynucleotide that encodes a positive selection marker and
comprises at least one selector codon.
[0222] Cells (e.g., a host cell) are selected or screened for those
that show an enhancement in suppression efficiency compared to
cells lacking or having a reduced amount of the member of the
plurality of RSs. These selected/screened cells comprise an active
RS that aminoacylates the O-tRNA. An orthogonal aminoacyl-tRNA
synthetase identified by the method is also a feature of the
invention.
[0223] Methods of producing a protein in a host cell (e.g., in a
mammalian cell such as a rodent cell or a primate cell, or the
like) having the unnatural amino acid at a selected position are
also a feature of the invention. For example, a method includes
growing, in an appropriate medium, a cell, where the cell comprises
a nucleic acid that comprises at least one selector codon and
encodes a protein, providing the unnatural amino acid, and
incorporating the unnatural amino acid into the specified position
in the protein during translation of the nucleic acid with the at
least one selector codon, thereby producing the protein. The cell
further comprises: an orthogonal-tRNA (O-tRNA) that functions in
the cell and recognizes the selector codon; and, an orthogonal
aminoacyl-tRNA synthetase (O--RS) that preferentially aminoacylates
the O-tRNA with the unnatural amino acid. A protein produced by
this method is also a feature of the invention.
[0224] The invention also provides compositions that include
proteins, where the proteins comprise an unnatural amino acid. In
certain embodiments, the protein comprises an amino acid sequence
that is at least 75% identical to that of a known protein, e.g., a
therapeutic protein, a diagnostic protein, an industrial enzyme, or
portions thereof. Optionally, the composition comprises a
pharmaceutically acceptable carrier.
Nucleic Acid and Polypeptide Sequences and Variants
[0225] As described herein, the invention provides for
polynucleotide sequences encoding, e.g., O-tRNAs and O-RSs, and
polypeptide amino acid sequences, e.g., O-RSs, and, e.g.,
compositions, systems and methods comprising said polynucleotide or
polypeptide sequences. Examples of said sequences, e.g., O-tRNA and
O-RS amino acid and nucleotide sequences are disclosed herein (see
FIG. 16, e.g., SEQ ID NOs: 3 and 8-101). However, one of skill in
the art will appreciate that the invention is not limited to those
sequences disclosed herein, e.g., in the Examples and sequence
listing. One of skill will appreciate that the invention also
provides many related sequences with the functions described
herein, e.g., polynucleotides and polypeptides encoding
conservative variants of an O-RS disclosed herein. General
methodology for the construction and analysis of orthogonal
synthetase species (O-RS) that are able to aminoacylate an O-tRNA
with an unnatural amino acid in a yeast host system are known in
the art.
[0226] The invention provides polypeptides (O-RSs) and
polynucleotides, e.g., O-tRNA, polynucleotides that encode O-RSs or
portions thereof, oligonucleotides used to isolate aminoacyl-tRNA
synthetase clones, etc. Polynucleotides of the invention include
those that encode proteins or polypeptides of interest of the
invention with one or more selector codon. In addition,
polynucleotides of the invention include, e.g., a polynucleotide
comprising a nucleotide sequence selected from SEQ ID NOS: 8-56,
and a polynucleotide that is complementary to or that encodes a
polynucleotide sequence thereof. A polynucleotide of the invention
also includes any polynucleotide that encodes an O-RS amino acid
sequence selected from SEQ ID NOS: 57-101. Similarly, an artificial
nucleic acid that hybridizes to a polynucleotide indicated above
under highly stringent conditions over substantially the entire
length of the nucleic acid (and is other than a naturally occurring
polynucleotide) is a polynucleotide of the invention. In one
embodiment, a composition includes a polypeptide of the invention
and an excipient (e.g., buffer, water, pharmaceutically acceptable
excipient, etc.). The invention also provides an antibody or
antisera specifically immunoreactive with a polypeptide of the
invention. An artificial polynucleotide is a polynucleotide that is
man made and is not naturally occurring.
[0227] A polynucleotide of the invention also includes an
artificial polynucleotide that is, e.g., at least 75%, at least
80%, at least 90%, at least 95%, at least 98% or more identical to
that of a naturally occurring tRNA, (but is other than a naturally
occurring tRNA). A polynucleotide also includes an artificial
polynucleotide that is, e.g., at least 75%, at least 80%, at least
90%, at least 95%, at least 98% or more identical (but not 100%
identical) to that of a naturally occurring tRNA.
[0228] In certain embodiments, a vector (e.g., a plasmid, a cosmid,
a phage, a virus, etc.) comprises a polynucleotide of the
invention. In one embodiment, the vector is an expression vector.
In another embodiment, the expression vector includes a promoter
operably linked to one or more of the polynucleotides of the
invention. In another embodiment, a cell comprises a vector that
includes a polynucleotide of the invention.
[0229] One of skill will also appreciate that many variants of the
disclosed sequences are included in the invention. For example,
conservative variations of the disclosed sequences that yield a
functionally identical sequence are included in the invention.
Variants of the nucleic acid polynucleotide sequences, wherein the
variants hybridize to at least one disclosed sequence, are
considered to be included in the invention. Unique subsequences of
the sequences disclosed herein, as determined by, e.g., standard
sequence comparison techniques, are also included in the
invention.
[0230] Conservative Variations
[0231] Owing to the degeneracy of the genetic code, "silent
substitutions" (i.e., substitutions in a nucleic acid sequence
which do not result in an alteration in an encoded polypeptide) are
an implied feature of every nucleic acid sequence that encodes an
amino acid sequence. Similarly, "conservative amino acid
substitutions," where one or a limited number of amino acids in an
amino acid sequence are substituted with different amino acids with
highly similar properties, are also readily identified as being
highly similar to a disclosed construct. Such conservative
variations of each disclosed sequence are a feature of the present
invention.
[0232] "Conservative variations" of a particular nucleic acid
sequence refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or, where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. One of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 4%, 2% or 1%) in an encoded
sequence are "conservatively modified variations" where the
alterations result in the deletion of an amino acid, addition of an
amino acid, or substitution of an amino acid with a chemically
similar amino acid. Thus, "conservative variations" of a listed
polypeptide sequence of the present invention include substitutions
of a small percentage, typically less than 5%, more typically less
than 2% or 1%, of the amino acids of the polypeptide sequence, with
an amino acid of the same conservative substitution group. Finally,
the addition of sequences which do not alter the encoded activity
of a nucleic acid molecule, such as the addition of a
non-functional sequence, is a conservative variation of the basic
nucleic acid.
[0233] Conservative substitution tables providing functionally
similar amino acids are well known in the art, where one amino acid
residue is substituted for another amino acid residue having
similar chemical properties (e.g., aromatic side chains or
positively charged side chains), and therefore does not
substantially change the functional properties of the polypeptide
molecule. The following sets forth example groups that contain
natural amino acids of like chemical properties, where
substitutions within a group is a "conservative substitution".
TABLE-US-00002 Conservative Amino Acid Substitutions Nonpolar
and/or Aliphatic Polar, Positively Negatively Side Uncharged
Aromatic Charged Charged Chains Side Chains Side Chains Side Chains
Side Chains Glycine Serine Phenylalanine Lysine Aspartate Alanine
Threonine Tyrosine Arginine Glutamate Valine Cysteine Tryptophan
Histidine Leucine Methionine Isoleucine Asparagine Proline
Glutamine
[0234] Nucleic Acid Hybridization
[0235] Comparative hybridization can be used to identify nucleic
acids of the invention, including conservative variations of
nucleic acids of the invention, and this comparative hybridization
method is a preferred method of distinguishing nucleic acids of the
invention. In addition, target nucleic acids which hybridize to a
nucleic acid represented by SEQ ID NOS: 8-56, under high,
ultra-high and ultra-ultra high stringency conditions are a feature
of the invention. Examples of such nucleic acids include those with
one or a few silent or conservative nucleic acid substitutions as
compared to a given nucleic acid sequence.
[0236] A test nucleic acid is said to specifically hybridize to a
probe nucleic acid when it hybridizes at least 50% as well to the
probe as to the perfectly matched complementary target, i.e., with
a signal to noise ratio at least half as high as hybridization of
the probe to the target under conditions in which the perfectly
matched probe binds to the perfectly matched complementary target
with a signal to noise ratio that is at least about
5.times.-10.times. as high as that observed for hybridization to
any of the unmatched target nucleic acids.
[0237] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of well
characterized physico-chemical forces, such as hydrogen bonding,
solvent exclusion, base stacking and the like. An extensive guide
to the hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, N.Y.), as well as in Current
Protocols in Molecular Biology, Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2004);
Hames and Higgins (1995) Gene Probes 1, IRL Press at Oxford
University Press, Oxford, England, and Hames and Higgins (1995)
Gene Probes 2, IRL Press at Oxford University Press, Oxford,
England, provide details on the synthesis, labeling, detection and
quantification of DNA and RNA, including oligonucleotides.
[0238] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C.
for 15 minutes (see, Sambrook et al., Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2001, for a description of
SSC buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example low
stringency wash is 2.times.SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 5.times. (or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0239] "Stringent hybridization wash conditions" in the context of
nucleic acid hybridization experiments such as Southern and
northern hybridizations are sequence dependent, and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, N.Y.), Hames and Higgins
(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,
England, and Hames and Higgins (1995) Gene Probes 2, IRL Press at
Oxford University Press, Oxford, England. Stringent hybridization
and wash conditions can easily be determined empirically for any
test nucleic acid. For example, in determining stringent
hybridization and wash conditions, the hybridization and wash
conditions are gradually increased (e.g., by increasing
temperature, decreasing salt concentration, increasing detergent
concentration and/or increasing the concentration of organic
solvents such as formalin in the hybridization or wash), until a
selected set of criteria are met. For example, in highly stringent
hybridization and wash conditions, the hybridization and wash
conditions are gradually increased until a probe binds to a
perfectly matched complementary target with a signal to noise ratio
that is at least 5.times. as high as that observed for
hybridization of the probe to an unmatched target.
[0240] "Very stringent" conditions are selected to be equal to the
thermal melting point (T.sub.m) for a particular probe. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the test sequence hybridizes to a perfectly matched probe.
For the purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. lower than the T.sub.m for the specific sequence
at a defined ionic strength and pH.
[0241] "Ultra high-stringency" hybridization and wash conditions
are those in which the stringency of hybridization and wash
conditions are increased until the signal to noise ratio for
binding of the probe to the perfectly matched complementary target
nucleic acid is at least 10.times. as high as that observed for
hybridization to any of the unmatched target nucleic acids. A
target nucleic acid which hybridizes to a probe under such
conditions, with a signal to noise ratio of at least 1/2 that of
the perfectly matched complementary target nucleic acid is said to
bind to the probe under ultra-high stringency conditions.
[0242] Similarly, even higher levels of stringency can be
determined by gradually increasing the hybridization and/or wash
conditions of the relevant hybridization assay. For example, those
in which the stringency of hybridization and wash conditions are
increased until the signal to noise ratio for binding of the probe
to the perfectly matched complementary target nucleic acid is at
least 10.times., 20.times., 50.times., 100.times., or 500.times. or
more as high as that observed for hybridization to any of the
unmatched target nucleic acids. A target nucleic acid which
hybridizes to a probe under such conditions, with a signal to noise
ratio of at least 1/2 that of the perfectly matched complementary
target nucleic acid is said to bind to the probe under
ultra-ultra-high stringency conditions.
[0243] Nucleic acids which do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
[0244] Unique Subsequences
[0245] In some aspects, the invention provides a nucleic acid that
comprises a unique subsequence in a nucleic acid selected from the
sequences of O-tRNAs and O-RSs disclosed herein. The unique
subsequence is unique as compared to a nucleic acid corresponding
to any known O-tRNA or O-RS nucleic acid sequence. Alignment can be
performed using, e.g., BLAST set to default parameters. Any unique
subsequence is useful, e.g., as a probe to identify the nucleic
acids of the invention or related nucleic acids.
[0246] Similarly, the invention includes a polypeptide which
comprises a unique subsequence in a polypeptide selected from the
sequences of O-RSs disclosed herein. Here, the unique subsequence
is unique as compared to a polypeptide corresponding to any of
known polypeptide sequence.
[0247] The invention also provides for target nucleic acids which
hybridizes under stringent conditions to a unique coding
oligonucleotide which encodes a unique subsequence in a polypeptide
selected from the sequences of O-RSs wherein the unique subsequence
is unique as compared to a polypeptide corresponding to any of the
control polypeptides (e.g., parental sequences from which
synthetases of the invention were derived, e.g., by mutation).
Unique sequences are determined as noted above.
[0248] Sequence Comparison, Identity, and Homology
[0249] The terms "identical" or "percent identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms described
below (or other algorithms available to persons of skill) or by
visual inspection.
[0250] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides (e.g., DNAs encoding an O-tRNA or
O-RS, or the amino acid sequence of an O-RS) refers to two or more
sequences or subsequences that have at least about 60%, about 80%,
about 90-95%, about 98%, about 99% or more nucleotide or amino acid
residue identity, when compared and aligned for maximum
correspondence, as measured using a sequence comparison algorithm
or by visual inspection. Such "substantially identical" sequences
are typically considered to be "homologous," without reference to
actual ancestry. Preferably, the "substantial identity" exists over
a region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably, the sequences are substantially
identical over at least about 150 residues, or over the full length
of the two sequences to be compared.
[0251] Proteins and/or protein sequences are "homologous" when they
are derived, naturally or artificially, from a common ancestral
protein or protein sequence. Similarly, nucleic acids and/or
nucleic acid sequences are homologous when they are derived,
naturally or artificially, from a common ancestral nucleic acid or
nucleic acid sequence. For example, any naturally occurring nucleic
acid can be modified by any available mutagenesis method to include
one or more selector codon. When expressed, this mutagenized
nucleic acid encodes a polypeptide comprising one or more unnatural
amino acid. The mutation process can, of course, additionally alter
one or more standard codon, thereby changing one or more standard
amino acid in the resulting mutant protein as well. Homology is
generally inferred from sequence similarity between two or more
nucleic acids or proteins (or sequences thereof). The precise
percentage of similarity between sequences that is useful in
establishing homology varies with the nucleic acid and protein at
issue, but as little as 25% sequence similarity is routinely used
to establish homology. Higher levels of sequence similarity, e.g.,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be
used to establish homology. Methods for determining sequence
similarity percentages (e.g., BLASTP and BLASTN using default
parameters) are described herein and are generally available.
[0252] For sequence comparison and homology determination,
typically one sequence acts as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated program parameters.
[0253] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by
the search for similarity method of Pearson and Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Current Protocols in Molecular Biology,
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
supplemented through 2006).
[0254] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al. (1990), J. Mol. Biol. 215:403-410).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always>0) and N
(penalty score for mismatching residues; always<0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0255] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul
(1993), Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0256] Mutagenesis and Other Molecular Biology Techniques
[0257] Polynucleotide and polypeptides of the invention and used in
the invention can be manipulated using molecular biological
techniques. General texts which describe molecular biological
techniques include Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology volume 152, Academic Press, Inc.,
San Diego, Calif.; Sambrook et al., Molecular Cloning--A Laboratory
Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 2001, and Current Protocols in Molecular
Biology, Ausubel et al., eds., Current Protocols, a joint venture
between Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc., (supplemented through 2006). These texts describe
mutagenesis, the use of vectors, promoters and many other relevant
topics related to, e.g., the generation of genes that include
selector codons for production of proteins that include unnatural
amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs
thereof.
[0258] Various types of mutagenesis are used in the invention,
e.g., to mutate tRNA molecules, to produce libraries of tRNAs, to
produce libraries of synthetases, to insert selector codons that
encode an unnatural amino acids in a protein or polypeptide of
interest. They include but are not limited to site-directed, random
point mutagenesis, homologous recombination, DNA shuffling or other
recursive mutagenesis methods, chimeric construction, mutagenesis
using uracil containing templates, oligonucleotide-directed
mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis
using gapped duplex DNA or the like, or any combination thereof.
Additional suitable methods include point mismatch repair,
mutagenesis using repair-deficient host strains,
restriction-selection and restriction-purification, deletion
mutagenesis, mutagenesis by total gene synthesis, double-strand
break repair, and the like. Mutagenesis, e.g., involving chimeric
constructs, is also included in the present invention. In one
embodiment, mutagenesis can be guided by known information of the
naturally occurring molecule or altered or mutated naturally
occurring molecule, e.g., sequence, sequence comparisons, physical
properties, crystal structure or the like.
[0259] Host cells are genetically engineered (e.g., transformed,
transduced or transfected) with the polynucleotides of the
invention or constructs which include a polynucleotide of the
invention, e.g., a vector of the invention, which can be, for
example, a cloning vector or an expression vector. For example, the
coding regions for the orthogonal tRNA, the orthogonal tRNA
synthetase, and the protein to be derivatized are operably linked
to gene expression control elements that are functional in the
desired host cell. Typical vectors contain transcription and
translation terminators, transcription and translation initiation
sequences, and promoters useful for regulation of the expression of
the particular target nucleic acid. The vectors optionally comprise
generic expression cassettes containing at least one independent
terminator sequence, sequences permitting replication of the
cassette in eukaryotes, or prokaryotes, or both (e.g., shuttle
vectors) and selection markers for both prokaryotic and eukaryotic
systems. Vectors are suitable for replication and/or integration in
prokaryotes, eukaryotes, or preferably both. See Giliman and Smith,
Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987);
Schneider et al., Protein Expr. Purif. 6435:10 (1995); Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152, Academic Press, Inc., San Diego, Calif.;
Sambrook et al., Molecular Cloning--A Laboratory Manual (3rd Ed.),
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
2001, and Current Protocols in Molecular Biology, Ausubel et al.,
eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2006). The vector can be, for example, in the form of a
plasmid, a bacterium, a virus, a naked polynucleotide, or a
conjugated polynucleotide. The vectors are introduced into cells
and/or microorganisms by standard methods including electroporation
(From et al. (1985), Proc. Natl. Acad. Sci. USA 82, 5824),
infection by viral vectors, high velocity ballistic penetration by
small particles with the nucleic acid either within the matrix of
small beads or particles, or on the surface (Klein et al. (1987),
Nature 327:70-73), and/or the like.
[0260] Bacteria and bacteriophage useful for cloning are widely
known to one of skill in the art, and are available from a variety
of sources. See, for example, the American Type Culture Collection
(ATCC; Manassas, Va.) and The ATCC Catalogue of Bacteria and
Bacteriophage (1996) Gherna et al. (eds) published by the ATCC.
Additional basic procedures for sequencing, cloning and other
aspects of molecular biology and underlying theoretical
considerations are also found in Sambrook et al., Molecular
Cloning--A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 2001, and Current
Protocols in Molecular Biology, Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2006),
and in Watson et al. (1992) Recombinant DNA Second Edition
Scientific American Books, NY. In addition, essentially any nucleic
acid (and virtually any labeled nucleic acid, whether standard or
non-standard) can be custom or standard ordered from any of a
variety of commercial sources, such as the Midland Certified
Reagent Company (Midland, Tex.), The Great American Gene Company
(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon
Technologies Inc. (Alameda, Calif.) and many others.
[0261] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for such activities as, for
example, screening steps, activating promoters or selecting
transformants. These cells can optionally be cultured into
transgenic organisms. Other useful references, e.g. for cell
isolation and culture (e.g., for subsequent nucleic acid isolation)
include Freshney (1994) Culture of Animal Cells, a Manual of Basic
Technique, Third Edition, Wiley-Liss, New York and the references
cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg
and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg N.Y.) and Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla.
Proteins and Polypeptides of Interest
[0262] Methods of producing a protein in a cell with an unnatural
amino acid at a specified position are also a feature of the
invention. For example, a method includes growing, in an
appropriate medium, the cell, where the cell comprises a nucleic
acid that comprises at least one selector codon and encodes a
protein; and, providing the unnatural amino acid; where the cell
further comprises: an orthogonal-tRNA (O-tRNA) that functions in
the cell and recognizes the selector codon; and, an orthogonal
aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates
the O-tRNA with the unnatural amino acid. A protein produced by
this method is also a feature of the invention.
[0263] In certain embodiments, the O-RS comprises a bias for the
aminoacylation of the cognate O-tRNA over any endogenous tRNA in an
expression system. The relative ratio between O-tRNA and endogenous
tRNA that is charged by the O-RS, when the O-tRNA and O-RS are
present at equal molar concentrations, is greater than 1:1,
preferably at least about 2:1, more preferably 5:1, still more
preferably 10:1, yet more preferably 20:1, still more preferably
50:1, yet more preferably 75:1, still more preferably 95:1, 98:1,
99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.
[0264] The invention also provides compositions that include
proteins, where the proteins comprise an unnatural amino acid. In
certain embodiments, the protein comprises an amino acid sequence
that is at least 75% identical to that of a therapeutic protein, a
diagnostic protein, an industrial enzyme, or portion thereof.
[0265] The compositions of the invention and compositions made by
the methods of the invention optionally are in a cell. The
O-tRNA/O-RS pairs or individual components of the invention can
then be used in a host system's translation machinery, which
results in an unnatural amino acid being incorporated into a
protein. International Publication Numbers WO 2004/094593, filed
Apr. 16, 2004, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE,"
and WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL
AMINO ACIDS," describe this process, and are incorporated herein by
reference. For example, when an O-tRNA/O-RS pair is introduced into
a host, e.g., a rodent or primate cell, the pair leads to the in
vivo incorporation of an unnatural amino acid (e.g., the unnatural
amino acids of FIG. 1) into a proteins in response to a selector
codon. The unnatural amino acid that is added to the system can be
a synthetic amino acid, such as a derivative of a phenylalanine or
tyrosine, which can be exogenously added to the growth medium.
Optionally, the compositions of the present invention can be in an
in vitro translation system, or in an in vivo system(s).
[0266] A cell of the invention provides the ability to synthesize
proteins that comprise unnatural amino acids in large useful
quantities. In some aspects, the composition optionally includes,
e.g., at least 10 micrograms, at least 50 micrograms, at least 75
micrograms, at least 100 micrograms, at least 200 micrograms, at
least 250 micrograms, at least 500 micrograms, at least 1
milligram, at least 10 milligrams or more of the protein that
comprises an unnatural amino acid, or an amount that can be
achieved with in vivo protein production methods (details on
recombinant protein production and purification are provided
herein). In another aspect, the protein is optionally present in
the composition at a concentration of, e.g., at least 10 micrograms
of protein per liter, at least 50 micrograms of protein per liter,
at least 75 micrograms of protein per liter, at least 100
micrograms of protein per liter, at least 200 micrograms of protein
per liter, at least 250 micrograms of protein per liter, at least
500 micrograms of protein per liter, at least 1 milligram of
protein per liter, or at least 10 milligrams of protein per liter
or more, in, e.g., a cell lysate, a buffer, a pharmaceutical
buffer, or other liquid suspension (e.g., in a volume of, e.g.,
anywhere from about 1 mL to about 100 L). The production of large
quantities (e.g., greater that that typically possible with other
methods, e.g., in vitro translation) of a protein in a cell
including at least one unnatural amino acid is a feature of the
invention.
[0267] The incorporation of an unnatural amino acid can be done to,
e.g., tailor changes in protein structure and/or function, e.g., to
change size, acidity, nucleophilicity, hydrogen bonding,
hydrophobicity, accessibility of protease target sites, target to a
moiety (e.g., for a protein array), incorporation of labels or
reactive groups, etc. Proteins that include an unnatural amino acid
can have enhanced or even entirely new catalytic or physical
properties. For example, the following properties are optionally
modified by inclusion of an unnatural amino acid into a protein:
toxicity, biodistribution, structural properties, spectroscopic
properties, chemical and/or photochemical properties, catalytic
ability, half-life (e.g., serum half-life), ability to react with
other molecules, e.g., covalently or noncovalently, and the like.
The compositions including proteins that include at least one
unnatural amino acid are useful for, e.g., novel therapeutics,
diagnostics, catalytic enzymes, industrial enzymes, binding
proteins (e.g., antibodies), and e.g., the study of protein
structure and function. See, e.g., Dougherty, (2000) Unnatural
Amino Acids as Probes of Protein Structure and Function, Current
Opinion in Chemical Biology, 4:645-652.
[0268] In some aspects of the invention, a composition includes at
least one protein with at least one, e.g., at least two, at least
three, at least four, at least five, at least six, at least seven,
at least eight, at least nine, or at least ten or more unnatural
amino acids. The unnatural amino acids can be the same or
different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or
more different sites in the protein that comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 or more different unnatural amino acids. In another
aspect, a composition includes a protein with at least one, but
fewer than all, of a particular amino acid present in the protein
is an unnatural amino acid. For a given protein with more than one
unnatural amino acids, the unnatural amino acids can be identical
or different (e.g., the protein can include two or more different
types of unnatural amino acids, or can include two of the same
unnatural amino acid). For a given protein with more than two
unnatural amino acids, the unnatural amino acids can be the same,
different or a combination of a multiple unnatural amino acid of
the same kind with at least one different unnatural amino acid.
[0269] Essentially any protein (or portion thereof) that includes
an unnatural amino acid (and any corresponding coding nucleic acid,
e.g., which includes one or more selector codons) can be produced
using the compositions and methods herein. No attempt is made to
identify the hundreds of thousands of known proteins, any of which
can be modified to include one or more unnatural amino acid, e.g.,
by tailoring any available mutation methods to include one or more
appropriate selector codon in a relevant translation system. Common
sequence repositories for known proteins include GenBank EMBL, DDBJ
and the NCBI. Other repositories can easily be identified by
searching the internet.
[0270] Typically, the proteins are, e.g., at least 60%, at least
70%, at least 75%, at least 80%, at least 90%, at least 95%, or at
least 99% or more identical to any available protein (e.g., a
therapeutic protein, a diagnostic protein, an industrial enzyme, or
portion thereof, and the like), and they comprise one or more
unnatural amino acid. Examples of therapeutic, diagnostic, and
other proteins that can be modified to comprise one or more
unnatural amino acid can be found, but not limited to, those in
International Publications WO 2004/094593, filed Apr. 16, 2004,
entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" and, WO
2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS." Examples of therapeutic, diagnostic, and other proteins
that can be modified to comprise one or more unnatural amino acids
include, but are not limited to, e.g., hirudin, Alpha-1
antitrypsin, Angiostatin, Antihemolytic factor, antibodies (further
details on antibodies are found below), Apolipoprotein, Apoprotein,
Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial
peptides, C-X-C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a,
Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC
chemokines (e.g., Monocyte chemoattractant protein-1, Monocyte
chemoattractant protein-2, Monocyte chemoattractant protein-3,
Monocyte inflammatory protein-1 alpha, Monocyte inflammatory
protein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065,
T64262), CD40 ligand, C-kit Ligand, Caspace, Collagen, Colony
stimulating factor (CSF), Complement factor 5a, Complement
inhibitor, Complement receptor 1, cytokines, (e.g., epithelial
Neutrophil Activating Peptide-78, GRO.alpha./MGSA, GRO.beta.,
GRO.gamma., MIP-1.alpha., MIP-1.delta., MCP-1), Epidermal Growth
Factor (EGF), Erythropoietin ("EPO"), Exfoliating toxins A and B,
Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth
Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF,
Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog proteins
(e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor
(HGF), Hirudin, Human serum albumin, Insulin, Insulin-like Growth
Factor (IGF), interferons (e.g., IFN-.alpha., IFN-.beta.,
IFN-.gamma.), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), Keratinocyte
Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor,
Luciferase, Neurturin, Neutrophil inhibitory factor (NIF),
oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF,
PDGF, peptide hormones (e.g., Human Growth Hormone), Pleiotropin,
Procaspace-3, Procaspace-9, Protein A, Protein G, Pyrogenic
exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complement
receptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-1,
2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor,
Somatomedin, Somatostatin, Somatotropin, Streptokinase,
Superantigens, i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1,
SEC2, SEC3, SED, SEE), Superoxide dismutase (SOD), Toxic shock
syndrome toxin (TSST-1), Thymosin alpha 1, Tissue plasminogen
activator, Tumor necrosis factor beta (TNF beta), Tumor necrosis
factor receptor (TNFR), Tumor necrosis factor-alpha (TNF alpha),
Vascular Endothelial Growth Factor (VEGEF), Urokinase and many
others.
[0271] One class of proteins that can be made using the
compositions and methods for in vivo incorporation of unnatural
amino acids described herein includes transcriptional modulators or
a portion thereof. Example transcriptional modulators include genes
and transcriptional modulator proteins that modulate cell growth,
differentiation, regulation, or the like. Transcriptional
modulators are found in prokaryotes, viruses, and eukaryotes,
including fungi, plants, yeasts, insects, and animals, including
mammals, providing a wide range of therapeutic targets. It will be
appreciated that expression and transcriptional activators regulate
transcription by many mechanisms, e.g., by binding to receptors,
stimulating a signal transduction cascade, regulating expression of
transcription factors, binding to promoters and enhancers, binding
to proteins that bind to promoters and enhancers, unwinding DNA,
splicing pre-mRNA, polyadenylating RNA, and degrading RNA.
[0272] One class of proteins of the invention (e.g., proteins with
one or more unnatural amino acids) include biologically active
proteins such as hirudin, cytokines, inflammatory molecules, growth
factors, their receptors, and oncogene products, e.g., interleukins
(e.g., IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II,
FGF, PDGF, TNF, TGF-.alpha., TGF-.beta., EGF, KGF, SCF/c-Kit,
CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44; signal
transduction molecules and corresponding oncogene products, e.g.,
Mos, Ras, Raf, and Met; and transcriptional activators and
suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid
hormone receptors such as those for estrogen, progesterone,
testosterone, aldosterone, the LDL receptor ligand and
corticosterone.
[0273] Enzymes (e.g., industrial enzymes) or portions thereof with
at least one unnatural amino acid are also provided by the
invention. Examples of enzymes include, but are not limited to,
e.g., amidases, amino acid racemases, acylases, dehalogenases,
dioxygenases, diarylpropane peroxidases, epimerases, epoxide
hydrolases, esterases, isomerases, kinases, glucose isomerases,
glycosidases, glycosyl transferases, haloperoxidases,
monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile
hydratases, nitrilases, proteases, phosphatases, subtilisins,
transaminase, and nucleases.
[0274] Many of these proteins are commercially available (See,
e.g., the Sigma BioSciences 2002 catalogue and price list), and the
corresponding protein sequences and genes and, typically, many
variants thereof, are well-known (see, e.g., Genbank). Any of them
can be modified by the insertion of one or more unnatural amino
acid according to the invention, e.g., to alter the protein with
respect to one or more therapeutic, diagnostic or enzymatic
properties of interest. Examples of therapeutically relevant
properties include serum half-life, shelf half-life, stability,
immunogenicity, therapeutic activity, detectability (e.g., by the
inclusion of reporter groups (e.g., labels or label binding sites)
in the unnatural amino acids), reduction of LD.sub.50 or other side
effects, ability to enter the body through the gastric tract (e.g.,
oral availability), or the like. Examples of diagnostic properties
include shelf half-life, stability, diagnostic activity,
detectability, or the like. Examples of relevant enzymatic
properties include shelf half-life, stability, enzymatic activity,
production capability, or the like.
[0275] A variety of other proteins can also be modified to include
one or more unnatural amino acid using compositions and methods of
the invention. For example, the invention can include substituting
one or more natural amino acids in one or more vaccine proteins
with an unnatural amino acid, e.g., in proteins from infectious
fungi, e.g., Aspergillus, Candida species; bacteria, particularly
E. coli, which serves a model for pathogenic bacteria, as well as
medically important bacteria such as Staphylococci (e.g., aureus),
or Streptococci (e.g., pneumoniae); protozoa such as sporozoa
(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates
(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such
as (+) RNA viruses (examples include Poxviruses e.g., vaccinia;
Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;
Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g.,
Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;
Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses),
dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e.,
Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses
such as Hepatitis B.
[0276] Agriculturally related proteins such as insect resistance
proteins (e.g., the Cry proteins), starch and lipid production
enzymes, plant and insect toxins, toxin-resistance proteins,
Mycotoxin detoxification proteins, plant growth enzymes (e.g.,
Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, "RUBISCO"),
lipoxygenase (LOX), and Phosphoenolpyruvate (PEP) carboxylase are
also suitable targets for unnatural amino acid modification.
[0277] In certain embodiments, the protein or polypeptide of
interest (or portion thereof) in the methods and/or compositions of
the invention is encoded by a nucleic acid. Typically, the nucleic
acid comprises at least one selector codon, at least two selector
codons, at least three selector codons, at least four selector
codons, at least five selector codons, at least six selector
codons, at least seven selector codons, at least eight selector
codons, at least nine selector codons, ten or more selector
codons.
[0278] Genes coding for proteins or polypeptides of interest can be
mutagenized using methods well-known to one of skill in the art and
described herein under "Mutagenesis and Other Molecular Biology
Techniques" to include, e.g., one or more selector codon for the
incorporation of an unnatural amino acid. For example, a nucleic
acid for a protein of interest is mutagenized to include one or
more selector codon, providing for the insertion of the one or more
unnatural amino acids. The invention includes any such variant,
e.g., mutant, versions of any protein, e.g., including at least one
unnatural amino acid. Similarly, the invention also includes
corresponding nucleic acids, i.e., any nucleic acid with one or
more selector codon that encodes one or more unnatural amino
acid.
[0279] To make a protein that includes an unnatural amino acid, one
can use host cells and organisms that are adapted for the in vivo
incorporation of the unnatural amino acid via orthogonal tRNA/RS
pairs. Host cells are genetically engineered (e.g., transformed,
transduced or transfected) with one or more vectors that express
the orthogonal tRNA, the orthogonal tRNA synthetase, and a vector
that encodes the protein to be derivatized. Each of these
components can be on the same vector, or each can be on a separate
vector, or two components can be on one vector and the third
component on a second vector. The vector can be, for example, in
the form of a plasmid, a bacterium, a virus, a naked
polynucleotide, or a conjugated polynucleotide.
[0280] Defining Polypeptides by Immunoreactivity
[0281] Because the polypeptides of the invention provide a variety
of new polypeptide sequences (e.g., polypeptides comprising
unnatural amino acids in the case of proteins synthesized in the
translation systems herein, or, e.g., in the case of the novel
synthetases, novel sequences of standard amino acids), the
polypeptides also provide new structural features which can be
recognized, e.g., in immunological assays. The generation of
antisera, which specifically bind the polypeptides of the
invention, as well as the polypeptides which are bound by such
antisera, are a feature of the invention. The term "antibody," as
used herein, includes, but is not limited to a polypeptide
substantially encoded by an immunoglobulin gene or immunoglobulin
genes, or fragments thereof which specifically bind and recognize
an analyte (antigen). Examples include polyclonal, monoclonal,
chimeric, and single chain antibodies, and the like. Fragments of
immunoglobulins, including Fab fragments and fragments produced by
an expression library, including phage display, are also included
in the term "antibody" as used herein. See, e.g., Paul, Fundamental
Immunology, 4th Ed., 1999, Raven Press, New York, for antibody
structure and terminology.
[0282] In order to produce antisera for use in an immunoassay, one
or more of the immunogenic polypeptides is produced and purified as
described herein. For example, recombinant protein can be produced
in a recombinant cell. An inbred strain of mice (used in this assay
because results are more reproducible due to the virtual genetic
identity of the mice) is immunized with the immunogenic protein(s)
in combination with a standard adjuvant, such as Freund's adjuvant,
and a standard mouse immunization protocol (see, e.g., Harlow and
Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York, for a standard description of antibody
generation, immunoassay formats and conditions that can be used to
determine specific immunoreactivity. Additional details on
proteins, antibodies, antisera, etc. can be found in International
Publication Numbers WO 2004/094593, entitled "EXPANDING THE
EUKARYOTIC GENETIC CODE;" WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/035605, entitled
"GLYCOPROTEIN SYNTHESIS;" and WO 2004/058946, entitled "PROTEIN
ARRAYS."
Use of O-tRNA and O-RS and O-tRNA/O-RS Pairs
[0283] The compositions of the invention and compositions made by
the methods of the invention optionally are in a cell. The
O-tRNA/O-RS pairs or individual components of the invention can
then be used in a host system's translation machinery, which
results in an unnatural amino acid being incorporated into a
protein. International Publication Number WO 2002/085923 by
Schultz, et al., entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS," describes this process and is incorporated herein by
reference. For example, when an O-tRNA/O-RS pair is introduced into
a host, e.g., Escherichia coli or yeast, the pair leads to the in
vivo incorporation of an unnatural amino acid, which can be
exogenously added to the growth medium, into a protein, e.g., a
myoglobin test protein or a therapeutic protein, in response to a
selector codon, e.g., an amber nonsense codon. Optionally, the
compositions of the invention can be in an in vitro translation
system, or in a cellular in vivo system(s). Proteins with the
unnatural amino acid can be used in any of a wide range of
applications. For example, the unnatural moiety incorporated into a
protein can serve as a target for any of a wide range of
modifications, for example, crosslinking with other proteins, with
small molecules such as labels or dyes and/or biomolecules. With
these modifications, incorporation of the unnatural amino acid can
result in improved therapeutic proteins and can be used to alter or
improve the catalytic function of enzymes. In some aspects, the
incorporation and subsequent modification of an unnatural amino
acid in a protein can facilitate studies on protein structure,
interactions with other proteins, and the like.
Kits
[0284] Kits are also a feature of the invention. For example, a kit
for producing a protein that comprises at least one unnatural amino
acid in a rodent or primate cell is provided, where the kit
includes at least one container containing a polynucleotide
sequence encoding an O-tRNA, and/or a polynucleotide sequence
encoding an O-RS, and/or an O-RS polypeptide. In one embodiment,
the kit further includes the unnatural amino acid. In another
embodiments, the kit further comprises instructional materials for
producing the protein comprising the unnatural amino acid.
EXAMPLES
[0285] The following examples are offered to illustrate, but not to
limit the claimed invention. One of skill will recognize a variety
of non-critical parameters that may be altered without departing
from the scope of the claimed invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1
Technical Limitations of Nonsense Suppression in Mammalian Cells by
Non-Mammalian Translation Components
[0286] One strategy to genetically encode unnatural amino acids in
mammalian cells would be to adapt the existing mutant tRNA/aaRS
pairs that have been generated in Escherichia coli or Saccharomyces
cerevisiae host systems. Because the tRNA.sup.Tyr identity elements
in E. coli, which include the variable arm and G1:C72 base pair in
the acceptor stem, are distinct from those in mammalian cells (Wang
and Schultz (2004), "Expanding the genetic code," Angew Chem Int Ed
Engl 44:34-66; and Bonnefond et al. (2005), "Evolution of the
tRNA(Tyr)/TyrRS aminoacylation systems," Biochimie 87:873-883), the
tRNA/aaRS pairs that have been used in bacteria are unfortunately
not likely to be orthogonal in eukaryotic cells. In contrast, tRNAs
in S. cerevisiae and mammalian cells are processed similarly and
have the same identity elements (Bonnefond et al. (2005),
"Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems,"
Biochimie 87:873-883). Moreover, because the translational
machinery of S. cerevisiae is also homologous to that of higher
eukaryotes, it is possible that one can transfer a modified
orthogonal tRNA/aaRS pair evolved in S. cerevisiae into mammalian
cells. Indeed, Yokoyama and coworkers used an EcTyrRS variant that
was evolved in S. cerevisiae to accept p-benzoyl-L-phenylalanine
(pBpa) to incorporate this photoreactive amino acid into human Grb2
protein in CHO cells (Hino et al. (2005), "Protein
photo-cross-linking in mammalian cells by site-specific
incorporation of a photoreactive amino acid," Nat Methods 2,
201-206). Unfortunately, functionally active E. coli
tRNA.sub.CUA.sup.Tyr does not express well in mammalian cells,
severely limiting the yields of mutant protein (Sakamoto et al.
(2002) "Site-specific incorporation of an unnatural amino acid into
proteins in mammalian cells," Nucleic Acids Res 30:4692-4699).
Example 2
Creation of Novel Nonsense Suppression Systems in Mammalian
Cells
[0287] To overcome the limitations in orthogonal tRNA expression
described above, the present invention provides a general system
that allows for the transfer of mutant suppressor tRNA/aaRS pairs
evolved in yeast to be used in mammalian cells. This method and the
novel orthogonal systems are demonstrated herein to efficiently
introduce a number of unnatural amino acids into green fluorescent
protein (GFP) in both rodent CHO host cells and primate (human)
293T host cells. Although these two cell types were used
experimentally, the invention is widely applicable to mammalian
host cells from diverse species.
Creation of a Novel Mammalian Nonsense Suppression System
[0288] The tRNAs in eukaryotes are transcribed by RNA polymerase
III, which recognizes two conserved intragenic transcriptional
control elements, namely, the A and B boxes (Sprague, Transcription
of Eukaryotic tRNA Genes, AMS Press, Washington, D.C.; 1994). E.
coli tRNA.sup.Tyr only has a B box element, and it has been shown
that the introduction of a pseudo-A box results in a non-functional
tRNA that is not recognized by EcTyrRS (Sakamoto et al.,
"Site-specific incorporation of an unnatural amino acid into
proteins in mammalian cells," Nucleic Acids Res 30:4692-4699
(2002)). Unlike E. coli tRNA.sup.Tyr, the tRNA.sup.Tyr from
Bacillus stearothermophilus (which has similar identity elements
and is still charged by EcTyrRS; Bedouelle, "Recognition of
tRNA(Tyr) by tyrosyl-tRNA synthetase," Biochimie 72, 589-598
(1990)) has naturally occurring internal A and B boxes. Thus, this
tRNA together with EcTyrRS are capable of functioning as an
orthogonal tRNA/aaRS pair in mammalian cells (Sakamoto et al.
(2002), "Site-specific incorporation of an unnatural amino acid
into proteins in mammalian cells," Nucleic Acids Res
30:4692-4699).
[0289] Construction and Expression of an Amber Suppressor
Orthogonal tRNA
[0290] To afford an amber suppressor BstRNA.sub.CUA.sup.Tyr, the
trinucleotide anticodon of BstRNA.sup.Tyr was changed to C(34)UA.
Since G34 of prokaryotic tRNA.sup.Tyr is only a weak identity
element of TyrRS (Hou and Schimmel (1989), "Modeling with in vitro
kinetic parameters for the elaboration of transfer RNA identity in
vivo," Biochemistry 28:4942-4947), the G34C mutant should not
significantly affect the binding of EcTyrRS to
BstRNA.sub.CUA.sup.Tyr. Furthermore, nonsense amber suppression
should be better tolerated in mammalian cells than opal or ochre
suppression due to the lower occurrence of the TAG stop codon in
mammalian genomes (TAG, 23%; TAA, 30%; TGA, 47% in Homo sapiens).
Since expression of the BstRNA.sub.CUA.sup.Tyr gene in eukaryotes
also depends on the 5' flanking sequence, the 5' flanking sequence
of human tRNA.sup.Tyr was added to BstRNA.sub.CUA.sup.Tyr to
enhance its transcription in mammalian cells. To further increase
transcription of BstRNA.sub.CUA.sup.Tyr, a gene cluster containing
three tandem repeats of the BstRNA.sub.CUA.sup.Tyr gene was
constructed and this gene cluster was inserted into the pUC18
plasmid to afford pUC18-3BstRNA.sub.CUA.sup.Tyr. The Northern blot
analysis of isolated total tRNAs from CHO cells transfected with
pUC18-3BstRNA.sub.CUA.sup.Tyr showed a two-fold higher level of
BstRNA.sub.CUA.sup.Tyr than cells transfected with a pUC18 plasmid
containing only one copy of BstRNA.sub.CUA.sup.Tyr.
[0291] Expression of an Orthogonal Synthetase
[0292] Next, the wild type EcTyrRS gene was inserted into the
mammalian expression vector pcDNA4/TO/myc-His A to afford
pcDNA4-EcTyrRS in which expression is controlled by a tetracycline
(Tet)-regulated CMV promoter. The use of an inducible expression
was intended to lower possible toxicity due to heterologous
expression of EcTyrRS in mammalian cells.
[0293] Demonstration of a Functional EcTyrRS/BstRNA.sub.CUA.sup.Tyr
Orthogonal Pair
[0294] The ability of the resulting suppressor
BstRNA.sub.CUA.sup.Tyr/EcTyrRS pair to efficiently suppress an
amber codon mutation at position Y37 of model protein green
fluorescent protein, GFP (GFP37TAG) was assayed in mammalian cells.
Because Y37 is located at the surface of GFP and distal from the
fluorophore, the introduction of an unnatural amino acid at this
position is not expected to affect the folding and fluorescent
properties of the protein (Ormo et al. (1996), "Crystal structure
of the Aequorea victoria green fluorescent protein," Science
273:1392-1395). Moreover, amber suppression of GFP37TAG results in
expression of full-length GFP, providing a rapid qualitative assay
of amber suppression efficiency. The mutant GFP37TAG gene was
inserted into pcDNA4/TO/myc-His A to afford pcDNA4-GFP37TAG in
which GFP is fused to myc and 6.times.His epitopes at C-terminus
and under the control of a Tet-regulated promoter.
[0295] The ability of the BstRNA.sub.CUA.sup.Tyr/EcTyrRS pair to
suppress the nonsense amber codon of GFP37TAG was examined in both
Invitrogen.TM. T-REx.TM. rodent CHO and human 293 cells. Both cell
lines constitutively express the tetracycline repressor and are
suitable for Tet-regulated expression of proteins using the
pcDNA4/TO/myc-His A plasmid. Cells were grown to 80-90% confluency
(Invitrogen.TM. T-REx.TM. CHO cells were grown in F12 media
containing 10% FBS, 1% Pen-Strep, 2 mM of L-glutamine, and 10
.mu.g/ml of blasticidin; Invitrogen.TM. T-REx.TM. 293 cells were
grown in DMEM containing 10% FBS, 1% Pen-Strep, 2 mM of
L-glutamine, and 5 .mu.g/ml of blasticidin) and transiently
transfected with 3 .mu.g of plasmids per 2.times.10.sup.6 cells
using FuGENE.RTM. 6 from Roche Applied Science (0.5 .mu.g of
pUC18-3BstRNA.sub.CUA.sup.Tyr, 0.5 .mu.g of pcDNA4-EcTyrRS, and 2
.mu.g of pcDNA4-GFP37TAG; pUC18-3BstRNA.sub.CUA.sup.Tyr or
pcDNA4-EcTyrRS was replaced with the same amount of pUC18 when they
are not present). Protein expression was induced by the addition of
1 .mu.g/ml tetracycline six hours after transfection, and cells
were grown at 37.degree. C. for two days. In both cell lines, the
expression of full length GFP is dependent upon the presence of
both BstRNA.sub.CUA.sup.Tyr and EcTyrRS genes (see FIGS. 13A and
13B). When transfected with pcDNA4-GFP37TAG and pcDNA4-EcTyrRS or
pUC18-3BstRNA.sub.CUA.sup.Tyr alone, cells did not exhibit green
fluorescence. These experiments demonstrate that
BstRNA.sub.CUA.sup.Tyr is charged only by EcTyrRS and that the
BstRNA.sub.CUA.sup.Tyr/EcTyrRS pair can function efficiently to
suppress a nonsense amber codon in primate cells and rodent
cells.
Example 3
Demonstration of System Universality Using Six Unnatural Amino
Acids
[0296] The generality of this orthogonal system was determined. In
this test system, the ability of BstRNA.sub.CUA.sup.Tyr and six
EcTyrRS variants to incorporate a variety of corresponding
unnatural amino acids into GFP37TAG in both Invitrogen.TM.
T-REx.TM. CHO and 293 cell lines was determined. The six EcTyrRS
variants were previously evolved in S. cerevisiae to encode the
following unnatural amino acids: [0297] p-methoxy-L-phenylalanine
(pMpa) [0298] p-acetyl-L-phenylalanine (pApa) [0299]
p-benzoyl-L-phenylalanine (pBpa) [0300] p-iodo-L-phenylalanine
(pIpa) [0301] p-azido-L-phenylalanine (pAzpa) [0302]
p-propargyloxyphenylalanine (pPpa)
[0303] The structures of these unnatural amino acids are shown in
FIG. 1; structures 1 through 6. The selection and isolation of the
synthetase variants are described in, for example, Chin et al.
(2003), "An expanded eukaryotic genetic code," Science 301:964-967;
Deiters et al. (2003), "Adding amino acids with novel reactivity to
the genetic code of Saccharomyces cerevisiae," J Am Chem Soc
125:11782-11783; WO 2005/003294 to Deiters et al., "UNNATURAL
REACTIVE AMINO ACID GENETIC CODE ADDITIONS," filed Apr. 16, 2004;
and WO 2006/034410 to Deiters et al., "ADDING PHOTOREGULATED AMINO
ACIDS TO THE GENETIC CODE," filed Sep. 21, 2005.
[0304] Each of the six EcTyrRS genes was inserted into
pcDNA4/TO/myc-His A to afford a pcDNA4-EcTyrRS derivative, and
expression of the aaRS in T-REx.TM. CHO and 293 cells was verified
by Western blot analysis (FIG. 14). All six EcTyrRS variants show
similar expression levels in both T-REx.TM. CHO and T-REx.TM. 293
cell lines.
[0305] The ability of the six EcTyrRS variants together with
BstRNA.sub.CUA.sup.Tyr to suppress the amber codon in GFP37TAG was
then tested in the presence and absence of unnatural amino acids in
the growth media. Transient transfection of cells with plasmids was
carried out as described for wild type EcTyrRS. Six hours after
transfection, the media were replaced by the fresh media containing
1 .mu.g/ml tetracycline and supplemented with the corresponding
unnatural amino acid (pMpa, 10 mM; pApa, 10 mM; pBpa, 1 mM; plpa, 8
mM; pAzpa, 5 mM; or pPpa, 1 mM). Cells were then grown 24 hours for
T-REx.TM. CHO cell lines and 48 hours for T-REx.TM. 293 cell lines
before harvesting. In both T-REx.TM. CHO and 293 cells, the
expression of full length GFP37TAG was dependent upon the presence
of unnatural amino acids in the growth media as indicated in FIG. 2
and FIG. 3. In the absence of unnatural amino acids, no GFP
expression (<1%) was detected, indicating that EcTyrRS variants
specifically charge BstRNA.sub.CUA.sup.Tyr with their cognate
unnatural amino acids with high fidelity. A low expression level of
GFP containing plpa was observed in T-REx.TM. CHO cells and no GFP
expression containing plpa was detected in T-REx.TM. 293 cells,
possibly due to the cellular toxicity of plpa (in the presence of 8
mM plpa, T-REx.TM. 293 cells die in 6 hours).
Example 4
Improved Plasmid Expression System for the Expression of Orthogonal
Components to Incorporate p-Methoxy-L-Phenylalanine
[0306] Because heterologous expression of
p-methoxy-L-phenylalanine-tRNA synthetase (pMpaRS) did not show any
apparent cellular toxicity under the growth conditions used, the
plasmid containing the pMpaRS gene was modified to encode (a) three
tandem repeats of the BstRNA.sub.CUA.sup.Tyr gene, and (b) the
pMpaRS gene (pSWAN-pMpaRS in FIG. 4A).
[0307] The pMpaRS gene was inserted directly after the
non-regulated CMV promoter for efficient and continuous expression
of pMpaRS. Another plasmid (pSWAN-GFP37TAG in FIG. 4B) containing
both three tandem repeats of the BstRNA.sub.CUA.sup.Tyr gene and
the GFP37TAG gene was also constructed. The gene encoding GFP37TAG
was inserted after a Tet-regulated CMV promoter to minimize
potential read-through of the nonsense amber codon caused by
endogenous amber suppression in mammalian cells. These two plasmids
were then assayed for their suppression efficiency. Because both
plasmids contain three tandem repeats of the BstRNA.sub.CUA.sup.Tyr
gene, varying the ratio of the two plasmids to increase the
suppression level is not expected to change the total amount of the
BstRNA.sub.CUA.sup.Tyr gene transfected into cells. Under optimized
transfection conditions (0.5 .mu.g of pSWAN-pMpaRS and 2.5 .mu.g
pSWAN-GFP37TAG per 2.times.10.sup.6 cells), the suppression level
is roughly two-fold higher than that using the three plasmids
pUC18-3BstRNA.sub.CUA.sup.Tyr, pcDNA4-pMpaRS, and pcDNA4-GFP37TAG
(the suppression level was determined by the number of fluorescent
cells). Cells were grown for 1-2 days after induction and kept
viable before harvesting. The yield of mutant GFP containing pMpa
(GFP-pMpa) was also quantified. In the presence of 10 mM pMpa in
the growth media, 1 .mu.g of mutant GFP can be obtained from
2.times.10.sup.7 adhesive T-REx.TM. CHO cells.
Example 5
Mass Spectroscopy and Fidelity Analysis
[0308] To further characterize GFP-pMpa, the mutant protein was
expressed in T-REx.TM. CHO cells transiently transfected with both
pSWAN-pMpaRS and pSWAN-GFP37TAG. The protein was purified using an
anti-myc antibody agarose column and then separated by SDS-PAGE.
The GFP-pMpa band from the SDS-PAGE gel was digested with trypsin
and analyzed by nanoscale reversed-phase liquid chromatography/mass
spectrometry/mass spectrometry (nano-RP LC/MS/MS). In parallel,
wild type GFP was purified from cells transfected with pcDNA4-GFP
and subjected to the same analysis.
[0309] The tandem mass spectra of the tryptic Y37 containing
fragments of:
TABLE-US-00003 (see FIG. 5 and SEQ ID NO: 103) FSVSGEGEGDATY*GK
where Y* denotes either tyrosine or pMpa) from the mutant protein
reveals intense pMpa peaks, indicating efficient incorporation of
pMpa (see FIG. 6 and FIG. 7). The Y* containing ions (y.sub.3 to
y.sub.14) all have a mass shift of 14 Da in comparison to wild type
GFP, which matches exactly the mass difference between tyrosine and
pMpa. The mass shift of y ion series together with the observation
of an identical mass shift of the b.sub.13 ion in FIG. 7
unambiguously assigns the site of pMpa incorporation to position 37
of GFP.
[0310] The ratios of the MS peaks of pMpa containing peptides to
those of tyrosine containing peptides were also obtained.
Integration of the single ion chromatograms of the precursor ions
of:
TABLE-US-00004 (see FIG. 5 and SEQ ID NO: 103) FSVSGEGEGDATY*GK
suggests high fidelity for incorporation of pMpa (>99.9%). An
estimate based on monitoring the three most abundant fragment ions
(y.sub.9, y.sub.11, and y.sub.12) in the tandem MS data suggests an
even greater purity (99.93%). To acquire the masses of the parent
protein, the mutant protein purified by anti-myc antibody column
was subjected to analysis with ESI TOF-MS. The theoretical mass of
the acetylated mutant protein missing the N-terminal methionine is
29,696.00 Da, which is in good agreement with the major component
of 29,696.0 Da observed in the charged-state deconvoluted ESI
TOF-MS spectrum in FIG. 8. A smaller feature at 29,654.0 Da is
assigned to the mass of GFP-pMpa lacking N-terminal acetylation. No
wild type GFP signals or signals indicating incorporation of
multiple pMpas were detected. This result further confirms the
selective incorporation of pMpa at position 37 of GFP.
Example 6
Improved Plasmid Expression Systems for the Expression of
Orthogonal Components that Incorporate pApa, pBpa, plpa, pAzpa and
pPpa
[0311] Plasmids were reengineered to afford other pSWAN-EcTyrRS
variant two-plasmid systems by subcloning the genes that encode the
various synthetase variants. These synthetase variants were as
follows: [0312] p-acetyl-L-phenylalanine-tRNA synthetase (pApaRS)
[0313] p-benzoyl-L-phenylalanine-tRNA synthetase (pBpaRS) [0314]
p-iodo-L-phenylalanine-tRNA synthetase (pIpaRS) [0315]
p-azido-L-phenylalanine-tRNA synthetase (pAzpaRS) [0316]
p-propargyloxyphenylalanine-tRNA synthetase (pPpaRS)
[0317] Transient transfection of T-REx.TM. CHO and 293 cells with
the pSWAN-EcTyrRS variants and pSWAN-GFP37TAG (0.5 .mu.g of
pSWAN-EcTyrRS variant and 2.5 .mu.g of pSWAN-GFP37TAG per
2.times.10.sup.6 cells) all afforded roughly two-fold higher
suppression levels than for the three plasmids
pUC18-3BstRNA.sub.CUA.sup.Tyr, pcDNA4-EcTyrRS variant, and
pcDNA4-GFP37TAG (with the exception of the pIpa specific variant).
Mutant GFPs containing pApa, pBpa, pAzpa or pPpa were also
expressed in T-REx.TM. CHO cells transfected with the pSWAN-EcTyrRS
variants and pSWAN-GFP37TAG and grown in media containing the
corresponding unnatural amino acid (pApa, 10 mM; pBpa, 1 mM; pAzpa,
5 mM; or pPpa, 1 mM) for 1-2 days, and purified using an anti-myc
antibody column. The yields of the mutant proteins containing pApa
and pAzpa were close to that for pMpa (.about.1 .mu.g per
2.times.10.sup.7 cells), and the yields of mutant proteins
containing pBpa and pPpa were somewhat lower (.about.0.7 .mu.g per
2.times.10.sup.7 cells). Because the expression levels of EcTyrRS
variants in T-REx.TM. CHO cells are similar, the lower yield of the
mutant proteins containing pBpa and pPpa may be due to the low
concentrations of unnatural amino acids in the media.
[0318] The mutant GFP proteins were also subjected to analysis with
nano-RP LC/MS/MS to obtain tandem MS of the tryptic fragments
of:
TABLE-US-00005 (FIG. 5 and SEQ ID NO: 103) FSVSGEGEGDATY*GK.
[0319] Tandem MS data (FIGS. 9-12) clearly showed the incorporation
of the unnatural amino acids at position 37 of GFP. The mutant GFP
containing pAzpa was only weakly detectable. A closer look at the
data revealed the presence of the p-aminophenylalanine (pAmpa)
containing peptide instead (FIG. 15), which is not surprising
considering the chemical reactivity and photo-instability of the
azido group (pAmpa has previously been observed from MS analysis of
pAzpa containing peptides; Chin et al. (2003), "Progress toward an
expanded eukaryotic genetic code," Chem Biol 10, 511-519). A trace
amount of the wild type peptide in the pBpa sample was identified,
but the signals were too weak for accurate quantitation of the
pBpa/tyrosine ratio. No wild type signals could be detected in the
pApa, pAzpa, and pPpa samples. The data from all five mutant
proteins indicate the high selectivity and fidelity of the
incorporation of unnatural amino acids.
Example 7
Discussion/Conclusions
[0320] In the mammalian genome, the occurrence of amber stop codons
is higher (23% in humans), in comparison to E. coli (7%).
Therefore, amber suppression might be toxic to cells if essential
proteins are not terminated correctly. Yokoyama and coworkers
showed that inducible expression of the mutant EcTyrRS that charges
its cognate tRNA with 3-iodo-L-tyrosine minimizes possible cellular
toxicity resulting from background incorporation of endogenous
tyrosine by the mutant aaRS in the absence of 3-iodo-L-tyrosine.
The present invention described herein provides a solution to this
problem. All EcTyrRS variants were previously evolved from a
two-step positive/negative selection scheme in S. cerevisiae, which
removes aaRS variants that incorporate endogenous amino acids.
Cotransfection of cells with a pSWAN-EcTyrRS variant and
pSWAN-GFP37TAG did not lead to observable read-through of the
nonsense amber codon in GFP37TAG in the absence of unnatural amino
acids, suggesting that the tRNA/aaRS pairs do not efficiently
suppress natural TAG stop codons in the absence of unnatural amino
acids. Cell lines stably expressing tRNA and aaRS proteins should
therefore be viable in the absence of unnatural amino acids. Since
there is no endogenous amber suppression observed, the expression
of the target protein containing unnatural amino acids also does
not require induction. Creation of stable cell lines maintaining
the tRNA, aaRS, and target protein genes will allow efficient
production of the target protein containing the unnatural amino
acid when cells are supplemented with the unnatural amino acid. In
the presently described proof-of-principle experiments, the host
mammalian cells survived three days after the addition of unnatural
amino acids, which is likely sufficient for recombinant protein
expression.
[0321] These studies have successfully been extended to other
unnatural amino acids, including 1,5-danyslalanine,
o-nitrobenzylcysteine and .alpha.-aminocaprylic acid. It is not
intended that the invention be limited to the cell lines or
unnatural amino acids described herein.
Example 8
Photoregulated Protein Sequences in Mammalian Cells
[0322] In some embodiments of the claimed translation systems and
methods, the unnatural amino acid is a photoregulated amino acid,
such as o-nitrobenzylserine, o-nitrobenzylcysteine,
.alpha.-aminocaprylic acid, and the like. Photoregulated amino
acids (e.g., photochromic, photocleavable, photoisomerizable, etc.)
can be used to spatially and temporally control a variety of
biological process, e.g., by directly regulating the activity of
enzymes, receptors, ion channels or the like, or by modulating the
intracellular concentrations of various signaling molecules. See,
e.g., Shigeri et al., Pharmacol. Therapeut., 2001, 91:85+; Curley,
et al., Pharmacol. Therapeut., 1999, 82:347+; Curley, et al., Curr.
Op. Chem. Bio., 1999, 3:84+; "Caged Compounds" Methods in
Enzymology, Marriott, G., Ed, Academic Press, NY, 1998, V. 291;
Adams, et al., Annu. Rev. Physiol., 1993, 55:755+; and Bochet, et
al., J. Chem. Soc., Perkin 1, 2002, 125+.
[0323] To increase the transcription level of Amber suppressor tRNA
derived from E. coli tRNALeu, the suppressor tRNA was put under
control of 5'- and 3'-flanking sequence of human initiator
tRNA.sup.Met. The over transcribed Amber suppressor tRNA together
with a mutant E. coli leucyl-tRNA synthetase specific for
o-nitrobenzylcysteine was used to site-specifically incorporate
this unnatural amino acid into a human procaspase-3 active site in
Chinese Hamster Ovary cells and human 293T cells. The expression of
the protein in the presence of o-nitrobenzylcysteine followed by
exposure of the cells to UV light for 5 min triggered cell death,
indicating that UV light induced cleavage of the nitrobenzyl group
from the unnatural cysteine residue and recovered the activity of
procaspase-3, which triggered cell apoptosis.
[0324] This experiment confirms that the selective substitution of
active site cysteine with o-nitrobenzylcysteine in a protein in
living mammalian cells can be used, for example, to spatially
control the protein activity by using, e.g., UV light. Similar
results can be obtained for proteins with different reactive site
groups (for example, employment of o-nitrobenzylserine in the
active site residues of serine proteases). Further details
regarding photocaged or photoregulated amino acids can be found in
PCT publication WO 2006/034410 by Dieters et al. (Mar. 30, 2006),
titled "Adding Photoregulated Amino Acids to the Genetic Code," the
contents of which are incorporated herein in their entirety.
Example 9
Materials and Methods
Mammalian Cell Transfection and Western Blot Analysis
[0325] Both T-REx.TM. CHO and T-REx.TM. 293 cells (Invitrogen.TM.)
constitutively express the tetracycline repressor, which regulates
the expression of genes inserted into the pcDNA4/TO/myc-His A
plasmid. T-REx.TM. CHO cells were grown in F-12 (Invitrogen.TM.),
10% FBS (Invitrogen.TM.), 1% Pen-Strep (Invitrogen.TM.), 2 mM
L-glutamine (Invitrogen.TM.), and 10 .mu.g/ml blasticidin
(Invitrogen.TM.) at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2; T-REx.TM. 293 cells were grown in Gibco D-MEM media
(Invitrogen.TM.), 10% FBS, 1% Pen-Strep, 2 mM L-glutamine, and 5
.mu.g/ml blasticidin at 37.degree. C. in a humidified atmosphere of
5% CO.sub.2. Cells were grown to 80-90% confluency in Costar.RTM.
6-well cell culture clusters and then transfected with plasmids
using FuGENE.RTM. 6 (Roche Applied Science) (9 .mu.l FuGENE.RTM.+3
.mu.g plasmids). Six hours after transfection, the media were
replaced by fresh media that contained 1 .mu.g/ml of
tetracycline.
[0326] To test amber suppression of EcTyrRS variants in the
presence of their corresponding unnatural amino acids, the
unnatural amino acids were also added to the fresh media. The
concentrations of unnatural amino acids in the media were 10 mM for
pMpa (Bachem, Inc), 10 mM for pApa (Synchem, Inc); 1 mM for pBpa
(Bachem, Inc), 8 mM for plpa (Bachem, Inc), 5 mM for pAzpa (Bachem,
Inc), and 1 mM for pPpa. Chiral pure pPpa was synthesized as
described previously (Deiters et al. (2003), "Adding amino acids
with novel reactivity to the genetic code of Saccharomyces
cerevisiae," J Am Chem Soc 125:11782-11783). T-REx.TM. CHO cells
were grown for 24 hours after addition of tetracycline and then
harvested; T-REx.TM. 293 cells were harvested after 48-hour
incubation.
[0327] For Western blot analysis, harvested cells were lysed in
RIPA buffer (Upstate Biotechnology/Millipore) with a 1:100 dilution
of protease inhibitor cocktail (Sigma). The supernatant was
fractionated by SDS-PAGE under denatured condition and transferred
to a 0.45 .mu.m nitrocellulose membrane (Invitrogen.TM.). For
T-REx.TM. CHO cells, the proteins immobilized on the membrane were
probed with anti-myc antibody (Invitrogen.TM.; 1:5000 dilution) as
the primary antibody and anti-mouse IgG-HRP (Invitrogen.TM.) as the
secondary antibody. Chemiluminescence was then detected with PIERCE
ECL Western Blotting substrate. For T-REx.TM. 293 cells, the
membranes were probed with anti-His-HRP (Invitrogen.TM.; 1:5000
dilution) and then detected with PIERCE ECL Western Blotting
substrate.
Materials and Methods
Bacterial Cell Transfection
[0328] Top 10 E. coli cells (Invitrogen.TM.) were used for cloning,
maintaining, and amplifying plasmids. Pfx high-fidelity DNA
polymerase (Invitrogen.TM.) was used for polymerase chain reaction
(PCR). FuGENE.RTM. 6 (Roche Applied Science) was used as the
transfection reagent. All plasmids were verified by sequencing.
Example 10
Materials and Methods
Plasmid Constructions
[0329] pUC18-3BstRNA.sub.CUA.sup.Tyr
[0330] The BstRNA.sub.CUA.sup.Tyr gene was constructed by annealing
four oligodeoxynucleotides (Integrated DNA Technologies, Inc). The
gene consists of the corresponding tRNA sequence lacking the 3'-CCA
and the 5'-flanking sequence of the human tRNA.sup.Tyr gene:
TABLE-US-00006 (SEQ ID NO: 104)
AGCGCTCCGGTTTTTCTGTGCTGAACCTCAGGGGACGCCGACACACGTAC ACGTC
[0331] Two restriction sites--EcoRI at the 5'-end and BamHI at the
3'-end, were incorporated into the synthetic DNA duplex, which was
then inserted into pUC18 to afford pUC18-BstRNA.sub.CUA.sup.Tyr.
The BstRNA.sub.CUA.sup.Tyr gene in pUC-BstRNA.sub.CUA.sup.Tyr was
then amplified by PCR. The amplified BstRNA.sub.CUA.sup.Tyr
containing a BglII restriction site at the 5'-end and a BamHI site
at the 3'-end was inserted into the BamHI restriction site of
pUC18-BstRNA.sub.CUA.sup.Tyr to afford
pUC18-2BstRNA.sub.CUA.sup.Tyr. The plasmid
pUC18-2BstRNA.sub.CUA.sup.Tyr only contains one BamHI restriction
site that was used to incorporate another copy of
BstRNA.sub.CUA.sup.Tyr to afford pUC18-3BstRNA.sub.CUA.sup.Tyr.
EcTyrRS and its six variants were all amplified from
pEcTyrRS/tRNA.sub.CUA (Chin et al. (2003), "An expanded eukaryotic
genetic code," Science 301:964-967) by PCR and then inserted into
the XbaI and ApaI sites of the pcDNA4/TO/myc-His A plasmid
(Invitrogen.TM.) to afford the pcDNA4-EcTyrRS vectors. EcTyrRS
genes were placed after two tetracycline operator sequences and the
CMV promoter, which confer the tetracycline regulated gene
expression in Invitrogen.TM. T-REx.TM. cell lines. They were also
linked to a c-myc epitope for Western blot analysis. The mutations
in six EcTyrRS variants were: [0332] pMpaRS: Y37V/D182S/F183M
[0333] pApaRS: Y371/D182G/F183M/L186A [0334] pBpaRS:
Y37G/D182G/F183Y/L186M [0335] pIpaRS: Y371/D183S/F183M [0336]
pAzpaRS: Y37L/D182S/F183M/L186A [0337] pPpaRS:
Y37S/D182T/F183M/L186V pcDNA4-EcTyrRS and pcDNA4-GFP37TAG
[0338] pcDNA4-GFP was created by inserting wild type enhanced GFP
gene into the XbaI and ApaI sites of vector pcDNA4/TO/myc-His A. In
pcDNA4-GFP, the GFP gene was ligated with a myc epitope and
6.times.His tags at the C-terminus for Western blot analysis and
affinity purification of the expressed protein. The plasmid
pcDNA4-GFP37TAG, in which the triple codon of Y37 is mutated to TAG
amber codon, was generated by a QuikChange.RTM. site-directed
mutagenesis kit (Stratagene).
pSWAN-EcTyrRS Variants and pSWAN-GFP37TAG
[0339] To construct pSWAN-EcTyrRS variants, 3BstRNA.sub.CUA.sup.Tyr
was isolated from pUC18-3BstRNA.sub.CUA.sup.Tyr using BamHI and
EcoRI restriction enzymes and inserted into the BglII and MfeI
sites of pcDNA3.1/hygro (+) (Invitrogen.TM.) to afford
pcDNA-3BstRNA.sub.CUA.sup.Tyr. The genes encoding the six EcTyrRS
variants were then inserted into the XbaI and ApaI sites of
pcDNA-3BstRNA.sub.CUA.sup.Tyr to afford pSWAN-EcTyrRS variants. To
create pSWAN-GFP37TAG, a pSWAN-EcTyrRS vector was amplified and
digested with the MluI and PciI restriction enzymes. The digested
fragments were separated by agarose gel electrophoresis and the
fragment containing 3BstRNA.sub.CUA.sup.Tyr was purified by
QIAquick.RTM. gel purification kit (QIAGEN.RTM.). The
pcDNA4-GFP37TAG fragment from the CMV promoter to the BGH polyA
site were amplified by PCR, digested with the MluI and PciI
restriction enzymes, and then ligated with the pSWAN-EcTyrRS
fragment containing 3BstRNA.sub.CUA.sup.Tyr to afford
pSWAN-GFP37TAG (.about.4000 bp).
Example 11
Materials and Methods
Protein Expression and Purification
[0340] pSWAN-EcTyrRS variants and pSWAN-GFP37TAG were used to
express mutant GFPs containing unnatural amino acids. T-REx.TM. CHO
cells (Invitrogen.TM.) were grown in 75 cm.sup.2 tissue culture
flasks (BD Biosciences) to 80-90% confluency and transfected with 2
.mu.g of pSWAN-EcTyrRS and 10 .mu.g pSWAN-GFP37TAG using 601
FuGENE.RTM. 6. After overnight incubation, the media were changed
to the fresh media containing the unnatural amino acid (pMpa, 10
mM; pApa, 10 mM; pBpa, 1 mM; pAzpa, 5 mM; and pPpa, 1 mM) and 1
.mu.g/ml tetracycline. Cells were then grown for 1-2 days at
37.degree. C. before harvesting, and then lysed with RIPA lysis
buffer. The supernatant from the cell lysate was dialyzed against
and equilibrated with PBS buffer before loading onto an anti-myc
antibody agarose column (Sigma). The column was washed with 15
column volumes of PBS buffer and then eluted with 0.1 M ammonium
hydroxide. The purified proteins were neutralized with 0.1 M acetic
acid and analyzed by SDS-PAGE. The bands corresponding to GFP were
excised for mass spectrometric analysis. To determine the yield and
acquire intact protein mass spectra, the protein was concentrated
to .about.0.1 mg/ml after elution.
Example 12
Materials and Methods
Proteolysis
[0341] Proteolysis of affinity purified proteins (performed for
wild-type and pBpa samples) was carried out by overnight incubation
of proteins at 37.degree. C. with sequencing-grade modified trypsin
(Promega) using a substrate/enzyme ratio of 10:1 (wt/wt) based on
the estimated protein concentration in 100 mM triethylammonium
bicarbonate buffer (pH 8.5). In-gel digestions (performed for pMpa,
pAzpa, pApa, and pPpa samples) were performed according to a
modified EMBL procedure (see the website for EMBL, Heidelberg,
Germany, Bioanalytical Research Group). Briefly, gel slices were
excised to approximately 1 mm.times.1 mm cubes, washed for 5
minutes with water, 15 minutes with acetonitrile, and dried in a
vacuum centrifuge before being rehydrated at 0.degree. C. for 30
minutes with 12.5 ng/.mu.l of sequencing-grade modified trypsin in
50 mM triethylammonium bicarbonate buffer (pH 8.5). Excess buffer
was removed and 20 .mu.l of 50 mM triethylammonium bicarbonate
buffer (pH 8.5) was added to the gel slices before incubating at
37.degree. C. overnight. Peptides were extracted from the gel
slices as described in the protocol.
Example 13
Materials and Methods
Nano-RP LC/MS/MS
[0342] Nano-RP LC/MS/MS was performed with an HPLC pump,
autosampler (Agilent Technologies, Palo Alto, Calif.), and an LTQ
Orbitrap hybrid mass spectrometer (ThermoElectron, San Jose,
Calif.). Tryptic digests were loaded with a pressure bomb onto a 75
.mu.m i.d. precolumn packed with 4 cm of 5-.mu.m Monitor C18
particles (Column Engineering, Ontario, Calif.) at a flow rate of
approximately 2 .mu.l/min. The precolumn was then connected to the
HPLC pump and after several minutes of washing with solvent A the
analytical column with integrated emitter tip (360 .mu.m
O.D..times.75 .mu.m i.d; 10 cm of 5 .mu.M C18, .about.5-.mu.m tip)
was connected in series. The chromatographic profile was from 100%
solvent A (0.1% aqueous acetic acid) to 50% solvent B (0.1% acetic
acid in acetonitrile) in 40 min; the flow rate through the
analytical column was ca. 100 nl/min.
[0343] For data-dependent experiments, the mass spectrometer was
programmed to first record a high-resolution Orbitrap scan then a
full-scan ion trap spectrum (m/z 500-2,000). This was followed by
10 data-dependent MS/MS scans (relative collision energy=35%; 3-Da
isolation window) triggered of the ion-trap scan and finally two
targeted MS/MS scans. The first targeted MS/MS scan was set to
isolate and fragment the doubly charged precursor ion of the
predicted unnatural amino acid modified peptide
TABLE-US-00007 (FSVSGEGEGDATY*GK; SEQ ID NO: 103).
[0344] The second targeted MS/MS scan was always set to isolate and
fragment the doubly-charge precursor ion of the wild-type
peptide:
TABLE-US-00008 (FSVSGEGEGDATYGK; SEQ ID NO: 102)
at m/z 752.3. The raw data were searched against the MSDB database
using MASCOT (Matrixscience, London, UK) for protein identification
and to find the scans containing the target peptides with the
unnatural amino acids as variable modification. To estimate
fidelity at Y*37 of GFP-pMpa, single ion chromatograms (doubly
charged precursor ions at m/z 752.3 for the wt and 759.3 for pMpa)
and selected ion chromatograms (y.sub.9, y.sub.11, and y.sub.12)
were integrated using the Xcalibur software package
(ThermoElectron, San Jose, Calif.).
[0345] Intact protein mass spectra were acquired on an automated
LC/MS system (Waters, Milford, Mass.) consisting of a capillary LC
with auto-sampler and a QTOF2 mass spectrometer. GFP-pMpa (0.1
mg/ml) was loaded onto a reversed-phase protein Captrap (Michrom
Bioresources, Auburn, Calif.) for desalting with 0.1% acetic acid
in water and eluted with 80% acetonitrile/0.1% acetic acid at 5
.mu.l/min into the ESI source of the mass spectrometer. Summing,
smoothing, and deconvolution of the spectra with the MaxEnt1
algorithm were performed using the MassLynx (Waters, Milford,
Mass.) software package.
Example 14
Promoters for Mammalian Transcription
[0346] Strong promoters for tRNA transcription in mammalian cells
include 5' flanking sequence GATCCGACCGTGTGCTTGGCA GAAC (SEQ ID NO:
105) and 3' flanking sequence GTCCTTTTTTTG (SEQ ID NO: 106) for the
E. coli suppressor tRNA.sup.Leu5(CUA).
[0347] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. All publications, patents,
patent applications, and/or other documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication,
patent, patent application, and/or other document were individually
indicated to be incorporated by reference for all purposes.
Sequence CWU 1
1
106187RNAArtificialmutant suppressor tRNA Leu5 CUA 1gcccggaugg
uggaaucggu agacacaagg gauucuaaau cccucggcgu ucgcgcugug 60cggguucaag
ucccgcuccg gguacca 87285DNAEscherichia coli 2ggtggggttc ccgagcggcc
aaagggagca gactctaaat ctgccgtcat cgacttcgaa 60ggttcgaatc cttcccccac
cacca 85382DNAArtificialmutant suppressor tyrosyl-tRNA(CUA)
3ggaggggtag cgaagtggct aaacgcggcg gactctaaat ccgctccctt tgggttcggc
60ggttcgaatc cgtccccctc ca 824860PRTEscherichia coli 4Met Gln Glu
Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp
Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu
Lys Tyr Tyr Cys Leu Ser Met Leu Pro Tyr Pro Ser Gly Arg Leu35 40
45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50
55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp
Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn
Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met
Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser
Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu
Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr
Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln
Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp
Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185
190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp
Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg
Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn
Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr
Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala
Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro
Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val
Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Tyr Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Tyr Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met His Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg
660 665 670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val
Ala Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala
Leu Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp
Asp Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala
Ala Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr
Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu
Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys
Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 86052583DNAEscherichia coli
5atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca ttgggatgag
60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg cctgtctatg
120cttccctatc cttctggtcg actacacatg ggccacgtac gtaactacac
catcggtgac 180gtgatcgccc gctaccagca tatgctgggc aaaaacgtcc
tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg
gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta
tatgaaaaac cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg
agctggcaac ctgtacgccg gaatactacc gttgggaaca gaaattcttc
420accgagctgt ataaaaaagg cctggtatat aagaagactt ctgcggtcaa
ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg
gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag
tggtttatca aaatcaatgc ttacgctgac 600gagctgctca acgatctgga
taaactggat cactggccag acaccgttaa aaccatgcag 660cgtaactgga
tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa cgactatgac
720aacacgctga ccgtttacac tacccgcccg gacaccttta tgggttgtac
ctacctggcg 780gtacgtgcgg gtcatccgct ggcgcagaaa gcggcggaaa
ataatcctga actggcggcc 840tttattgacg aatgccgtaa caccaaagtt
gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt
taaagcggtt cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa
acttcgtatt gatggagtac ggcacgggcg cagttatggc ggtaccgggg
1020cacgaccagc gcgactacga gtttgcctct aaatacggcc tgaacatcaa
accggttatc 1080ctggcagctg acggctctga gccagatctt tctcagcaag
ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt
gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg
cgttggcgag cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc
gtcagcgtta ctggggcgcg ccgattccga tggtgacgct ggaagacggt
1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc tgccggaaga
tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat ccggagtggg
cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc
gacaccttta tggagtcctc ctggtactat 1500gcgcgctaca cttgcccgca
gtacaaagaa ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg
tggatatcta cattggtggt attgaacacg ccattatgca cctgctctac
1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg tgaactctga
cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct
tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat
gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc
ggcaggccat gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga
agaacaacgg tatcgacccg caggtgatgg ttgaacgtta cggcgcggac
1920accgttcgtc tgtttatgat gtttgcttct ccggctgata tgactctcga
atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct
ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac
gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa
aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca
ccgcaattgc ggcgattatg gagctgatga acaaactggc gaaagcacca
2220accgatggcg agcaggaccg cgctctgatg caggaagcac tgctggccgt
tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc acgctgtggc
aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct
gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa
cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac
aggttcgcga acgtgctggc caggaacatc tggtagcaaa atatcttgat
2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac tcctcaatct
ggtcgttggc 2580taa 25836424PRTEscherichia coli 6Met Ala Ser Ser Asn
Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr
Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala
Leu Tyr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly
His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly
His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Asp Phe Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42071275DNAEscherichia coli 7atggcaagca
gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg 60gacgaggaag
cgttagcaga gcgactggcg caaggcccga tcgcgctcta ttgcggcttc
120gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg
cctgaaacgc 180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg
gcgcgacggg tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag
ctgaacaccg aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca
ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360gctatcgcgg
cgaacaacta tgactggttc ggcaatatga atgtgctgac cttcctgcgc
420gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt
taagcagcgt 480ctcaaccgtg aagatcaggg gatttcgttc actgagtttt
cctacaacct gttgcagggt 540tatgacttcg cctgtctgaa caaacagtac
ggtgtggtgc tgcaaattgg tggttctgac 600cagtggggta acatcacttc
tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660tttggcctga
ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa
720ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta
ccagttctgg 780atcaacactg cggatgccga cgtttaccgc ttcctgaagt
tcttcacctt tatgagcatt 840gaagagatca acgccctgga agaagaagat
aaaaacagcg gtaaagcacc gcgcgcccag 900tatgtactgg cggagcaggt
gactcgtctg gttcacggtg aagaaggttt acaggcggca 960aaacgtatta
ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc
1020gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc
agacctgatg 1080caggcactgg tcgattctga actgcaacct tcccgtggtc
aggcacgtaa aactatcgcc 1140tccaatgcca tcaccattaa cggtgaaaaa
cagtccgatc ctgaatactt ctttaaagaa 1200gaagatcgtc tgtttggtcg
ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260atttgctgga aataa
127581275DNAArtificialmutant synthetase 8atggcaagca gtaacttgat
taaacaattg caagagcggg ggctggtagc ccaggtgacg 60gacgaggaag cgttagcaga
gcgactggcg caaggcccga tcgcactcgt gtgtggcttc 120gatcctaccg
ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc
180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg
tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg
aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca ggttgccccg
ttcctcgatt tcgactgtgg agaaaactct 360gctatcgcgg ccaataatta
tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420gatattggca
aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt
480ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct
gctgcagggt 540tatagtatgg cctgtttgaa caaacagtac ggtgtggtgc
tgcaaattgg tggttctgac 600cagtggggta acatcacttc tggtatcgac
ctgacccgtc gtctgcatca gaatcaggtg 660tttggcctga ccgttccgct
gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720ggcggcgcag
tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg
780atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt
tatgagcatt 840gaagagatca acgccctgga agaagaagat aaaaacagcg
gtaaagcacc gcgcgcccag 900tatgtactgg cggagcaggt gactcgtctg
gttcacggtg aagaaggttt acaggcggca 960aaacgtatta ccgaatgcct
gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020gaacagctgg
cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg
1080caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa
aactatcgcc 1140tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc
ctgaatactt ctttaaagaa 1200gaagatcgtc tgtttggtcg ttttacctta
ctgcgtcgcg gtaaaaagaa ttactgtctg 1260atttgctgga aataa
127591275DNAArtificialmutant synthetase 9atggcaagca gtaacttgat
taaacaattg caagagcggg ggctggtagc ccaggtgacg 60gacgaggaag cgttagcaga
gcgactggcg caaggcccga tcgcactcac ttgtggcttc 120gatcctaccg
ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc
180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg
tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg
aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca ggttgccccg
ttcctcgatt tcgactgtgg agaaaactct 360gctatcgcgg ccaataatta
tgactggttc agcaatatga atgtgctgac cttcctgcgc 420gatattggca
aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt
480ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct
gctgcagggt 540tatacgtatg cctgtctgaa caaacagtac ggtgtggtgc
tgcaaattgg tggttctgac 600cagtggggta acatcacttc tggtatcgac
ctgacccgtc gtctgcatca gaatcaggtg 660tttggcctga ccgttccgct
gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720ggcggcgcag
tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg
780atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt
tatgagcatt 840gaagagatca acgccctgga agaagaagat aaaaacagcg
gtaaagcacc gcgcgcccag 900tatgtactgg cggagcaggt gactcgtctg
gttcacggtg aagaaggttt acaggcggca 960aaacgtatta ccgaatgcct
gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020gaacagctgg
cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg
1080caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa
aactatcgcc 1140tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc
ctgaatactt ctttaaagaa 1200gaagatcgtc tgtttggtcg ttttacctta
ctgcgtcgcg gtaaaaagaa ttactgtctg 1260atttgctgga aataa
1275101275DNAArtificialmutant synthetase 10atggcaagca gtaacttgat
taaacaattg caagagcggg ggctggtagc ccaggtgacg 60gacgaggaag cgttagcaga
gcgactggcg caaggcccga tcgcactcgt gtgtggcttc 120gatcctaccg
ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc
180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg
tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg
aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca ggttgccccg
ttcctcgatt tcgactgtgg agaaaactct
360gctatcgcgg ccaataatta tgactggttc ggcaatatga atgtgctgac
cttcctgcgc 420gatattggca aacacttctc cgttaaccag atgatcaaca
aagaagcggt taagcagcgt 480ctcaaccgtg aagatcaggg gatttcgttc
actgagtttt cctacaacct gctgcagggt 540tatagtatgg cctgtttgaa
caaacagtac ggtgtggtgc tgcaaattgg tggttctgac 600cagtggggta
acatcacttc tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg
660tttggcctga ccgttccgct gatcactaaa gcagatggca ccaaatttgg
taaaactgaa 720ggcggcgcag tctggttgga tccgaagaaa accagcccgt
acaaattcta ccagttctgg 780atcaacactg cggatgccga cgtttaccgc
ttcctgaagt tcttcacctt tatgagcatt 840gaagagatca acgccctgga
agaagaagat aaaaacagcg gtaaagcacc gcgcgcccag 900tatgtactgg
cggagcaggt gactcgtctg gttcacggtg aagaaggttt acaggcggca
960aaacgtatta ccgaatgcct gttcagcggt tctttgagtg cgctgagtga
agcggacttc 1020gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg
aaaagggcgc agacctgatg 1080caggcactgg tcgattctga actgcaacct
tcccgtggtc aggcacgtaa aactatcgcc 1140tccaatgcca tcaccattaa
cggtgaaaaa cagtccgatc ctgaatactt ctttaaagaa 1200gaagatcgtc
tgtttggtcg ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg
1260atttgctgga aataa 1275111275DNAArtificialmutant synthetase
11atggcaagca gtaacttgat taaacaattg caagagcggg ggctggtagc ccaggtgacg
60gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcgt gtgtggcttc
120gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg
cctgaaacgc 180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg
gcgcgacggg tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag
ctgaacaccg aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca
ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 360gctatcgcgg
ccaataatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc
420gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt
taagcagcgt 480ctcaaccgtg aagatcaggg gatttcgttc actgagtttt
cctacaacct gctgcagggt 540tatagtatgg cctgtttgaa caaacagtac
ggtgtggtgc tgcaaattgg tggttctgac 600cagtggggta acatcacttc
tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg 660tttggcctga
ccgttccgct gatcactaaa gcagatggca ccaaatttgg taaaactgaa
720ggcggcgcag tctggttgga tccgaagaaa accagcccgt acaaattcta
ccagttctgg 780atcaacactg cggatgccga cgtttaccgc ttcctgaagt
tcttcacctt tatgagcatt 840gaagagatca acgccctgga agaagaagat
aaaaacagcg gtaaagcacc gcgcgcccag 900tatgtactgg cggagcaggt
gactcgtctg gttcacggtg aagaaggttt acaggcggca 960aaacgtatta
ccgaatgcct gttcagcggt tctttgagtg cgctgagtga agcggacttc
1020gaacagctgg cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc
agacctgatg 1080caggcactgg tcgattctga actgcaacct tcccgtggtc
aggcacgtaa aactatcgcc 1140tccaatgcca tcaccattaa cggtgaaaaa
cagtccgatc ctgaatactt ctttaaagaa 1200gaagatcgtc tgtttggtcg
ttttacctta ctgcgtcgcg gtaaaaagaa ttactgtctg 1260atttgctgga aataa
1275121275DNAArtificialmutant synthetase 12atggcaagca gtaacttgat
taaacaattg caagagcggg ggctggtagc ccaggtgacg 60gacgaggaag cgttagcaga
gcgactggcg caaggcccga tcgcactcac gtgtggcttc 120gatcctaccg
ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc
180ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg
tctgattggc 240gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg
aagaaactgt tcaggagtgg 300gtggacaaaa tccgtaagca ggttgccccg
ttcctcgatt tcgactgtgg agaaaactct 360gctatcgcgg ccaataatta
tgactggttc ggcaatatga atgtgctgac cttcctgcgc 420gatattggca
aacacttctc cgttaaccag atgatcaaca aagaagcggt taagcagcgt
480ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacagcct
gctgcagggt 540tatacgatgg cctgtctgaa caaacagtac ggtgtggtgc
tgcaaattgg tggttctgac 600cagtggggta acatcacttc tggtatcgac
ctgacccgtc gtctgcatca gaatcaggtg 660tttggcctga ccgttccgct
gatcactaaa gcagatggca ccaaatttgg taaaactgaa 720ggcggcgcag
tctggttgga tccgaagaaa accagcccgt acaaattcta ccagttctgg
780atcaacactg cggatgccga cgtttaccgc ttcctgaagt tcttcacctt
tatgagcatt 840gaagagatca acgccctgga agaagaagat aaaaacagcg
gtaaagcacc gcgcgcccag 900tatgtactgg cggagcaggt gactcgtctg
gttcacggtg aagaaggttt acaggcggca 960aaacgtatta ccgaatgcct
gttcagcggt tctttgagtg cgctgagtga agcggacttc 1020gaacagctgg
cgcaggacgg cgtaccgatg gttgagatgg aaaagggcgc agacctgatg
1080caggcactgg tcgattctga actgcaacct tcccgtggtc aggcacgtaa
aactatcgcc 1140tccaatgcca tcaccattaa cggtgaaaaa cagtccgatc
ctgaatactt ctttaaagaa 1200gaagatcgtc tgtttggtcg ttttacctta
ctgcgtcgcg gtaaaaagaa ttactgtctg 1260atttgctgga aataa
127513540DNAArtificialmutant synthetase 13cgggggctgg tagcccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcacttgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcagcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttatacg tatgcctgtc tgaacaaaca
gtacggtgtg 54014540DNAArtificialmutant synthetase 14cgggggctgg
taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac
tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcagcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatacg tatgcctgtc
tgaacaaaca gtacggtgtg 54015540DNAArtificialmutant synthetase
15cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcagcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatacg tatgcctgtc
tgaacaaaca gtacggtgtg 54016540DNAArtificialmutant synthetase
16cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttattcg tatgcctgtg
cgaacaaaca gtacggtgtg 54017540DNAArtificialmutant synthetase
17cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcacttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcagcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatacg tatgcctgtc
tgaacaaaca gtacggtgtg 54018540DNAArtificialmutant synthetase
18cgggggctgg taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcctttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttattct attgcctgtt
cgaacaaaca gtacggtgtg 54019540DNAArtificialmutant synthetase
19cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcgtgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatagt attgcctgtt
tgaacaaaca gtacggtgtg 54020540DNAArtificialmutant synthetase
20cgggggctgg taccccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcgtgtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatagt attgcctgtt
tgaacaaaca gtacggtgtg 54021540DNAArtificialmutant synthetase
21cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tctggtgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaagg
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaatt gttatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatatg cgtgcctgtg
agaacaaaca gtacggtgtg 54022624DNAArtificialmutant synthetase
22cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact ggcgcaaggc
60ccgatcgcac tcatttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaaggtc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatggt atggcctgtg
ctaacaaaca gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccaatgg
ggtaacatca cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62423609DNAArtificialmutant synthetase 23caggtgacgg acgaggaagc
gttagcagag cgactggcgc aaggcccgat cgcactcggt 60tgtggcttcg atcctaccgc
tgacagcttg catttggggc atcttgttcc attgttatgc 120ctgaaacgct
tccagcaggc gggccacaag ccggttgcgc tggtaggcgg cgcgacgggt
180ctgattggcg acccgagctt caaagctgcc gagcgtaagc tgaacaccga
agaaactgtt 240caggagtggg tggacaaaat ccgtaagcag gttgccccgt
tcctcgattt cgactgtgga 300gaaaactctg ctatcgcggc caataattat
gactggttcg gcaatatgaa tgtgctgacc 360ttcctgcgcg atattggcaa
acacttctcc gttaaccaga tgatcaacaa agaagcggtt 420aagcagcgtc
tcaaccgtga agatcagggg atttcgttca ctgagttttc ctacaacctg
480ctgcagggtt atggttttgc ctgtttgaac aaacagtacg gtgtggtgct
gcaaattggt 540ggttctgacc agtggggtaa catcacttct ggtatcgacc
tgacccgtcg tctgcatcag 600aatcaggtg 60924591DNAArtificialmutant
synthetase 24gcgttagcag agcgactggc gcaaggcccg atcgcactcg ggtgtggctt
cgatcctacc 60gctgacagct tgcatttggg gcatcttgtt ccattgttat gcctgaaacg
cttccagcag 120gcgggccaca agccggttgc gctggtaggc ggcgcgacgg
gtctgattgg cgacccgagc 180ttcaaagctg ccgagcgtaa gctgaacacc
gaagaaactg ttcaggagtg ggtggacaaa 240atccgtaagc aggttgcccc
gttcctcgat ttcgactgtg gagaaaactc tgctatcgcg 300gccaataatt
atgactggtt cggcaatatg aatgtgctga ccttcctgcg cgatattggc
360aaacacttct ccgttaacca gatgatcaac aaagaagcgg ttaagcagcg
tctcaaccgt 420gaagatcagg ggatttcgtt cactgagttt tcctacaacc
tgctgcaggg ttatggttat 480gcctgtatga acaaacagta cggtgtggtg
ctgcaaattg gtggttctga ccagtggggt 540aacatcactt ctggtatcga
cctgacccgt cgtctgcatc agaatcaggt g 59125621DNAArtificialmutant
synthetase 25gggctggtag cccaggtgac ggacgnagaa gcgttagcag agcgactggc
gcaaggcccg 60atcgcactcc tttgtggctt cgatcctacc gctgacagct tgcatttggg
gcatcttgtt 120ccattgttat gcctgaaacg cttccagcag gcgggccaca
agccggttgc gctggtaggc 180ggcgcgacgg gtctgattgg cgacccgagc
ttcaaagctg ccgagcgtaa gctgaacacc 240gaagaaactg ttcaggagtg
ggtggacaaa atccgtaagc aggttgcccc gttcctcgat 300ttcgactgtg
gagaaaactc tgctatcgcg gccaataatt atgactggtt cggcaatatg
360aatgtgctga ccttcctgcg cgatattggc aaacacttct ccgttaacca
gatgatcaac 420aaagaagcgg ttaagcagcg tctcaaccgt gaagatcagg
ggatttcgtt cactgagttt 480tcctacaacc tgctgcaggg ttattctatg
gcctgtgcga acaaacagta cggtgtggtg 540ctgcaaattg gtggttctga
ccagtggggt aacatcactt ctggtatcga cctgacccgt 600cgtctgcatc
anaatcangt g 62126588DNAArtificialmutant synthetase 26ttagcagagc
gactggcgca aggcccgatc gcactcgttt gtggcttcga tcctaccgct 60gacagcttgc
atttggggca tcttgttcca ttgttatgcc tgaaacgctt ccagcaggcg
120ggccacaagc cggttgcgct ggtaggcggc gcgacgggtc tgattggcga
cccgagcttc 180aaagctgccg agcgtaagct gaacaccgaa gaaactgttc
aggagtgggt ggacaaaatc 240cgtaagcagg ttgccccgtt cctcgatttc
gactgtggag aaaactctgc tatcgcggcc 300aataattatg actggttcgg
caatatgaat gtgctgacct tcctgcgcga tattggcaaa 360cacttctccg
ttaaccagat gatcaacaaa gaagcggtta agcagcgtct caaccgtgaa
420gatcagggga tttcgttcac tgagttttcc tacaacctgc tgcagggtta
ttctgcggcc 480tgtgcgaaca aacagtacgg tgtggtgctg caaattggtg
gttctgacca gtggggtaac 540atcacttctg gtatcgacct gacccgtcgt
ctgcatcaga atcaggtg 58827600DNAArtificialmutant synthetase
27gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcct gtgtggcttc
60gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg cctgaaacgc
120ttccagcagg cgggccacaa gccggttgcg ctggtaggcg gcgcgacggg
tctgattggc 180gacccgagct tcaaagctgc cgagcgtaag ctgaacaccg
aagaaactgt tcaggagtgg 240gtggacaaaa tccgtaagca ggttgccccg
ttcctcgatt tcgactgtgg agaaaactct 300gctatcgcgg ccaataatta
tgactggttc ggcaatatga atgtgctgac cttcctgcgc 360gatattggca
aacacttctc cgttaaccag atgatcaaca aanaagcggt taagcagcgt
420ctcaaccgtg aagatcaggg gatttcgttc actgagtttt cctacaacct
gctgcagggt 480tattcggctg cctgtgcgaa caaacagtac ggngnggngc
tgcaaattgg nggttctgac 540caggggggta acatcacttc tggtatcgac
ctgacccgtc gtctgcatca aaatcaggtg 60028591DNAArtificialmutant
synthetase 28gcgttagcag agcgactggc gcaaggcccg atcgcactcg tttgtggctt
cgatcctacc 60gctgacagct tgcatttggg gcatcttgtt ccattgttgt gcctgaaacg
cttccagcag 120gcgggccaca agccggttgc gctggtaggc ggcgcgacgg
gtctgattgg cgacccgagc 180ttcaaagctg ccgagcgtaa gctgaacacc
gaagaaactg ttcaggagtg ggtggacaaa 240atccgtaagc aggttgcccc
gttcctcgat ttcgactgtg gagaaaactc tgctatcgcg 300gccaataatt
atgactggtt cggcaatatg aatgtgctga ccttcctgcg cgatattggc
360aaacacttct ccgttaacca gatgatcaac aaagaagcgg ttaagcagcg
tctcaaccgt 420gaagatcagg ggatttcgtt cactgagttt tcctacaacc
tgctgcaggg ttatagtgcg 480gcctgtgtta acaaacagta cggtgtggtg
ctgcaaattg gtggttctga ccagtggggt 540aacatcactt ctggtatcga
cctgacccgt cgtctgcatc agaatcangt g 59129600DNAArtificialmutant
synthetase 29gacgaggaag cgttagcaga gcgactggcg caaggcccga tcgcactcat
ttgtggcttc 60gatcctaccg ctgacagctt gcatttgggg catcttgttc cattgttatg
cctgaaacgc 120ttccagcagg cgggccacaa gccggttgcg ctggtaggcg
gcgcgacggg tctgattggc 180gacccgagct tcaaagctgc cgagcgtaag
ctgaacaccg aagaaactgt tcaggagtgg 240gtggacaaaa tccgtaagca
ggttgccccg ttcctcgatt tcgactgtgg agaaaactct 300gctatcgcgg
ccaatgatta tgactggttc ggcaatatga atgtgctgac cttcctgcgc
360gatattggca aacacttctc cgttaaccag atgatcaaca aagaagcggt
taagcagcgt 420ctcaaccgtg aagatcaggg gatttcgttc actgagtttt
cctacaacct gctgcagggt 480tataattttg cctgtgtgaa caaacagtac
ggtgtggtgc tgcaaattgg tggttctgac 540cagtggggta acatcacttc
tggtatcgac ctgacccgtc gtctgcatca gaatcaggtg
60030579DNAArtificialmutant synthetase 30cgactggcgc aaggcccgat
cgcactcacg tgtggcttcg atcctaccgc tgacagcttg 60catttggggc atcttgttcc
attgttatgc ctgaaacgct tccagcaggc gggccacaag 120ccggttgcgc
tggtaggcgg cgcgacgggt ctgattggcg acccgagctt caaagctgcc
180gagcgtaagc tgaacaccga agaaactgtt caggagtggg tggacaaaat
ccgtaagcag 240gttgccccgt tcctcgattt cgactgtgga gaaaactctg
ctatcgcggc caataattat 300gactggttcg gcaatatgaa tgtgctgacc
ttcctgcgcg atattggcaa acacttctcc 360gttaaccaga tgatcaacaa
agaagcggtt aagcagcgtc tcaaccgtga agatcagggg 420atttcgttca
ctgagttttc ctacaatctg ctgcagggtt attcggctgc ctgtcttaac
480aaacagtacg gtgtggtgct gcaaattggt ggttctgacc agtggggtaa
catcacttct 540ggtatcgacc tgacccgtcg tctgcatcag aatcaggtg
57931624DNAArtificialmutant synthetase 31cgggggctgg tancccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcgggtgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttattct atggcctgtt tgaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctganc 600cgtcgtctgc atcagaatca ggtg
62432625DNAArtificialmutant synthetase 32cgggggctgg tagcccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcacgtgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca atctgctgca gggttattcg gctgcctgtc ttaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgaacctgan 600ccgtcgtctg catcaaaatc aagtg
62533624DNAArtificialmutant synthetase 33cgggggctgg taccccaagt
gacggacgag gaaacgttag cagagcgact ggcgcaaggc 60ccgatcgcac tctcttgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcaggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttatacg atggcctgtg tgaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62434624DNAArtificialmutant synthetase 34cgggggctgg tagcccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcgcgtgcgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaagg ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttattct tatgcctgtc ttaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62435624DNAArtificialmutant synthetase 35cgggggctgg tagcccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcgcgtgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttatacg atggcctgtt gtaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62436624DNAArtificialmutant synthetase 36cgggggctgg taccccaagt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcacgtgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcgctgag
480ttttcctaca acctgctgca gggttatacg tttgcctgta tgaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62437606DNAArtificialmutant synthetase 37gtgacggacg aggaagcgtt
agcagagcga ctggcgcaag gcccgatcgc actcacgtgt 60ggcttcgatc ctaccgctga
cagcttgcat ttggggcatc ttgttccatt gttatgcctg 120aaacgcttcc
agcaggcggg ccacaagccg gttgcgctgg taggcggcgc gacgggtctg
180attggcgacc cgagcttcaa agctgccgag cgtaagctga acaccgaaga
aactgttcag 240gagtgggtgg acaaaatccg taagcaggtt gccccgttcc
tcgatttcga ctgtggagaa 300aactctgcta tcgcggccaa taattatgac
tggttcggca atatgaatgt gctgaccttc 360ctgcgcgata ttggcaaaca
cttctccgtt aaccagatga tcaacaaaga agcggttaag 420cagcgtctca
accgtgaaga tcaggggatt tcgttcactg agttttccta caatctgctg
480cagggttatt cggctgcctg tcttaacaaa cagtacggtg tggtgctgca
aattggtggt 540tctgaccagt ggggtaacat cacttctggt atcgacctga
cccgtcgtct gcatcagaat 600caggtg 60638624DNAArtificialmutant
synthetase 38cgggggctgg tagcccaggt gacggacgag gaagcgttag cagagcgact
ggcgcaaggc 60ccgatcgcac tcgtttgtgg cttcgatcct accgctgaca gcttgcattt
ggggcatctt 120gttccattgt tatgcctgaa acgcttccag caggcgggcc
acaagccggt tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg
agcttcaaag ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga
gtgggtggac aaaatccgta agcaggttgc cccgttcctc 300gatttcgact
gtggagaaaa ctctgctatc gcggccaata attatgactg gttcggcaat
360atgaatgtgc tgaccttcct gcgcgatatt ggcaaacact tctccgttaa
ccagatgatc 420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc
aggggatttc gttcactgag 480ttttcctaca acctgctgca gggttattcg
atggcctgta cgaacaaaca gtacggtgtg 540gtgctgcaaa ttggtggttc
tgaccagtgg ggtaacatca cttctggtat cgacctgacc 600cgtcgtctgc
atcagaatca ggtg 62439624DNAArtificialmutant synthetase 39cgggggctgg
tancccaagt gacggacggg gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac
tcagttgtgg cttcgatcct accgctgaca gcttgcattt ggggcatctt
120gttccattgt tatgcctgaa acgcttccag caggcgggcc acaagccggt
tgcgctggta 180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag
ctgccgagcg taagctgaac 240accgaagaaa ctgttcagga gtgggtggac
aaaatccgta agcaggttgc cccgttcctc 300gatctcgact gtggagaaaa
ctctgctatc gcggccaata attatgactg gttcggcaat 360atgaatgtgc
tgaccttcct gcgcgatatt ggcaaacact tctccgttaa ccagatgatc
420aacaaagaag cggttaagca gcgtctcaac cgtgaagatc aggggatttc
gttcactgag 480ttttcctaca acctgctgca gggttatagt tttgcctgtc
tgaacaaaca gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg
ggtaacatca cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
62440624DNAArtificialmutant synthetase 40cgggggctgg tagcccaggt
gacggacgag gaagcgttag cagagcgact ggcgcaaggc 60ccgatcgcac tcacgtgtgg
cttcgatcct accgctgaca gcttgcattt ggggcatctt 120gttccattgt
tatgcctgaa acgcttccag caggcgggcc acaagccggt tgcgctggta
180ggcggcgcga cgggtctgat tggcgacccg agcttcaaag ctgccgagcg
taagctgaac 240accgaagaaa ctgttcagga gtgggtggac aaaatccgta
agcaggttgc cccgttcctc 300gatttcgact gtggagaaaa ctctgctatc
gcggccaata attatgactg gttcggcaat 360atgaatgtgc tgaccttcct
gcgcgatatt ggcaaacact tctccgttaa ccagatgatc 420aacaaagaag
cggttaagca gcgtctcaac cgtgaagatc aggggatttc gttcactgag
480ttttcctaca acctgctgca gggttatacg tttgcctgta ctaacaaaca
gtacggtgtg 540gtgctgcaaa ttggtggttc tgaccagtgg ggtaacatca
cttctggtat cgacctgacc 600cgtcgtctgc atcagaatca ggtg
624412583DNAArtificialmutant synthetase 41atggaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctgct 120aatccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgtccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgactct agaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctg ctggatttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatcgg
tattggtggt attgaacacg ccattatgac gctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583422589DNAArtificialmutant synthetase 42atggaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctgct 120aatccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgtccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacgcgttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgactct agaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctg ctggatttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatcgg
tattggtggt attgaacacg ccattatgac gctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc
2580taagcggcc 2589432589DNAArtificialmutant synthetase 43atggaagagc
aataccgccc ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat
ttgaagtaac cgaagacgag agcaaagaga agtattactg cctgtctgct
120aatccctatc cttctggtcg actacacatg ggccacgtac gtaactacac
catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc
tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg
gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta
tatgaaaaac cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg
agctggcaac ctgtacgccg gaatactacc gttgggaaca gaaattcttc
420accgagctgt ataaaaaagg cctggtatat aagaagactt ctgcggtcaa
ctggtgtccg 480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg
gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag
tggtttatca aaatcactgc ttacgctgac 600gagctgctca acgatctgga
taaactggat cactggccag acaccgttaa aaccatgcag 660cgtaactgga
tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa cgactatgac
720aacacgctga ccgtttacac tacccgcccg gacaccttta tgggttgtac
ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa
ataatcctga actggcggcc 840tttattgacg aatgccgtaa caccaaagtt
gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt
taaagcggtt cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa
acttcgtatt gatggagtac ggcacgggcg cagttatggc ggcgccgggg
1020cacgaccagc gcgactacga gtttgcctct aaatacggcc tgaacatcaa
accggttatc 1080ctggcagctg acggctctga gccagatctt tctcagcaag
ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt
gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg
cgttggcgag cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc
gtcagcgtta ctggggcgcg ccgattccga tggtgactct agaagacggt
1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc tgccggaaga
tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat ccggagtggg
cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc
gacaccttta tggagtcctg ctggatttat 1500gcgcgctaca cttgcccgca
gtacaaagaa ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg
tggatatcgg tattggtggt attgaacacg ccattatgac gctgctctac
1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg tgaactctga
cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct
tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat
gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc
ggcaggccat gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga
agaacaacgg tatcgacccg caggtgatgg ttgaacgtta cggcgcggac
1920accgttcgtc tgtttatgat gtttgcttct ccggctgata tgactctcga
atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct
ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac
gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa
aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca
ccgcaattgc ggcgattatg gagctgatga acaaactggc gaaagcacca
2220accgatggcg agcaggatcg cgctctgatg caggaagcac tgctggccgt
tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc acgctgtggc
aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct
gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa
cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac
aggttcgcga acgtgctggc caggaacatc tggtagcaaa atatcttgat
2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac tcctcaatct
ggtcgttggc 2580taagcggcc 2589442844DNAArtificialmutant synthetase
44atctcgaagc acacgaaact ttttccttcc ttcattcacg cacactactc tctaatgagc
60aacggtatac ggccttcctt ccagttactt gaatttgaaa taaaaaaaag tttgctgtct
120tgctatcaag tataaataga cctgcaatta ttaatctttt gtttcctcgt
cattgttctc 180gttccctttc ttccttgttt ctttttctgc acaatatttc
aagctatacc aagcatacaa 240tcaactgaat tcagtatgga agagcaatac
cgcccggaag agatagaatc caaagtacag 300cttcattggg atgagaagcg
cacatttgaa gtaaccgaag acgagggcaa agagaagtat 360tactgcctgt
cttggtcgcc ctatccttct ggtcgactac acatgggcca cgtacgtaac
420tacaccatcg gtgacgtgat cgcccgctac cagcgtatgc tgggcaaaaa
cgtcctgcag 480ccgatcggct gggacgcgtt tggtctgcct gcggaaggcg
cggcggtgaa aaacaacacc 540gctccggcac cgtggacgta cgacaacatc
gcgtatatga aaaaccagct caaaatgctg 600ggctttggtt atgactggag
ccgcgagctg gcaacctgta cgccggaata ctaccgttgg 660gaacagaaat
tcttcaccga gctgtataaa aaaggcctgg tatataagaa gacttctgcg
720gtcaactggt gtccgaacga ccagaccgta ctggcgaacg aacaagttat
cgacggctgc 780tgctggcgct gcgataccaa agttgaacgt aaagagatcc
cgcagtggtt tatcaaaatc 840actgcttacg ctgacgagct gctcaacgat
ctggataaac tggatcactg gccagacacc 900gttaaaacca tgcagcgtaa
ctggatcggt cgttccgaag gcgtggagat caccttcaac 960gttaacgact
atgacaacac gctgaccgtt tacgcttccc gcccggacac ctttatgggt
1020tgtacctacc tggcggtagc tgcgggtcat ccgctggcgc agaaagcggc
ggaaaataat 1080cctgaactgg cggcctttat tgacgaatgc cgtaacacca
aagttgccga agctgaaatg 1140gcgacgatgg agaaaaaagg cgtcgatact
ggctttaaag cggttcaccc attaacgggc 1200gaagaaattc ccgtttgggc
agcaaacttc gtattgatgg agtacggcac gggcgcagtt 1260atggcggtac
cggggcacga ccagcgcgac tacgagtttg cctctaaata cggcctgaac
1320atcaaaccgg ttatcctggc agctgacggc tctgagccag atctttctca
gcaagccctg 1380actgaaaaag gcgtgctgtt caactctggc gagttcaacg
gtcttgacca tgaagcggcc 1440ttcaacgcca tcgccgataa actgactgcg
atgggcgttg gcgagcgtaa agtgaactac 1500cgcctgcgcg actggggtgt
ttcccgtcag cgttactggg gcgcgccgat tccgatggtg 1560actctagaag
acggtaccgt aatgccgacc ccggacgacc agctgccggt gatcctgccg
1620gaagatgtgg taatggacgg cattaccagc ccgattaaag cagatccgga
gtgggcgaaa 1680actaccgtta acggtatgcc agcactgcgt gaaaccgaca
ctttcgacac ctttatggag 1740tcctgctgga tttatgcgcg ctacacttgc
ccgcagtaca aagaaggtat gctggattcc 1800gaagcggcta actactggct
gccggtggat atcgcgattg gtggtattga acacgccatt 1860atggggctgc
tctacttccg cttcttccac aaactgatgc gtgatgcagg catggtgaac
1920tctgacgaac cagcgaaaca gttgctgtgt cagggtatgg tgctggcaga
tgccttctac 1980tatgttggcg aaaacggcga acgtaactgg gtttccccgg
ttgatgctat cgttgaacgt 2040gacgagaaag gccgtatcgt gaaagcgaaa
gatgcggcag gccatgaact ggtttatacc 2100ggcataagca aaatgtccaa
gtcgaagaac aacggtatcg acccgcaggt gatggttgaa 2160cgttacggcg
cggacaccgt tcgtctgttt atgatgtttg cttctccggc tgatatgact
2220ctcgaatggc aggaatccgg tgtggaaggg gctaaccgct tcctgaaacg
tgcctggaaa 2280ctggtttacg agcacacagc aaaaggtgat gttgcggcac
tgaacgttga tgcgctgact 2340gaaaatcaga aagcgctgcg tcgcgatgtg
cataaaacga tcgctaaagt gaccgatgat 2400atcggccgtc gtcagacctt
caacaccgca attgcggcga ttatggagct gatgaacaaa 2460ctggcgaaag
caccaaccga tggcgagcag gatcgcgctc tgatgcagga agcactgctg
2520gccgttgtcc gtatgcttaa cccgttcacc ccgcacatct gcttcacgct
gtggcaggaa 2580ctgaaaggcg aaggcgatat cgacaacgcg ccgtggccgg
ttgctgacga aaaagcgatg 2640gtggaagact ccacgctggt cgtggtgcag
gttaacggta aagtccgtgc caaaatcacc 2700gttccggtgg acgcaacgga
agaacaggtt cgcgaacgtg ctggccagga acatctggta 2760gcaaaatatc
ttgatggcgt tactgtacgt aaagtgattt acgtaccagg taaactcctc
2820aatctggtcg ttggctaagc ggcc 2844452583DNAArtificialmutant
synthetase 45atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctgct 120gcgccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggccttat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcgt tattggtggt attgaacacg ccattatggg
gctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 2583462583DNAArtificialmutant
synthetase 46atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctgtg 120atgccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggctgtat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcct gattggtggt attgaacacg ccattatggg
gctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 2583472583DNAArtificialmutant
synthetase 47atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctcat 120cctccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctgggcgtat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcat gattggtggt attgaacacg ccattatggg
tctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 2583482583DNAArtificialmutant
synthetase 48atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctgtg 120tatccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggctgtat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcct gattggtggt attgaacacg ccattatggg
tctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 2583492583DNAArtificialmutant
synthetase 49atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctttg 120gagccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggcgttat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcgc tattggtggt attgaacacg ccattatggg
tctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583502583DNAArtificialmutant synthetase 50atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctatg 120gagccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgacgct ggaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctc ctggcgttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatctt
tattggtggt attgaacacg ccattatggg gctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583512583DNAArtificialmutant synthetase 51atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctttg 120gagccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgacgct ggaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctc ctggcgttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatctg
tattggtggt attgaacacg ccattatggg tctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583522583DNAArtificialmutant synthetase 52atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctttt 120gagccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgacgct ggaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctc ctggcgttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatcac
gattggtggt attgaacacg ccattatggg tctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583532583DNAArtificialmutant synthetase 53atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctggg 120gagccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgacgct ggaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctc ctggcggtat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatcct
gattggtggt attgaacacg ccattatggg tctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583542583DNAArtificialmutant synthetase 54atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtctggt 120tggccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa cgactatgac 720aacacgctga
ccgtttacac tacccgcccg gacaccttta tgggttgtac ctacctggcg
780gtagctgcgg gtcatccgct ggcgcagaaa gcggcggaaa ataatcctga
actggcggcc 840tttattgacg aatgccgtaa caccaaagtt gccgaagctg
aaatggcgac gatggagaaa 900aaaggcgtcg atactggctt taaagcggtt
cacccattaa cgggcgaaga aattcccgtt 960tgggcagcaa acttcgtatt
gatggagtac ggcacgggcg cagttatggc ggtaccgggg 1020cacgaccagc
gcgactacga gtttgcctct aaatacggcc tgaacatcaa accggttatc
1080ctggcagctg acggctctga gccagatctt tctcagcaag ccctgactga
aaaaggcgtg 1140ctgttcaact ctggcgagtt caacggtctt gaccatgaag
cggccttcaa cgccatcgcc 1200gataaactga ctgcgatggg cgttggcgag
cgtaaagtga actaccgcct gcgcgactgg 1260ggtgtttccc gtcagcgtta
ctggggcgcg ccgattccga tggtgacgct ggaagacggt 1320accgtaatgc
cgaccccgga cgaccagctg ccggtgatcc tgccggaaga tgtggtaatg
1380gacggcatta ccagcccgat taaagcagat ccggagtggg cgaaaactac
cgttaacggt 1440atgccagcac tgcgtgaaac cgacactttc gacaccttta
tggagtcctc ctgggcttat 1500gcgcgctaca cttgcccgca gtacaaagaa
ggtatgctgg attccgaagc ggctaactac 1560tggctgccgg tggatatcct
tattggtggt attgaacacg ccattatggg tctgctctac 1620ttccgcttct
tccacaaact gatgcgtgat gcaggcatgg tgaactctga cgaaccagcg
1680aaacagttgc tgtgtcaggg tatggtgctg gcagatgcct tctactatgt
tggcgaaaac 1740ggcgaacgta actgggtttc cccggttgat gctatcgttg
aacgtgacga gaaaggccgt 1800atcgtgaaag cgaaagatgc ggcaggccat
gaactggttt ataccggcat gagcaaaatg 1860tccaagtcga agaacaacgg
tatcgacccg caggtgatgg ttgaacgtta cggcgcggac 1920accgttcgtc
tgtttatgat gtttgcttct ccggctgata tgactctcga atggcaggaa
1980tccggtgtgg aaggggctaa ccgcttcctg aaacgtgtct ggaaactggt
ttacgagcac 2040acagcaaaag gtgatgttgc ggcactgaac gttgatgcgc
tgactgaaaa tcagaaagcg 2100ctgcgtcgcg atgtgcataa aacgatcgct
aaagtgaccg atgatatcgg ccgtcgtcag 2160accttcaaca ccgcaattgc
ggcgattatg gagctgatga acaaactggc gaaagcacca 2220accgatggcg
agcaggatcg cgctctgatg caggaagcac tgctggccgt tgtccgtatg
2280cttaacccgt tcaccccgca catctgcttc acgctgtggc aggaactgaa
aggcgaaggc 2340gatatcgaca acgcgccgtg gccggttgct gacgaaaaag
cgatggtgga agactccacg 2400ctggtcgtgg tgcaggttaa cggtaaagtc
cgtgccaaaa tcaccgttcc ggtggacgca 2460acggaagaac aggttcgcga
acgtgctggc caggaacatc tggtagcaaa atatcttgat 2520ggcgttactg
tacgtaaagt gatttacgta ccaggtaaac tcctcaatct ggtcgttggc 2580taa
2583552583DNAArtificialmutant synthetase 55atgcaagagc aataccgccc
ggaagagata gaatccaaag tacagcttca ttgggatgag 60aagcgcacat ttgaagtaac
cgaagacgag agcaaagaga agtattactg cctgtcttgg 120tcgccctatc
cttctggtcg actacacatg ggccacgtac gtaactacac catcggtgac
180gtgatcgccc gctaccagcg tatgctgggc aaaaacgtcc tgcagccgat
cggctgggac 240gcgtttggtc tgcctgcgga aggcgcggcg gtgaaaaaca
acaccgctcc ggcaccgtgg 300acgtacgaca acatcgcgta tatgaaaaac
cagctcaaaa tgctgggctt tggttatgac 360tggagccgcg agctggcaac
ctgtacgccg gaatactacc gttgggaaca gaaattcttc 420accgagctgt
ataaaaaagg cctggtatat aagaagactt ctgcggtcaa ctggtgcccg
480aacgaccaga ccgtactggc gaacgaacaa gttatcgacg gctgctgctg
gcgctgcgat 540accaaagttg aacgtaaaga gatcccgcag tggtttatca
aaatcactgc ttacgctgac 600gagctgctca acgatctgga taaactggat
cactggccag acaccgttaa aaccatgcag 660cgtaactgga tcggtcgttc
cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggatttat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcgc gattggtggt attgaacacg ccattatggg
gctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 2583562583DNAArtificialmutant
synthetase 56atgcaagagc aataccgccc ggaagagata gaatccaaag tacagcttca
ttgggatgag 60aagcgcacat ttgaagtaac cgaagacgag agcaaagaga agtattactg
cctgtctggt 120acgccctatc cttctggtcg actacacatg ggccacgtac
gtaactacac catcggtgac 180gtgatcgccc gctaccagcg tatgctgggc
aaaaacgtcc tgcagccgat cggctgggac 240gcgtttggtc tgcctgcgga
aggcgcggcg gtgaaaaaca acaccgctcc ggcaccgtgg 300acgtacgaca
acatcgcgta tatgaaaaac cagctcaaaa tgctgggctt tggttatgac
360tggagccgcg agctggcaac ctgtacgccg gaatactacc gttgggaaca
gaaattcttc 420accgagctgt ataaaaaagg cctggtatat aagaagactt
ctgcggtcaa ctggtgcccg 480aacgaccaga ccgtactggc gaacgaacaa
gttatcgacg gctgctgctg gcgctgcgat 540accaaagttg aacgtaaaga
gatcccgcag tggtttatca aaatcactgc ttacgctgac 600gagctgctca
acgatctgga taaactggat cactggccag acaccgttaa aaccatgcag
660cgtaactgga tcggtcgttc cgaaggcgtg gagatcacct tcaacgttaa
cgactatgac 720aacacgctga ccgtttacac tacccgcccg gacaccttta
tgggttgtac ctacctggcg 780gtagctgcgg gtcatccgct ggcgcagaaa
gcggcggaaa ataatcctga actggcggcc 840tttattgacg aatgccgtaa
caccaaagtt gccgaagctg aaatggcgac gatggagaaa 900aaaggcgtcg
atactggctt taaagcggtt cacccattaa cgggcgaaga aattcccgtt
960tgggcagcaa acttcgtatt gatggagtac ggcacgggcg cagttatggc
ggtaccgggg 1020cacgaccagc gcgactacga gtttgcctct aaatacggcc
tgaacatcaa accggttatc 1080ctggcagctg acggctctga gccagatctt
tctcagcaag ccctgactga aaaaggcgtg 1140ctgttcaact ctggcgagtt
caacggtctt gaccatgaag cggccttcaa cgccatcgcc 1200gataaactga
ctgcgatggg cgttggcgag cgtaaagtga actaccgcct gcgcgactgg
1260ggtgtttccc gtcagcgtta ctggggcgcg ccgattccga tggtgacgct
ggaagacggt 1320accgtaatgc cgaccccgga cgaccagctg ccggtgatcc
tgccggaaga tgtggtaatg 1380gacggcatta ccagcccgat taaagcagat
ccggagtggg cgaaaactac cgttaacggt 1440atgccagcac tgcgtgaaac
cgacactttc gacaccttta tggagtcctc ctggtggtat 1500gcgcgctaca
cttgcccgca gtacaaagaa ggtatgctgg attccgaagc ggctaactac
1560tggctgccgg tggatatcct tattggtggt attgaacacg ccattatggg
tctgctctac 1620ttccgcttct tccacaaact gatgcgtgat gcaggcatgg
tgaactctga cgaaccagcg 1680aaacagttgc tgtgtcaggg tatggtgctg
gcagatgcct tctactatgt tggcgaaaac 1740ggcgaacgta actgggtttc
cccggttgat gctatcgttg aacgtgacga gaaaggccgt 1800atcgtgaaag
cgaaagatgc ggcaggccat gaactggttt ataccggcat gagcaaaatg
1860tccaagtcga agaacaacgg tatcgacccg caggtgatgg ttgaacgtta
cggcgcggac 1920accgttcgtc tgtttatgat gtttgcttct ccggctgata
tgactctcga atggcaggaa 1980tccggtgtgg aaggggctaa ccgcttcctg
aaacgtgtct ggaaactggt ttacgagcac 2040acagcaaaag gtgatgttgc
ggcactgaac gttgatgcgc tgactgaaaa tcagaaagcg 2100ctgcgtcgcg
atgtgcataa aacgatcgct aaagtgaccg atgatatcgg ccgtcgtcag
2160accttcaaca ccgcaattgc ggcgattatg gagctgatga acaaactggc
gaaagcacca 2220accgatggcg agcaggatcg cgctctgatg caggaagcac
tgctggccgt tgtccgtatg 2280cttaacccgt tcaccccgca catctgcttc
acgctgtggc aggaactgaa aggcgaaggc 2340gatatcgaca acgcgccgtg
gccggttgct gacgaaaaag cgatggtgga agactccacg 2400ctggtcgtgg
tgcaggttaa cggtaaagtc cgtgccaaaa tcaccgttcc ggtggacgca
2460acggaagaac aggttcgcga acgtgctggc caggaacatc tggtagcaaa
atatcttgat 2520ggcgttactg tacgtaaagt gatttacgta ccaggtaaac
tcctcaatct ggtcgttggc 2580taa 258357424PRTArtificialmutant
synthetase 57Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Met Ala Cys Leu
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42058424PRTArtificialmutant synthetase 58Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Thr Met Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42059424PRTArtificialmutant synthetase 59Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Thr Tyr Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42060424PRTArtificialmutant
synthetase 60Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Met Ala Cys Ser
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42061424PRTArtificialmutant synthetase 61Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala
Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val
Asp Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp
Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp
Phe Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42062424PRTArtificialmutant synthetase 62Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Arg Met Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42063424PRTArtificialmutant synthetase 63Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Gly Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42064424PRTArtificialmutant
synthetase 64Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Ile Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Gly Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Gly Met Ala Cys Ala
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42065424PRTArtificialmutant synthetase 65Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Gly Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Gly Phe Ala Cys Ala Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42066424PRTArtificialmutant synthetase 66Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Gly Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Gly Tyr Ala Cys Met Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42067424PRTArtificialmutant
synthetase 67Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly
Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly
Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His
Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150
155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr
Asn 165 170 175Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala Asn Lys Gln
Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly
Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln
Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala
Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val
Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln
Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265
270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu
Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr
Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu
Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe
Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln
Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly
Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro
Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42068424PRTArtificialmutant synthetase 68Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Ser Ala Ala Cys Ala Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42069424PRTArtificialmutant synthetase 69Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Leu Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Ser Ala Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42070424PRTArtificialmutant
synthetase 70Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Ala Ala Cys Val
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42071424PRTArtificialmutant synthetase 71Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asp Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Asn Phe Ala Cys Val Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42072424PRTArtificialmutant synthetase 72Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Ser Ala Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42073424PRTArtificialmutant
synthetase 73Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Gly Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met
Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu
Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu
Gln Gly Tyr Ser Met Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185
190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser
Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe
Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys
Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro
Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn
Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr
Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp
Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42074424PRTArtificialmutant
synthetase 74Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Ala Ala Cys Leu
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42075424PRTArtificialmutant synthetase 75Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Ser Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Thr Met Ala Cys Val Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42076424PRTArtificialmutant synthetase 76Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Ala Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Ser Tyr Ala Cys Leu Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42077424PRTArtificialmutant
synthetase 77Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Ala Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Thr Met Ala Cys Cys
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42078424PRTArtificialmutant synthetase 78Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Thr Phe Ala Cys Met Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42079424PRTArtificialmutant synthetase 79Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu
Gln Gly Tyr Ser Val Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185
190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser
Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe
Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys
Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro
Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn
Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr
Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp
Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42080424PRTArtificialmutant
synthetase 80Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Met Ala Cys Thr
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42081424PRTArtificialmutant synthetase 81Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Ser Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Ser Phe Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42082424PRTArtificialmutant synthetase 82Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Thr Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Thr Phe Ala Cys Thr Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val
Phe Gly Leu Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr
Lys Phe Gly Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp
Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile
Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe
Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu
Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295
300Glu Gln Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala
Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu
Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp
Gly Val Pro Met Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met
Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln
Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly
Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu
Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410
415Asn Tyr Cys Leu Ile Cys Trp Lys 42083424PRTArtificialmutant
synthetase 83Met Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg
Gly Leu Val1 5 10 15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg
Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr
Ala Asp Ser Leu His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu
Lys Arg Phe Gln Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly
Gly Ala Thr Gly Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala
Glu Arg Lys Leu Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp
Lys Ile Arg Lys Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys
Gly Glu Asn Ser Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe
Gly Asn Met Asn Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135
140His Phe Ser Val Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln
Arg145 150 155 160Leu Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu
Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly Tyr Ser Tyr Ala Cys Leu
Asn Lys Gln Tyr Gly Val 180 185 190Val Leu Gln Ile Gly Gly Ser Asp
Gln Trp Gly Asn Ile Thr Ser Gly195 200 205Ile Asp Leu Thr Arg Arg
Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val Pro Leu Ile
Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230 235 240Gly
Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys Phe 245 250
255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr Arg Phe Leu
260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile Asn Ala Leu
Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro Arg Ala Gln
Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val His Gly Glu
Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr Glu Cys Leu
Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala Asp Phe Glu
Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345 350Met Glu Lys
Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu Leu355 360 365Gln
Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser Asn Ala Ile370 375
380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu Tyr Phe Phe Lys
Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe Thr Leu Leu Arg
Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys Trp Lys
42084424PRTArtificialmutant synthetase 84Met Ala Ser Ser Asn Leu
Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10 15Ala Gln Val Thr Asp
Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly 20 25 30Pro Ile Ala Leu
Ile Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu His35 40 45Leu Gly His
Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln Gln Ala50 55 60Gly His
Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly Leu Ile Gly65 70 75
80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu Asn Thr Glu Glu Thr
85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys Gln Val Ala Pro Phe
Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser Ala Ile Ala Ala Asn
Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn Val Leu Thr Phe Leu
Arg Asp Ile Gly Lys130 135 140His Phe Ser Val Asn Gln Met Ile Asn
Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu Asn Arg Glu Asp Gln
Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170 175Leu Leu Gln Gly
Tyr Ser Met Ala Cys Leu Asn Lys Gln Tyr Gly Val 180 185 190Val Leu
Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ser Gly195 200
205Ile Asp Leu Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu
Thr210 215 220Val Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly
Lys Thr Glu225 230 235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys
Thr Ser Pro Tyr Lys Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala
Asp Ala Asp Val Tyr Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met
Ser Ile Glu Glu Ile Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn
Ser Gly Lys Ala Pro Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln
Val Thr Arg Leu Val His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315
320Lys Arg Ile Thr Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser
325 330 335Glu Ala Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met
Val Glu 340 345 350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val
Asp Ser Glu Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr
Ile Ala Ser Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser
Asp Pro Glu Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe
Gly Arg Phe Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys
Leu Ile Cys Trp Lys 42085424PRTArtificialmutant synthetase 85Met
Ala Ser Ser Asn Leu Ile Lys Gln Leu Gln Glu Arg Gly Leu Val1 5 10
15Ala Gln Val Thr Asp Glu Glu Ala Leu Ala Glu Arg Leu Ala Gln Gly
20 25 30Pro Ile Ala Leu Val Cys Gly Phe Asp Pro Thr Ala Asp Ser Leu
His35 40 45Leu Gly His Leu Val Pro Leu Leu Cys Leu Lys Arg Phe Gln
Gln Ala50 55 60Gly His Lys Pro Val Ala Leu Val Gly Gly Ala Thr Gly
Leu Ile Gly65 70 75 80Asp Pro Ser Phe Lys Ala Ala Glu Arg Lys Leu
Asn Thr Glu Glu Thr 85 90 95Val Gln Glu Trp Val Asp Lys Ile Arg Lys
Gln Val Ala Pro Phe Leu 100 105 110Asp Phe Asp Cys Gly Glu Asn Ser
Ala Ile Ala Ala Asn Asn Tyr Asp115 120 125Trp Phe Gly Asn Met Asn
Val Leu Thr Phe Leu Arg Asp Ile Gly Lys130 135 140His Phe Ser Val
Asn Gln Met Ile Asn Lys Glu Ala Val Lys Gln Arg145 150 155 160Leu
Asn Arg Glu Asp Gln Gly Ile Ser Phe Thr Glu Phe Ser Tyr Asn 165 170
175Leu Leu Gln Gly Tyr Ser Met Ala Cys Ala Asn Lys Gln Tyr Gly Val
180 185 190Val Leu Gln Ile Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr
Ser Gly195 200 205Ile Asp Leu
Thr Arg Arg Leu His Gln Asn Gln Val Phe Gly Leu Thr210 215 220Val
Pro Leu Ile Thr Lys Ala Asp Gly Thr Lys Phe Gly Lys Thr Glu225 230
235 240Gly Gly Ala Val Trp Leu Asp Pro Lys Lys Thr Ser Pro Tyr Lys
Phe 245 250 255Tyr Gln Phe Trp Ile Asn Thr Ala Asp Ala Asp Val Tyr
Arg Phe Leu 260 265 270Lys Phe Phe Thr Phe Met Ser Ile Glu Glu Ile
Asn Ala Leu Glu Glu275 280 285Glu Asp Lys Asn Ser Gly Lys Ala Pro
Arg Ala Gln Tyr Val Leu Ala290 295 300Glu Gln Val Thr Arg Leu Val
His Gly Glu Glu Gly Leu Gln Ala Ala305 310 315 320Lys Arg Ile Thr
Glu Cys Leu Phe Ser Gly Ser Leu Ser Ala Leu Ser 325 330 335Glu Ala
Asp Phe Glu Gln Leu Ala Gln Asp Gly Val Pro Met Val Glu 340 345
350Met Glu Lys Gly Ala Asp Leu Met Gln Ala Leu Val Asp Ser Glu
Leu355 360 365Gln Pro Ser Arg Gly Gln Ala Arg Lys Thr Ile Ala Ser
Asn Ala Ile370 375 380Thr Ile Asn Gly Glu Lys Gln Ser Asp Pro Glu
Tyr Phe Phe Lys Glu385 390 395 400Glu Asp Arg Leu Phe Gly Arg Phe
Thr Leu Leu Arg Arg Gly Lys Lys 405 410 415Asn Tyr Cys Leu Ile Cys
Trp Lys 42086860PRTArtificialmutant synthetase 86Met Gln Glu Gln
Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp Asp
Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu Lys
Tyr Tyr Cys Leu Ser Ala Ala Pro Tyr Pro Ser Gly Arg Leu35 40 45His
Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50 55
60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp Asp65
70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn Asn Thr
Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met Lys Asn
Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser Arg Glu
Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu Gln Lys
Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr Lys Lys
Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln Thr Val
Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp Arg Cys
Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185 190Ile
Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp Lys195 200
205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg Asn Trp
Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn Val Asn
Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr Arg Pro
Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala Ala Gly
His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro Glu Leu
Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val Ala Glu
Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295 300Thr Gly
Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro Val305 310 315
320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr Gly Ala Val Met
325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr Glu Phe Ala Ser
Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile Leu Ala Ala Asp
Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala Leu Thr Glu Lys
Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn Gly Leu Asp His
Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp Lys Leu Thr Ala
Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410 415Leu Arg Asp
Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile 420 425 430Pro
Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro Asp Asp435 440
445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met Asp Gly Ile
Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala Lys Thr Thr
Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr Asp Thr Phe
Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Pro Tyr Ala Arg Tyr Thr
Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser Glu Ala Ala
Asn Tyr Trp Leu Pro Val Asp Ile Val Ile515 520 525Gly Gly Ile Glu
His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535 540His Lys
Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro Ala545 550 555
560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp Ala Phe Tyr Tyr
565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val Ser Pro Val Asp
Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg Ile Val Lys Ala
Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr Thr Gly Met Ser
Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile Asp Pro Gln Val
Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr Val Arg Leu Phe
Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650 655Glu Trp Gln
Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665 670Val
Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala Ala675 680
685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg
Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp Ile Gly
Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met
Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp Gly Glu
Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala Val Val
Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe Thr Leu
Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775 780Ala Pro
Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser Thr785 790 795
800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val
805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly
Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val
Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val
Val Gly850 855 86087860PRTArtificialmutant synthetase 87Met Gln Glu
Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp
Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu
Lys Tyr Tyr Cys Leu Ser Val Met Pro Tyr Pro Ser Gly Arg Leu35 40
45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50
55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp
Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn
Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met
Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser
Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu
Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr
Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln
Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp
Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185
190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp
Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg
Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn
Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr
Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala
Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro
Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val
Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Leu Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Leu Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg
660 665 670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val
Ala Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala
Leu Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp
Asp Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala
Ala Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr
Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu
Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys
Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 86088860PRTArtificialmutant
synthetase 88Met Gln Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser His Pro Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Thr Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Ser Trp Ala Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Met Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645
650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys
Arg 660 665 670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp
Val Ala Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys
Ala Leu Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr
Asp Asp Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile
Ala Ala Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro
Thr Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu
Leu Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760
765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp
Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu
Asp Ser Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val
Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln
Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr
Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly
Lys Leu Leu Asn Leu Val Val Gly850 855 86089860PRTArtificialmutant
synthetase 89Met Gln Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser Val Tyr Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Thr Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Ser Trp Leu Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Leu Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645 650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala
Asn Arg Phe Leu Lys Arg 660 665 670Val Trp Lys Leu Val Tyr Glu His
Thr Ala Lys Gly Asp Val Ala Ala675 680 685Leu Asn Val Asp Ala Leu
Thr Glu Asn Gln Lys Ala Leu Arg Arg Asp690 695 700Val His Lys Thr
Ile Ala Lys Val Thr Asp Asp Ile Gly Arg Arg Gln705 710 715 720Thr
Phe Asn Thr Ala Ile Ala Ala Ile Met Glu Leu Met Asn Lys Leu 725 730
735Ala Lys Ala Pro Thr Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu
740 745 750Ala Leu Leu Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro
His Ile755 760 765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly
Asp Ile Asp Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala
Met Val Glu Asp Ser Thr785 790 795 800Leu Val Val Val Gln Val Asn
Gly Lys Val Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr
Glu Glu Gln Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val
Ala Lys Tyr Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr
Val Pro Gly Lys Leu Leu Asn Leu Val Val Gly850 855
86090860PRTArtificialmutant synthetase 90Met Gln Glu Gln Tyr Arg
Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp Asp Glu Lys
Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr
Cys Leu Ser Leu Glu Pro Tyr Pro Ser Gly Arg Leu35 40 45His Met Gly
His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50 55 60Tyr Gln
Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp Asp65 70 75
80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn Asn Thr Ala
85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met Lys Asn Gln
Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser Arg Glu Leu
Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu Gln Lys Phe
Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr Lys Lys Thr
Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln Thr Val Leu
Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp
Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys
Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp Lys195 200
205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg Asn Trp
Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn Val Asn
Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr Arg Pro
Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala Ala Gly
His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro Glu Leu
Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val Ala Glu
Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295 300Thr Gly
Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro Val305 310 315
320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr Gly Ala Val Met
325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr Glu Phe Ala Ser
Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile Leu Ala Ala Asp
Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala Leu Thr Glu Lys
Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn Gly Leu Asp His
Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp Lys Leu Thr Ala
Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410 415Leu Arg Asp
Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile 420 425 430Pro
Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro Asp Asp435 440
445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met Asp Gly Ile
Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala Lys Thr Thr
Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr Asp Thr Phe
Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Arg Tyr Ala Arg Tyr Thr
Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser Glu Ala Ala
Asn Tyr Trp Leu Pro Val Asp Ile Ala Ile515 520 525Gly Gly Ile Glu
His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535 540His Lys
Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro Ala545 550 555
560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp Ala Phe Tyr Tyr
565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val Ser Pro Val Asp
Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg Ile Val Lys Ala
Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr Thr Gly Met Ser
Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile Asp Pro Gln Val
Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr Val Arg Leu Phe
Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650 655Glu Trp Gln
Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665 670Val
Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala Ala675 680
685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg
Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp Ile Gly
Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met
Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp Gly Glu
Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala Val Val
Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe Thr Leu
Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775 780Ala Pro
Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser Thr785 790 795
800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val
805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly
Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val
Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val
Val Gly850 855 86091860PRTArtificialmutant synthetase 91Met Gln Glu
Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp
Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu
Lys Tyr Tyr Cys Leu Ser Met Glu Pro Tyr Pro Ser Gly Arg Leu35 40
45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50
55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp
Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn
Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met
Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser
Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu
Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr
Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln
Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp
Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185
190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp
Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg
Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn
Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr
Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala
Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro
Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val
Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Arg Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Phe Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp
Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665
670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala
Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu
Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp
Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala
Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp
Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala
Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe
Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 86092860PRTArtificialmutant
synthetase 92Met Gln Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser Leu Glu Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Thr Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Ser Trp Arg Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Cys Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645 650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala
Asn Arg Phe Leu Lys Arg 660 665 670Val Trp Lys Leu Val Tyr Glu His
Thr Ala Lys Gly Asp Val Ala Ala675 680 685Leu Asn Val Asp Ala Leu
Thr Glu Asn Gln Lys Ala Leu Arg Arg Asp690 695 700Val His Lys Thr
Ile Ala Lys Val Thr Asp Asp Ile Gly Arg Arg Gln705 710 715 720Thr
Phe Asn Thr Ala Ile Ala Ala Ile Met Glu Leu Met Asn Lys Leu 725 730
735Ala Lys Ala Pro Thr Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu
740 745 750Ala Leu Leu Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro
His Ile755 760 765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly
Asp Ile Asp Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala
Met Val Glu Asp Ser Thr785 790 795 800Leu Val Val Val Gln Val Asn
Gly Lys Val Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr
Glu Glu Gln Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val
Ala Lys Tyr Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr
Val Pro Gly Lys Leu Leu Asn Leu Val Val Gly850 855
86093860PRTArtificialmutant synthetase 93Met Gln Glu Gln Tyr Arg
Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp Asp Glu Lys
Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr
Cys Leu Ser Phe Glu Pro Tyr Pro Ser Gly Arg Leu35 40 45His Met Gly
His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50 55 60Tyr Gln
Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp Asp65 70 75
80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn Asn Thr Ala
85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met Lys Asn Gln
Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser Arg Glu Leu
Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu Gln Lys Phe
Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr Lys Lys Thr
Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln Thr Val Leu
Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp
Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys
Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp Lys195 200
205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg Asn Trp
Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn Val Asn
Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr Arg Pro
Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala Ala Gly
His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro Glu Leu
Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val Ala Glu
Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295 300Thr Gly
Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro Val305 310 315
320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr Gly Ala Val Met
325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr Glu Phe Ala Ser
Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile Leu Ala Ala Asp
Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala Leu Thr Glu Lys
Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn Gly Leu Asp His
Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp Lys Leu Thr Ala
Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410 415Leu Arg Asp
Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile 420 425 430Pro
Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro Asp Asp435 440
445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met Asp Gly Ile
Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala Lys Thr Thr
Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr Asp Thr Phe
Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Arg Tyr Ala Arg Tyr Thr
Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser Glu Ala Ala
Asn Tyr Trp Leu Pro Val Asp Ile Thr Ile515 520 525Gly Gly Ile Glu
His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535 540His Lys
Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro Ala545 550 555
560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp Ala Phe Tyr Tyr
565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val Ser Pro Val Asp
Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg Ile Val Lys Ala
Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr Thr Gly Met Ser
Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile Asp Pro Gln Val
Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr Val Arg Leu Phe
Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650 655Glu Trp Gln
Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665 670Val
Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala Ala675 680
685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg
Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp Ile Gly
Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met
Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp Gly Glu
Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala Val Val
Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe Thr Leu
Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775 780Ala Pro
Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser Thr785 790 795
800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val
805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly
Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val
Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val
Val Gly850 855 86094860PRTArtificialmutant synthetase 94Met Gln Glu
Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp
Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu
Lys Tyr Tyr Cys Leu Ser Gly Glu Pro Tyr Pro Ser Gly Arg Leu35 40
45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50
55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp
Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn
Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met
Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser
Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu
Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr
Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln
Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp
Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185
190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp
Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg
Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn
Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr
Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala
Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro
Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val
Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Arg Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Leu Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp Gln Glu Ser Gly Val Glu Gly
Ala Asn Arg Phe Leu Lys Arg 660 665 670Val Trp Lys Leu Val Tyr Glu
His Thr Ala Lys Gly Asp Val Ala Ala675 680 685Leu Asn Val Asp Ala
Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg Asp690 695 700Val His Lys
Thr Ile Ala Lys Val Thr Asp Asp Ile Gly Arg Arg Gln705 710 715
720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met Glu Leu Met Asn Lys Leu
725 730 735Ala Lys Ala Pro Thr Asp Gly Glu Gln Asp Arg Ala Leu Met
Gln Glu 740 745 750Ala Leu Leu Ala Val Val Arg Met Leu Asn Pro Phe
Thr Pro His Ile755 760 765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly
Glu Gly Asp Ile Asp Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu
Lys Ala Met Val Glu Asp Ser Thr785 790 795 800Leu Val Val Val Gln
Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp
Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His
Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840
845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val Val Gly850 855
86095860PRTArtificialmutant synthetase 95Met Gln Glu Gln Tyr Arg
Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp Asp Glu Lys
Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr
Cys Leu Ser Gly Trp Pro Tyr Pro Ser Gly Arg Leu35 40 45His Met Gly
His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50 55 60Tyr Gln
Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp Asp65 70 75
80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn Asn Thr Ala
85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met Lys Asn Gln
Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser Arg Glu Leu
Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu Gln Lys Phe
Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr Lys Lys Thr
Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln Thr Val Leu
Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp
Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys
Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp Lys195 200
205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg Asn Trp
Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn Val Asn
Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr Arg Pro
Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala Ala Gly
His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro Glu Leu
Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val Ala Glu
Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295 300Thr Gly
Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro Val305 310 315
320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr Gly Ala Val Met
325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr Glu Phe Ala Ser
Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile Leu Ala Ala Asp
Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala Leu Thr Glu Lys
Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn Gly Leu Asp His
Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp Lys Leu Thr Ala
Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410 415Leu Arg Asp
Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile 420 425 430Pro
Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro Asp Asp435 440
445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met Asp Gly Ile
Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala Lys Thr Thr
Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr Asp Thr Phe
Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Ala Tyr Ala Arg Tyr Thr
Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser Glu Ala Ala
Asn Tyr Trp Leu Pro Val Asp Ile Leu Ile515 520 525Gly Gly Ile Glu
His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535 540His Lys
Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro Ala545 550 555
560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp Ala Phe Tyr Tyr
565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val Ser Pro Val Asp
Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg Ile Val Lys Ala
Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr Thr Gly Met Ser
Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile Asp Pro Gln Val
Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr Val Arg Leu Phe
Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650 655Glu Trp Gln
Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665 670Val
Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala Ala675 680
685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg
Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp Ile Gly
Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met
Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp Gly Glu
Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala Val Val
Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe Thr Leu
Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775 780Ala Pro
Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser Thr785 790 795
800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val
805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly
Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val
Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val
Val Gly850 855 86096860PRTArtificialmutant synthetase 96Met Gln Glu
Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp
Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu
Lys Tyr Tyr Cys Leu Ser Trp Ser Pro Tyr Pro Ser Gly Arg Leu35 40
45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50
55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp
Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn
Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met
Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser
Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu
Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr
Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln
Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp
Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185
190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp
Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg
Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn
Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr
Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala
Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro
Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val
Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Ser Trp Ile Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Ala Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg
660 665 670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val
Ala Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala
Leu Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp
Asp Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala
Ala Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr
Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu
Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys
Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 86097860PRTArtificialmutant
synthetase 97Met Gln Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser Gly Thr Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Thr Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Ser Trp Trp Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Leu Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645 650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala
Asn Arg Phe Leu Lys Arg 660
665 670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala
Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu
Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp
Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala
Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp
Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala
Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe
Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 86098860PRTArtificialmutant
synthetase 98Met Glu Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser Ala Asn Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Thr Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Cys Trp Ile Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Gly Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Thr Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645 650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala
Asn Arg Phe Leu Lys Arg 660 665 670Val Trp Lys Leu Val Tyr Glu His
Thr Ala Lys Gly Asp Val Ala Ala675 680 685Leu Asn Val Asp Ala Leu
Thr Glu Asn Gln Lys Ala Leu Arg Arg Asp690 695 700Val His Lys Thr
Ile Ala Lys Val Thr Asp Asp Ile Gly Arg Arg Gln705 710 715 720Thr
Phe Asn Thr Ala Ile Ala Ala Ile Met Glu Leu Met Asn Lys Leu 725 730
735Ala Lys Ala Pro Thr Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu
740 745 750Ala Leu Leu Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro
His Ile755 760 765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly
Asp Ile Asp Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala
Met Val Glu Asp Ser Thr785 790 795 800Leu Val Val Val Gln Val Asn
Gly Lys Val Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr
Glu Glu Gln Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val
Ala Lys Tyr Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr
Val Pro Gly Lys Leu Leu Asn Leu Val Val Gly850 855
86099860PRTArtificialmutant synthetase 99Met Glu Glu Gln Tyr Arg
Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His Trp Asp Glu Lys
Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25 30Glu Lys Tyr Tyr
Cys Leu Ser Ala Asn Pro Tyr Pro Ser Gly Arg Leu35 40 45His Met Gly
His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala Arg50 55 60Tyr Gln
Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly Trp Asp65 70 75
80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys Asn Asn Thr Ala
85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr Met Lys Asn Gln
Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp Ser Arg Glu Leu
Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp Glu Gln Lys Phe
Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val Tyr Lys Lys Thr
Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp Gln Thr Val Leu
Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp
Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys
Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu Asp Lys195 200
205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln Arg Asn Trp
Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe Asn Val Asn
Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr Thr Arg Pro
Asp Ala Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val Ala Ala Gly
His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn Pro Glu Leu
Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys Val Ala Glu
Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295 300Thr Gly
Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro Val305 310 315
320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr Gly Ala Val Met
325 330 335Ala Val Pro Gly His Asp Gln Arg Asp Tyr Glu Phe Ala Ser
Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile Leu Ala Ala Asp
Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala Leu Thr Glu Lys
Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn Gly Leu Asp His
Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp Lys Leu Thr Ala
Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410 415Leu Arg Asp
Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile 420 425 430Pro
Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro Asp Asp435 440
445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met Asp Gly Ile
Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala Lys Thr Thr
Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr Asp Thr Phe
Asp Thr Phe Met Glu Ser 485 490 495Cys Trp Ile Tyr Ala Arg Tyr Thr
Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser Glu Ala Ala
Asn Tyr Trp Leu Pro Val Asp Ile Gly Ile515 520 525Gly Gly Ile Glu
His Ala Ile Met Thr Leu Leu Tyr Phe Arg Phe Phe530 535 540His Lys
Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro Ala545 550 555
560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp Ala Phe Tyr Tyr
565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val Ser Pro Val Asp
Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg Ile Val Lys Ala
Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr Thr Gly Met Ser
Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile Asp Pro Gln Val
Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr Val Arg Leu Phe
Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650 655Glu Trp Gln
Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg 660 665 670Val
Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala Ala675 680
685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu Arg Arg
Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp Ile Gly
Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala Ile Met
Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp Gly Glu
Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala Val Val
Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe Thr Leu
Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775 780Ala Pro
Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser Thr785 790 795
800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala Lys Ile Thr Val
805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg Glu Arg Ala Gly
Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp Gly Val Thr Val
Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu Leu Asn Leu Val
Val Gly850 855 860100860PRTArtificialmutant synthetase 100Met Glu
Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys Val Gln Leu1 5 10 15His
Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu Asp Glu Ser Lys 20 25
30Glu Lys Tyr Tyr Cys Leu Ser Ala Asn Pro Tyr Pro Ser Gly Arg Leu35
40 45His Met Gly His Val Arg Asn Tyr Thr Ile Gly Asp Val Ile Ala
Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val Leu Gln Pro Ile Gly
Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu Gly Ala Ala Val Lys
Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr Asp Asn Ile Ala Tyr
Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly Phe Gly Tyr Asp Trp
Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro Glu Tyr Tyr Arg Trp
Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135 140Lys Lys Gly Leu Val
Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys Pro145 150 155 160Asn Asp
Gln Thr Val Leu Ala Asn Glu Gln Val Ile Asp Gly Cys Cys 165 170
175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys Glu Ile Pro Gln Trp Phe
180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp Glu Leu Leu Asn Asp Leu
Asp Lys195 200 205Leu Asp His Trp Pro Asp Thr Val Lys Thr Met Gln
Arg Asn Trp Ile210 215 220Gly Arg Ser Glu Gly Val Glu Ile Thr Phe
Asn Val Asn Asp Tyr Asp225 230 235 240Asn Thr Leu Thr Val Tyr Thr
Thr Arg Pro Asp Thr Phe Met Gly Cys 245 250 255Thr Tyr Leu Ala Val
Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala 260 265 270Glu Asn Asn
Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg Asn Thr275 280 285Lys
Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys Lys Gly Val Asp290 295
300Thr Gly Phe Lys Ala Val His Pro Leu Thr Gly Glu Glu Ile Pro
Val305 310 315 320Trp Ala Ala Asn Phe Val Leu Met Glu Tyr Gly Thr
Gly Ala Val Met 325 330 335Ala Ala Pro Gly His Asp Gln Arg Asp Tyr
Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn Ile Lys Pro Val Ile
Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp Leu Ser Gln Gln Ala
Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375 380Gly Glu Phe Asn
Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile Ala385 390 395 400Asp
Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys Val Asn Tyr Arg 405 410
415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg Tyr Trp Gly Ala Pro Ile
420 425 430Pro Met Val Thr Leu Glu Asp Gly Thr Val Met Pro Thr Pro
Asp Asp435 440 445Gln Leu Pro Val Ile Leu Pro Glu Asp Val Val Met
Asp Gly Ile Thr450 455 460Ser Pro Ile Lys Ala Asp Pro Glu Trp Ala
Lys Thr Thr Val Asn Gly465 470 475 480Met Pro Ala Leu Arg Glu Thr
Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490 495Cys Trp Ile Tyr Ala
Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met 500 505 510Leu Asp Ser
Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile Gly Ile515 520 525Gly
Gly Ile Glu His Ala Ile Met Thr Leu Leu Tyr Phe Arg Phe Phe530 535
540His Lys Leu Met Arg Asp Ala Gly Met Val Asn Ser Asp Glu Pro
Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly Met Val Leu Ala Asp
Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly Glu Arg Asn Trp Val
Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg Asp Glu Lys Gly Arg
Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly His Glu Leu Val Tyr
Thr Gly Met Ser Lys Met Ser Lys Ser Lys610 615 620Asn Asn Gly Ile
Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala Asp625 630 635 640Thr
Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala Asp Met Thr Leu 645 650
655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala Asn Arg Phe Leu Lys Arg
660 665
670Val Trp Lys Leu Val Tyr Glu His Thr Ala Lys Gly Asp Val Ala
Ala675 680 685Leu Asn Val Asp Ala Leu Thr Glu Asn Gln Lys Ala Leu
Arg Arg Asp690 695 700Val His Lys Thr Ile Ala Lys Val Thr Asp Asp
Ile Gly Arg Arg Gln705 710 715 720Thr Phe Asn Thr Ala Ile Ala Ala
Ile Met Glu Leu Met Asn Lys Leu 725 730 735Ala Lys Ala Pro Thr Asp
Gly Glu Gln Asp Arg Ala Leu Met Gln Glu 740 745 750Ala Leu Leu Ala
Val Val Arg Met Leu Asn Pro Phe Thr Pro His Ile755 760 765Cys Phe
Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly Asp Ile Asp Asn770 775
780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala Met Val Glu Asp Ser
Thr785 790 795 800Leu Val Val Val Gln Val Asn Gly Lys Val Arg Ala
Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr Glu Glu Gln Val Arg
Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val Ala Lys Tyr Leu Asp
Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr Val Pro Gly Lys Leu
Leu Asn Leu Val Val Gly850 855 860101860PRTArtificialmutant
synthetase 101Met Glu Glu Gln Tyr Arg Pro Glu Glu Ile Glu Ser Lys
Val Gln Leu1 5 10 15His Trp Asp Glu Lys Arg Thr Phe Glu Val Thr Glu
Asp Glu Gly Lys 20 25 30Glu Lys Tyr Tyr Cys Leu Ser Trp Ser Pro Tyr
Pro Ser Gly Arg Leu35 40 45His Met Gly His Val Arg Asn Tyr Thr Ile
Gly Asp Val Ile Ala Arg50 55 60Tyr Gln Arg Met Leu Gly Lys Asn Val
Leu Gln Pro Ile Gly Trp Asp65 70 75 80Ala Phe Gly Leu Pro Ala Glu
Gly Ala Ala Val Lys Asn Asn Thr Ala 85 90 95Pro Ala Pro Trp Thr Tyr
Asp Asn Ile Ala Tyr Met Lys Asn Gln Leu 100 105 110Lys Met Leu Gly
Phe Gly Tyr Asp Trp Ser Arg Glu Leu Ala Thr Cys115 120 125Thr Pro
Glu Tyr Tyr Arg Trp Glu Gln Lys Phe Phe Thr Glu Leu Tyr130 135
140Lys Lys Gly Leu Val Tyr Lys Lys Thr Ser Ala Val Asn Trp Cys
Pro145 150 155 160Asn Asp Gln Thr Val Leu Ala Asn Glu Gln Val Ile
Asp Gly Cys Cys 165 170 175Trp Arg Cys Asp Thr Lys Val Glu Arg Lys
Glu Ile Pro Gln Trp Phe 180 185 190Ile Lys Ile Thr Ala Tyr Ala Asp
Glu Leu Leu Asn Asp Leu Asp Lys195 200 205Leu Asp His Trp Pro Asp
Thr Val Lys Thr Met Gln Arg Asn Trp Ile210 215 220Gly Arg Ser Glu
Gly Val Glu Ile Thr Phe Asn Val Asn Asp Tyr Asp225 230 235 240Asn
Thr Leu Thr Val Tyr Ala Ser Arg Pro Asp Thr Phe Met Gly Cys 245 250
255Thr Tyr Leu Ala Val Ala Ala Gly His Pro Leu Ala Gln Lys Ala Ala
260 265 270Glu Asn Asn Pro Glu Leu Ala Ala Phe Ile Asp Glu Cys Arg
Asn Thr275 280 285Lys Val Ala Glu Ala Glu Met Ala Thr Met Glu Lys
Lys Gly Val Asp290 295 300Thr Gly Phe Lys Ala Val His Pro Leu Thr
Gly Glu Glu Ile Pro Val305 310 315 320Trp Ala Ala Asn Phe Val Leu
Met Glu Tyr Gly Thr Gly Ala Val Met 325 330 335Ala Val Pro Gly His
Asp Gln Arg Asp Tyr Glu Phe Ala Ser Lys Tyr 340 345 350Gly Leu Asn
Ile Lys Pro Val Ile Leu Ala Ala Asp Gly Ser Glu Pro355 360 365Asp
Leu Ser Gln Gln Ala Leu Thr Glu Lys Gly Val Leu Phe Asn Ser370 375
380Gly Glu Phe Asn Gly Leu Asp His Glu Ala Ala Phe Asn Ala Ile
Ala385 390 395 400Asp Lys Leu Thr Ala Met Gly Val Gly Glu Arg Lys
Val Asn Tyr Arg 405 410 415Leu Arg Asp Trp Gly Val Ser Arg Gln Arg
Tyr Trp Gly Ala Pro Ile 420 425 430Pro Met Val Thr Leu Glu Asp Gly
Thr Val Met Pro Thr Pro Asp Asp435 440 445Gln Leu Pro Val Ile Leu
Pro Glu Asp Val Val Met Asp Gly Ile Thr450 455 460Ser Pro Ile Lys
Ala Asp Pro Glu Trp Ala Lys Thr Thr Val Asn Gly465 470 475 480Met
Pro Ala Leu Arg Glu Thr Asp Thr Phe Asp Thr Phe Met Glu Ser 485 490
495Cys Trp Ile Tyr Ala Arg Tyr Thr Cys Pro Gln Tyr Lys Glu Gly Met
500 505 510Leu Asp Ser Glu Ala Ala Asn Tyr Trp Leu Pro Val Asp Ile
Ala Ile515 520 525Gly Gly Ile Glu His Ala Ile Met Gly Leu Leu Tyr
Phe Arg Phe Phe530 535 540His Lys Leu Met Arg Asp Ala Gly Met Val
Asn Ser Asp Glu Pro Ala545 550 555 560Lys Gln Leu Leu Cys Gln Gly
Met Val Leu Ala Asp Ala Phe Tyr Tyr 565 570 575Val Gly Glu Asn Gly
Glu Arg Asn Trp Val Ser Pro Val Asp Ala Ile 580 585 590Val Glu Arg
Asp Glu Lys Gly Arg Ile Val Lys Ala Lys Asp Ala Ala595 600 605Gly
His Glu Leu Val Tyr Thr Gly Ile Ser Lys Met Ser Lys Ser Lys610 615
620Asn Asn Gly Ile Asp Pro Gln Val Met Val Glu Arg Tyr Gly Ala
Asp625 630 635 640Thr Val Arg Leu Phe Met Met Phe Ala Ser Pro Ala
Asp Met Thr Leu 645 650 655Glu Trp Gln Glu Ser Gly Val Glu Gly Ala
Asn Arg Phe Leu Lys Arg 660 665 670Ala Trp Lys Leu Val Tyr Glu His
Thr Ala Lys Gly Asp Val Ala Ala675 680 685Leu Asn Val Asp Ala Leu
Thr Glu Asn Gln Lys Ala Leu Arg Arg Asp690 695 700Val His Lys Thr
Ile Ala Lys Val Thr Asp Asp Ile Gly Arg Arg Gln705 710 715 720Thr
Phe Asn Thr Ala Ile Ala Ala Ile Met Glu Leu Met Asn Lys Leu 725 730
735Ala Lys Ala Pro Thr Asp Gly Glu Gln Asp Arg Ala Leu Met Gln Glu
740 745 750Ala Leu Leu Ala Val Val Arg Met Leu Asn Pro Phe Thr Pro
His Ile755 760 765Cys Phe Thr Leu Trp Gln Glu Leu Lys Gly Glu Gly
Asp Ile Asp Asn770 775 780Ala Pro Trp Pro Val Ala Asp Glu Lys Ala
Met Val Glu Asp Ser Thr785 790 795 800Leu Val Val Val Gln Val Asn
Gly Lys Val Arg Ala Lys Ile Thr Val 805 810 815Pro Val Asp Ala Thr
Glu Glu Gln Val Arg Glu Arg Ala Gly Gln Glu 820 825 830His Leu Val
Ala Lys Tyr Leu Asp Gly Val Thr Val Arg Lys Val Ile835 840 845Tyr
Val Pro Gly Lys Leu Leu Asn Leu Val Val Gly850 855
86010215PRTArtificialtryptic peptide from enhanced GFP 102Phe Ser
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys1 5 10
1510315PRTArtificialtryptic peptide from enhanced GFP 103Phe Ser
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Xaa Gly Lys1 5 10
1510455DNAHomo sapiens 104agcgctccgg tttttctgtg ctgaacctca
ggggacgccg acacacgtac acgtc 5510525DNAArtificial5'-flanking
sequence 105gatccgaccg tgtgcttggc agaac
2510612DNAArtificial3'-flanking sequence 106gtcctttttt tg 12
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