WO2009064416A2 - Genetic incorporation of an alpha-hydroxy acid into proteins to generate ester backbone linkages at defined sites - Google Patents

Abstract

Orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the α-hydroxy acid p-hydroxy-L-phenyllactic acid into proteins produced in eubacterial host cells such as E. coli are described. Novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the α-hydroxy acid p-hydroxy-L-phenyllactic acid, and translation systems are provided. Methods of selectively hydrolyzing or modifying proteins including α-hydroxy acid residues, and therefore ester bonds in their backbones, are also provided.

Description

GENETIC INCORPORATION OF AN ALPHA-HYDROXY ACID INTO PROTEINS TO GENERATE ESTER BACKBONE LINKAGES

AT DEFINED SITES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of United States provisional patent application USSN 61/003,371, filed November 15, 2007, entitled "GENETIC INCORPORATION OF AN ALPHA-HYDROXY ACID INTO PROTEINS TO GENERATE ESTER BACKBONE LINKAGES AT DEFINED SITES" by Jiantao Guo et al., which is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant No. 5ROl

GM 62159 from the National Institutes of Health and support under Grant No. ER46051 from the Department of Energy. The government may have certain rights to this invention.

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 α-hydroxy acids into proteins. The invention also relates to methods of producing proteins including α-hydroxy acids using such pairs, as well as methods of modifying such proteins.

BACKGROUND OF THE INVENTION

[0004] Site-directed mutagenesis techniques in which one of the twenty naturally occurring amino acids is replaced by another in a protein of interest facilitate elucidation of the roles particular side chains play in events such as folding, catalysis, and molecular recognition. The polypeptide backbone can also play a key role in such events. However, since the backbone cannot be altered using conventional site-directed mutagenesis techniques, the contribution of particular backbone hydrogen bond donors and acceptors has been more difficult to assess.

[0005] Replacement of an α-amino acid in a protein of interest with an α-hydroxy acid replaces an amide bond in the polypeptide backbone with an ester bond. Like an amide, the ester can serve as a hydrogen bond acceptor (albeit a weaker acceptor), but it can not serve as a hydrogen bond donor. Such replacement can therefore alter hydrogen bonding interactions within the protein.

[0006] Replacement of amino acids with α-hydroxy acids has been achieved in translation systems where a nonsense suppressor tRNA was charged in vitro with an α- hydroxy acid. See, e.g., England et al. (1999) Cell 96:89-98, Ellman et al. (1992) Science 255: 197-200, and Lu et al. (2001) Nat Neurosci 4:239-246. However, the requirement that the suppressor tRNA be chemically aminoacylated with the α-hydroxy acid in vitro limits the usefulness of this method. For example, the acylated tRNA is consumed as a stoichiometric reagent during translation and cannot be regenerated, limiting suppression efficiency and protein yields.

[0007] Another approach involves the use of "orthogonal" tRNAs and corresponding novel "orthogonal" aminoacyl-tRNA synthetases to add unnatural amino acids to proteins using the in vivo protein biosynthetic machinery of the eubacteria Escherichia coli (E. colϊ) and other organisms. A general methodology has been developed for the in vivo site-specific incorporation of diverse unnatural amino acids into proteins in both prokaryotic and eukaryotic organisms. 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 during polypeptide translation in vivo. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon and a corresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS) that charges the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the endogenous tRNAs, RSs, amino acids or codons in the host organism (i.e., they must be orthogonal). The use of such orthogonal tRNA-RS pairs has made it possible to genetically encode a large number of structurally diverse unnatural amino acids. See, e.g., International Patent Publications WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINO ACYL-tRNA SYNTHETASE PAIRS," WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS," and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE" for general description of incorporation of unnatural amino acids using orthogonal synthetase/tRNA pairs; see also, e.g., Wang et al. (2001) Science 292:498-500; Chin et al. (2002) Journal of the American Chemical Society 124:9026-9027; Chin and Schultz (2002) ChemBioChem 11: 1135-1137; Chin et al. (2002) PNAS United States of America 99:11020-11024; Wang and Schultz (2002) Chem. Comm. 1-10); and International Patent Publications WO 2005/019415, filed July 7, 2004; WO 2005/007870, filed July 7, 2004; and WO 2005/007624, filed July 7, 2004.

[0008] There is need in the art for additional orthogonal synthetase/tRNA pairs, e.g., pairs that can incorporate specific α-hydroxy acids of interest. Among other aspects, the present invention provides a novel orthogonal synthetase/tRNA pair that facilitates incorporation of the α-hydroxy acidp-hydroxy-L-phenyllactic acid, and therefore of an ester bond, at defined positions in proteins of interest. A complete understanding of the invention will be obtained upon review of the following.

SUMMARY OF THE INVENTION

[0009] One aspect of the invention provides a general approach for selective incorporation of the α-hydroxy acidp-hydroxy-L-phenyllactic acid into proteins by genetically encoding the α-hydroxy acid. This general approach to the biosynthesis of proteins including ester bonds at selected positions facilitates further study and application of this backbone modification.

[0010] In one aspect, the invention provides compositions and methods for selectively incorporating the α-hydroxy acid/7-hydroxy-L-phenyllactic acid into a growing polypeptide chain in response to a selector codon, e.g., an amber stop codon, in vitro or in vivo (e.g., in a host cell). These compositions and methods involve pairs of orthogonal- tRNAs (O-tRNAs) and orthogonal aminoacyl-tRNA synthetases (O-RSs) that do not cross- react with the host cell's endogenous 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-RS does not charge any endogenous tRNA with an amino acid (natural or unnatural) to a significant or detectable level. These novel compositions and methods facilitate production of large quantities of proteins having translationally incorporated p-hydroxy-L-phenyllactic acid, and thus having ester bonds at selected positions in the polypeptide backbone.

[0011] A first general class of embodiments provides a translation system. The translation system comprises a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), and a first α-hydroxy acid that is p-hydroxy-L-phenyllactic acid, where the first O-RS preferentially charges the first O-tRNA with the /p-hydroxy-L- phenyllactic acid. In some embodiments, the first O-RS preferentially charges the first O- tRNA with p-hydroxy-L-phenyllactic acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising that same O-tRNA, the p-hydroxy- L-phenyllactic acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID NO: 1 (e.g., at least 60%, 70%, 75%, 80%, 90%, 95%, or 99% or more efficiency).

[0012] The translation systems can use components derived from a variety of sources. In one embodiment, the first O-RS is derived from or homologous to a Methanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase (e.g., SEQ ID NO:5). Optionally, the first O-RS comprises an Arg residue at position 155, a GIy residue at position 173, a VaI residue at position 176, or a combination thereof, wherein amino acid position numbering corresponds to amino acid position numbering of the wild-type tyrosyl tRNA synthetase; the O-RS optionally also includes one or more of a GIu residue at position 36, an He residue at position 137, and a Tyr residue at position 151. The O-RS used in the system can comprise the amino acid sequence of SEQ ID NO: 1 or a conservative variant of that sequence. In some embodiments, the first O-tRNA is an amber suppressor tRNA. In some embodiments, the first O-tRNA comprises or is encoded by SEQ ID NO: 3.

[0013] The translation system optionally also includes 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.

[0014] In one class of embodiments, the translation system includes a second orthogonal pair (that is, a second O-RS and a second O-tRNA) that utilizes a second α- hydroxy or other unnatural amino acid that is different from the first 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 charges the second O-tRNA with the second α-hydroxy or 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.

[0015] In some embodiments, the translation system resides in a host cell (and includes the host cell). The host cell used is not particularly limited, as long as the O-RS and O-tRNA retain their orthogonality in their host cell environment. For example, the host cell can be a eubacterial cell, such as E. coll The host cell can be engineered to reduce or eliminate undesired metabolism of the α-hydroxy acid; for example, the host cell can be an E. coli cell having disruptions in tyrB and aspC. The host cell can comprise one or more polynucleotides that encode components of the translation system, such as the O-RS and/or O-tRNA. In some embodiments, the polynucleotide encoding the O-RS comprises a nucleotide sequence of SΕQ ID NO:2.

[0016] Another general class of embodiments provides methods for producing in a translation system a protein having at least a first α-hydroxy acid residue at a selected position in the protein. In the methods, a translation system is provided that comprises (i) a first α-hydroxy acid that is p-hydroxy-L-phenyllactic acid, (ii) a first orthogonal aminoacyl- tRNA synthetase (O-RS), (iii) a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the p-hydroxy-L-phenyllactic acid, and (iv) a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one selector codon that is recognized by the first O-tRNA. The α-hydroxy acid is then incorporated at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the α-hydroxy acid at the selected position (and thus comprising an ester bond at a selected location in the polypeptide backbone). In some embodiments, the first O-RS preferentially charges the first O-tRNA with /?-hydroxy-L-phenyllactic acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising that same O-tRNA, the^»-hydroxy-L- phenyllactic acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SΕQ ID NO:1 (e.g., at least 60%, 70%, 75%, 80%, 90%, 95%, or 99% or more efficiency).

[0017] These methods can be widely applied using a variety of reagents and steps.

In some embodiments, a polynucleotide encoding the first O-RS is provided. Optionally, the polynucleotide comprises the nucleotide sequence of SΕQ ID NO:2. In some embodiments, an O-RS derived from or homologous to a Methanococcus jannaschii aminoacyl-tRNA synthetase (e.g., a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase, e.g., SΕQ ID NO:5) is provided. For example, in one class of embodiments, providing the first orthogonal aminoacyl-tRNA synthetase 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 charges the O-tRNA with the α-hydroxy acid. The selecting step can comprise positively selecting and negatively selecting for the O-RS from a pool of resulting aminoacyl-tRNA synthetase molecules following site-directed mutagenesis. Optionally, the first O-RS comprises an Arg residue at position 155, a GIy residue at position 173, a VaI residue at position 176, or a combination thereof, wherein amino acid position numbering corresponds to amino acid position numbering of the wild-type tyrosyl tRNA synthetase; the O-RS optionally also includes one or more of a GIu residue at position 36, an He residue at position 137, and a Tyr residue at position 151. Optionally, the first O-RS comprises an amino acid sequence selected from the group consisting SEQ ID NO:1 and conservative variants thereof. In some embodiments, a polynucleotide encoding the O-tRNA is provided. In some embodiments, the first O-tRNA is an amber suppressor tRNA. In some embodiments, the first O-tRNA comprises or is encoded by SEQ ID NO:3. In such embodiments, the nucleic acid encoding the protein comprises an amber selector codon that is utilized by the translation system.

[0018] These methods can also be employed to incorporate more than one unnatural amino acid into a protein. In such methods, a second orthogonal pair is employed in conjunction with the first orthogonal pair, where the second pair has different amino acid and selector codon specificities. For example, the translation system can also include a second O-RS and a second O-tRNA, where the second O-RS preferentially charges the second O-tRNA with a second α-hydroxy or other unnatural amino acid that is different from the first α-hydroxy 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. The resulting protein thus comprises the first and second unnatural amino acids.

[0019] The methods for producing a protein with an α-hydroxy acid can also be conducted in the context of a host cell. In these cases, a host cell is provided, where the host cell comprises the first α-hydroxy acid, the first O-RS, the first O-tRNA, and the nucleic acid that encodes the protein. Culturing the host cell results in incorporation of the unnatural amino acid. As for the embodiments described above, the host cell used is not particularly limited, as long as the O-RS and O-tRNA retain their orthogonality in their host cell environment. For example, the host cell can be a eubacterial cell, such as E. coli. The host cell can be engineered to reduce or eliminate undesired metabolism of the α-hydroxy acid; for example, the host cell can be an E. coli cell having disruptions in tyrB and aspC. The host cell can comprise one or more polynucleotides that encode components of the translation system, such as the O-RS and/or O-tRNA. In some embodiments, the polynucleotide encoding the O-RS comprises a nucleotide sequence of SEQ ID NO:2. In some embodiments, the step of providing a translation system is accomplished by providing a cell extract.

[0020] The ester bond introduced into the protein's backbone by incorporation of the p-hydroxy-L-phenyllactic acid residue is subject to attack by nucleophiles. Accordingly, the methods can include reacting the protein with a nucleophilic compound, optionally after purification of the protein from the translation system. In one example, the nucleophilic compound is water and the reaction involves hydrolysis of the protein at the ester bond. In another example, the nucleophilic compound is ammonia and the reaction involves ammoniolysis of the protein at the ester bond. In general, the reaction involves transacylation. Other exemplary nucleophilic compounds comprise one or more of a label, a fluorophore, an affinity tag, a biotin moiety, an oligonucleotide, a carbohydrate, a toxin, a drug, a polyethylene glycol, a synthetic peptide, or a metal ion chelator and/or comprise an alkoxyamine, a hydroxylamine, a hydrazine, a hydrazide, an amine, a thiol, or a hydroxyl.

[0021] Other aspects of the invention provide a variety of compositions, including nucleic acids and proteins. The nature of the composition is not particularly limited, other than that the composition comprises the specified nucleic acid or protein. The compositions of the invention can comprise any number of additional components of any nature.

[0022] For example, one general class of embodiments provides a composition comprising an O-RS polypeptide, where the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a conservative variant thereof. In some aspects, the conservative variant polypeptide charges a cognate orthogonal tRNA (O-tRNA) with an unnatural amino acid (e.g., an α-hydroxy acid, e.g.,/?-hydroxy-L-phenyllactic acid) with an efficiency that is at least 50% of the efficiency observed for a translation system comprising the O-tRNA, the unnatural amino acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID NO: 1 (e.g., at least 60%, 70%, 75%, 80%, 90%, 95%, or 99% or more efficiency). In some embodiments, the polypeptides are in a cell.

[0023] Polynucleotides that encode any of the above polypeptides are also a feature of the invention. In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or the complement thereof. Vectors comprising the polynucleotides of the invention (e.g., expression vectors) are also a feature of the invention, as are cells comprising the vectors.

[0024] As noted, site-specific incorporation of an α-hydroxy acid and the concomitant introduction of an ester bond at a defined position in the polypeptide backbone are of particular interest herein. In one aspect, since replacement of an amide bond with an ester bond alters hydrogen bonding patterns, site-specific incorporation of an α-hydroxy acid (e.g., /?-hydroxy-L-phenyllactic acid) facilitates study of the roles of backbone hydrogen bonding in protein folding, thermodynamics, and kinetics, biomolecular interactions, ion channel gating, and enzyme mechanisms, for example. In other aspects, reactivity of the ester bond is exploited, for example, for removal of C-terminal affinity tags after affinity purification or for modification of the C-terminus through a selective transacylation reaction.

[0025] In one exemplary application, the ester bond is introduced between two polypeptides from different parental polypeptides (e.g., two different proteins, two homologous proteins from different species, an affinity tag and a protein of interest, etc.). The resulting fusion protein can then be fragmented by hydrolysis of the ester bond.

[0026] Thus, one general class of embodiments provides methods of producing a first polypeptide comprising a first polypeptide sequence. The methods include providing a translation system comprising an α-hydroxy acid (e.g., /?-hydroxy-L-phenyllactic acid), a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the α-hydroxy acid, and a nucleic acid encoding a fusion protein. The nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by the first O-tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon. The α-hydroxy acid is incorporated at a selected position in the fusion protein during translation of the fusion protein in response to the selector codon, thereby producing the fusion protein comprising the α-hydroxy acid at the selected position and an ester bond in the protein backbone (between the first and second polypeptide sequences). The first polypeptide sequence is then released from the second polypeptide sequence by hydrolysis of the ester bond, producing the first polypeptide.

[0027] The methods can be employed to remove polypeptide tags after affinity purification of the fusion protein. Accordingly, the methods can include isolating the fusion protein from the translation system, for example, by providing a solid support comprising a binding moiety, binding the second polypeptide sequence to the binding moiety, and separating materials not captured on the solid support from the solid support, prior to hydrolysis of the ester bond. After hydrolysis, the first polypeptide can be isolated from the second polypeptide sequence. Hydrolysis of the ester bond can be conveniently achieved, for example, by incubating the fusion protein in an alkaline aqueous solution.

[0028] The second polypeptide sequence can be N-terminal of the first polypeptide sequence in the fusion protein. However, preferably, the second polypeptide sequence is C- terminal of the first polypeptide sequence in the fusion protein.

[0029] The second polypeptide optionally is or includes an affinity tag such as those well known in the art. For example, the second polypeptide sequence can comprise one or more of a polyhistidine tag, a polyarginine tag, a polycysteine tag, a polyphenyalanine tag, a polyaspartic acid tag, a glutathione-S-transferase (GST) sequence, an S tag, an epitope tag, a maltose binding protein sequence, a galactose-binding protein sequence, and a cellulose binding domain.

[0030] A related general class of embodiments provides methods of covalently attaching a first moiety to the C-terminus of a first polypeptide sequence. The methods include providing a translation system comprising an α-hydroxy acid (e.g.,/?-hydroxy-L- phenyllactic acid), a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the α- hydroxy acid, and a nucleic acid encoding a precursor protein. The nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by the first O-tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon. The α-hydroxy acid is incorporated at a selected position in the precursor protein during translation of the precursor protein in response to the selector codon, thereby producing the precursor protein comprising an ester bond in the protein backbone and the α-hydroxy acid at the selected position. The precursor protein is contacted with a nucleophilic compound comprising the first moiety, and the nucleophilic compound reacts with the ester bond in the precursor protein to attach the first moiety to the C-terminus of the first polypeptide sequence and release the second polypeptide sequence from the first polypeptide sequence.

[0031] In a preferred aspect, the nucleophilic compound is a compound other than water. For example, in one embodiment, the nucleophilic compound is ammonia, the first moiety comprises a nitrogen atom, and reacting the ammonia with the ester bond comprises ammoniolysis of the ester bond. As other examples, the nucleophilic compound can comprise an alkoxyamine, a hydroxylamine, a hydrazine, a hydrazide, an amine, a thiol, or a hydroxyl.

[0032] The first moiety that is transferred to the first polypeptide optionally comprises one or more of a label (e.g., a fluorophore, spin label, or other biophysical probe), an affinity tag, a biotin moiety, an oligonucleotide or derivative (e.g., a tag, an antisense nucleic acid, or an siRNA, e.g., a chemically synthesized polynucleotide), a carbohydrate, a toxin, a drug, a polyethylene glycol (PEG) or derivative, a polypeptide (e.g., a synthetic peptide), a metal ion chelator, a cross-linking agent, or a fatty acid. A variety of useful nucleophilic compounds are commercially available, and others can be readily produced by one of skill.

[0033] As for the embodiments above, the second polypeptide optionally comprises an affinity tag that is employed in purification as described above prior to its removal during the transacylation reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 depicts the structure of p-hydroxy-L-phenyllactic acid and schematically illustrates its incorporation into a polypeptide.

[0035] Figure 2 Panels A and B illustrate metabolic engineering of amino acid synthesis pathways in E. coli. Panel A illustrates Tyr and Phe biosynthesis in E. coli. TyrA, chorismate mutase/prephenate dehydrogenase; TyrB, tyrosine aminotransferase; AspC, aspartate aminotransferase. Panel B depicts a growth curve of GWAPOl cells in GMML medium with different nutrient supplementation. Diamonds (O), no additional nutrient; squares (D), p-hydroxy-L-phenyllactic acid and aspartic acid (1 mM each); triangles (Δ),/?-hydroxyphenylpyruvate and aspartic acid (1 mM each); asterisks (*), tyrosine and aspartic acid (1 mM each); and circles (O), tyrosine, phenylalanine, and aspartic acid (1 mM each).

[0036] Figure 3 Panel A depicts Coomassie-stained SDS-PAGE of Lys99 → 1 mutant myoglobin expressed in the presence (lane 1 , full-length protein and cleaved fragments due to the basic SDS-PAGE buffer) and absence (lane 2) of 1 mM 1. Lane 3 shows the myoglobin Lys99Tyr mutant. The same samples in lane 1 , 2 and 3 were treated with 0.67 M NaOH for 20 minutes at 4 °C, neutralized to pH 7.0, and analyzed in lanes 4 (cleaved fragments), 5, and 6, respectively. Panel B depicts LC-ESI spectra of the Lys99 — » 1 mutant myoglobin before (top panel) and after (bottom panel) base hydrolysis. Panel C depicts ESI-MS spectra of the two fragments of Lys99 — > 1 mutant myoglobin after hydrolysis. The insert shows the deconvoluted spectra. Expected masses of the two fragments are 11049 Da and 7360 Da; observed masses are 11048 Da and 7360 Da (with N- terminal methionine).

[0037] Figure 4 depicts Coomassie-stained SDS-PAGE of Ser4 → 1, Ala75 → 1,

Lys99 — » 1, and Tyrl04 → 1 mutant myoglobins before base hydrolysis (lanes 1, 2, 3 and 4, respectively), and after base hydrolysis (lanes 5, 6, 7 and 8, respectively). Some fragmentation is observed in the SDS-PAGE for the mutants before base treatment. Because no fragmentation is observed in the LC-ESI experiments, this is likely due to hydrolysis in the basic SDS-PAGE buffer (pH 8.8).

[0038] Figure 5 Panels A and B illustrate efficient C-terminal His₆ tag cleavage of

Ser63 — > 1 mutant Z-domain protein. Panel A depicts SDS-PAGE gel of the Ser63 — > 1 mutant Z-domain protein. Molecular weight marker, lane M; Z-domain protein with C- terminal His₆ tag before base hydrolysis, lane 1 ; Z-domain protein with C-terminal His₆ tag after base hydrolysis, lane 2. Panel B depicts ESI-MS spectra of Ser63 → 1 mutant Z- domain protein. Calculated mass before hydrolysis: 7961 Da (without the N-terminal methionine); observed mass 7960 Da. Calculated mass after hydrolysis: 6975 Da (without the N-terminal methionine); observed mass: 6974 Da.

[0039] Figure 6 Panel A depicts the structure of sperm whale myoglobin. Residues

Ala75 and TyrlO4 are shown as sticks. Panel B depicts guanidine hydrochloride (GuHCl) induced denaturation of wild type and mutant apo-myoglobin proteins monitored by circular dichroism. Wild type (squares, ■), TyrlO4 — > 1 (triangles, A), Ala75 — » Tyr (circles, •), Ala75 — » 1 (diamonds, ♦) samples were assayed in 10 mM sodium phosphate, pH 7.3, with various concentrations of GuHCl as indicated. Panel C depicts the free energy change (ΔG) between folded (F) and unfolded (U) states in various concentrations of GuHCl. The insert shows the calculated unfolding energy of the wild type and mutant myoglobin in 0 M GuHCl.

[0040] Figure 7 Panel A schematically illustrates hydrolysis of a polypeptide including an α-hydroxy acid and thus an ester bond in its backbone. Panel B schematically illustrates ammoniolysis of a polypeptide including an α-hydroxy acid. Panel C schematically illustrates a generalized acyl transfer reaction involving a polypeptide including an α-hydroxy acid and thus having an ester bond in its backbone.

[0041] Schematic figures are not necessarily to scale.

DEFINITIONS

[0042] Unless defined otherwise, 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. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0043] As used in this specification and the appended claims, the singular forms "a,"

"an" and "the" include plural referents unless the context 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, etc.

[0044] Orthogonal: As used herein, the term "orthogonal" refers to functional molecules, e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl-tRNA synthetase (O-RS), that function poorly or not at all with endogenous components of a cell or translation system, when compared to a corresponding molecule (tRNA or RS) that is endogenous to the cell or translation system. Orthogonal components are usefully provided as cognate components that function well with each other, e.g., an O-RS can be provided that can efficiently aminoacylate a cognate O-tRNA in a cell, even though the O-tRNA functions poorly or not at all as a substrate for the endogenous RS of the cell, and the ORS functions poorly or not at all with endogenous tRNAs of the cell. Various comparative efficiencies of the orthogonal and endogenous components can be evaluated. For example, an O-tRNA will typically display poor or non-existent activity as a substrate, under typical physiological conditions, with endogenous RSs, e.g., the O-tRNA is less than 20% or less than 10% as efficient as a substrate as endogenous tRNAs for any endogenous RS, and will typically be less than 5%, and usually less than 1% as efficient a substrate. At the same time, the O-tRNA can be highly efficient as a substrate for the O-RS, e.g., at least 50%, and often 75%, 90%, 95%, 99%, or even 100% or more as efficient as an aminoacylation substrate as any endogenous tRNA is for its endogenous RS. Similarly, an O-RS will typically display poor or non-existent activity, under typical physiological conditions, with endogenous tRNAs, e.g., showing less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency with an endogenous tRNA compared to an endogenous tRNA synthetase with the endogenous tRNA.

[0045] Orthogonal tRNA: As used herein, an "orthogonal tRNA" (O-tRNA) is a tRNA that is orthogonal to a translation system of interest. The O-tRNA can exist charged with an amino acid (including, e.g., an unnatural amino acid such as an α-hydroxy acid), or in an uncharged state. It is also to be understood that an O-tRNA is optionally charged (aminoacylated) by a cognate orthogonal aminoacyl-tRNA synthetase, e.g., with an α- hydroxy acid. It will be appreciated that an O-tRNA of the invention is advantageously used to insert essentially any amino acid, whether natural or unnatural (e.g., an α-hydroxy acid), into a growing polypeptide during translation in response to a selector codon. As an example, 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) the same as or homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence to) the exemplary tRNA of SEQ ID NO:3, or (6) a conservative variant of the exemplary tRNA of SEQ ID NO:3. 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, e.g., with an unnatural amino acid such as an α-hydroxy acid.

[0046] Orthogonal aminoacyl tRNA synthetase: As used herein, an "orthogonal aminoacyl tRNA synthetase" (O-RS) is an enzyme that preferentially charges (aminoacylates) an O-tRNA with an amino acid (whether natural or unnatural, e.g., an α- hydroxy acid) in a translation system of interest. As an example, as used herein, an "orthogonal tyrosyl amino acid synthetase" (tyrosyl-O-RS) is an enzyme that preferentially charges (e.g., aminoacylates) a 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. For example, the O-RS can charge the O-tRNA with an α-hydroxy acid, e.g., with/7-hydroxy-L- phenyllactic acid. The tyrosyl-O-RS is optionally: (1) identical or substantially similar to a naturally occurring tyrosyl-RS, (2) derived from a naturally occurring tyrosyl-RS by natural or artificial mutagenesis, (3) derived by any process that takes a sequence of a wild-type or mutant tyrosyl-RS sequence of (1) or (2) into account, (4) homologous to a wild- type or mutant tyrosyl-RS; (5) the same as or homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence to) the exemplary O-RS of SEQ ID NO: 1, or (6) a conservative variant of the exemplary O-RS of SEQ ID NO: 1.

[0047] 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.

[0048] Preferentially charges: As used herein in reference to orthogonal translation systems, an O-RS "preferentially charges" or "preferentially aminoacylates" a cognate O- tRNA when the O-RS charges the O-tRNA with an amino acid (whether natural or unnatural, e.g., an α-hydroxy 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. The terms "charges" and "aminoacylates" are used interchangeably herein to refer to loading of a tRNA with a natural or unnatural amino acid by a tRNA synthetase, regardless of whether the reaction is technically an aminoacylation reaction (as for one of the natural amino acids) or is an analogous acylation reaction (as for an α- hydroxy acid).

[0049] The O-RS "preferentially charges an O-tRNA with an unnatural amino acid"

(or "preferentially aminoacylates an O-tRNA with an unnatural amino acid") when (a) the O-RS preferentially charges (aminoacylates) the O-tRNA compared to an endogenous tRNA, and (b) where that charging (aminoacylation) is specific for the unnatural amino acid, as compared to charging (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.

[0050] 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, noncoding codons, codons derived from natural or unnatural base pairs, and/or the like.

[0051] Suppressor tRNA: A "suppressor tRNA" is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by providing a mechanism for incorporating an amino acid into a polypeptide chain in response to a selector codon. For example, a suppressor tRNA can read through, e.g., a stop codon (e.g., an amber, ocher or opal codon), a four base codon, a rare codon, etc. For example, a typical suppressor tRNA allows the incorporation of an amino acid in response to a stop codon (i.e., "read- through") during the translation of a polypeptide.

[0052] 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.

[0053] 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.

[0054] Suppression efficiency can be determined by any of a number of assays known in the art. For example, a /?-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_60O of about 0.5, and β-galactosidase assays are performed, e.g., using the BetaFluor™ β-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.

[0055] Translation system: The term "translation system" refers to the components that incorporate an amino acid (whether natural or unnatural) 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 non-eukaryotic cell, e.g., a bacterium (such as E. coli), or in a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell, and/or the like.

[0056] Unnatural amino acid: As used herein, the term "unnatural amino acid" refers to any amino acid, modified amino acid, and/or amino acid analog that is not one of the 20 common naturally occurring amino acids or selenocysteine or pyrrolysine. As used broadly herein, unnatural amino acids include amino acid analogs having modified backbone structures, such as α-hydroxy acids. For example, the α-hydroxy acid/?-hydroxy- L-phenyllactic acid (see Figure 1) finds use with the invention.

[0057] 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.

[0058] 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 OtRNA 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 charge (aminoacylate) a cognate O-tRNA with an unnatural amino acid, e.g., p-hydroxy-L-phenyllactic acid. 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.

[0059] 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, optionally at least 60%, 70%, 75%, 80%, 90%, 95%, or 99% or more. 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Nucleic acid: The term "nucleic acid" or "polynucleotide" encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. The polymer can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The nucleotides of the polynucleotide can, e.g., be deoxyribonucleotides, ribonucleotides or nucleotide analogs, can be natural or non-natural, and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like. A nucleic acid can be e.g., single-stranded or double-stranded. Unless otherwise indicated, a particular nucleic acid sequence of this invention encompasses complementary sequences, in addition to the sequence explicitly indicated.

[0064] Polynucleotide sequence: A "polynucleotide sequence" or "nucleotide sequence" is a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.

[0065] Polypeptide: A "polypeptide" is a polymer comprising two or more amino acid residues (e.g., a peptide or a protein). The polymer can additionally comprise non- amino acid elements such as labels, quenchers, blocking groups, or the like and can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural or unnatural and can be unsubstituted, unmodified, substituted or modified.

[0066] Amino acid sequence: An "amino acid sequence" or "polypeptide sequence" is a polymer of amino acid residues (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.

[0067] Fusion protein: The term "fusion protein" indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein (or a peptide purification tag etc.)- The fusion gene can then be expressed by a cell or translation system as a single polypeptide.

[0068] Host cell: The term "host cell" means a cell which contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such as E. coli or eukaryotic cells

[0069] Vector: The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome- conjugated DNA, or the like, that are not autonomously replicating. Most commonly, the vectors of the present invention are plasmids.

[0070] An "expression vector" is a vector, such as a plasmid, which is capable of promoting expression as well as replication of a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.

[0071] Label: A "label" is a moiety that facilitates detection of a molecule.

Common labels in the context of the present invention include fluorescent, luminescent, and/or colorimetric labels. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labels are commercially available and can be used in the context of the invention.

[0072] Hydroxylamines: "Hydroxylamines" include hydroxylamine (H₂N-OH) and derivatives thereof.

[0073] Alkoxyamines: "Alkoxyamines" include O-alkyl hydroxylamines, with or without substitution on N (R₂NOR', R' ≠ H), and derivatives thereof. [0074] Hydrazines: "Hydrazines" include hydrazine (H₂NNH₂) and derivatives thereof. When one or more substituents are acyl groups, the compound is referred to as a "hydrazide."

[0075] 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.

[0076] 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).

[0077] 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., β-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, β-gal/lacZ (β- galactosidase), ADH (alcohol dehydrogenase), his3, ura3, Ieu2, Iys2, or the like.

[0078] 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.

[0079] Therapeutic protein: A "therapeutic protein" is a protein that can be administered to a patient to treat a disease or disorder.

[0080] A variety of additional terms are defined or otherwise characterized herein. DETAILED DESCRIPTION

[0081] In one aspect, the invention provides methods and compositions for the selective incorporation of the α-hydroxy acid/?-hydroxy-L-phenyllactic acid into proteins by genetically encoding the α-hydroxy acid. This general approach to the biosynthesis of proteins including ester bonds at selected positions facilitates further study and application of this backbone modification.

[0082] As a general method for the site-specific incorporation of ester bonds, in one aspect the present invention describes inter alia the evolution of an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allows the efficient, selective incorporation of p-hydroxy-L-phenyllactic acid (also called 4-hydroxy-L-phenyllactic acid; Figure 1) into proteins with good yield and high fidelity, e.g., in prokaryotes such as E. coli in response to the amber nonsense codon TAG. Briefly, novel orthogonal aminoacyl-tRNA synthetase (O- RS) polypeptides derived from the Methanococcus jannaschii tyrosyl-tRNA synthetase that specifically charge a cognate orthogonal tRNA (O-tRNA) with the unnatural amino acid/?- hydroxy-L-phenyllactic acid, e.g., in an E. coli host cell, are provided. These evolved tRNA- synthetase pairs can be used to site-specifically incorporate the unnatural α-hydroxy acid into a protein. The incorporation of the α-hydroxy acid into the protein can be programmed to occur at any desired position by engineering the polynucleotide encoding the protein of interest to contain a selector codon that signals the incorporation of the α- hydroxy acid.

[0083] In orthogonal pairs for the genetic encoding and incorporation of the unnatural amino acidp-hydroxy-L-phenyllactic acid into proteins, e.g., in a eubacteria such as E. coli, the orthogonal components do not cross-react with endogenous E. coli components of the translational machinery of the host cell, but recognize the desired unnatural amino acid and incorporate it into proteins in response to a selector codon (e.g., an amber nonsense codon, TAG). The orthogonal components provided by the invention include orthogonal aminoacyl-tRNA synthetases derived from Methanococcus jannaschii tyrosyl tRNA-synthetase and the mutant tyrosyl tRNAcuA amber suppressor O-tRNA, which function as an orthogonal pair in a eubacterial host cell.

[0084] Among other aspects, this invention provides compositions of and methods for identifying and producing additional orthogonal tRNA-aminoacyl-tRNA synthetase pairs, e.g., 0-tRNA/O-RS pairs that can be used to incorporate /j-hydroxy-L-phenyllactic acid into proteins. An 0-tRNA/O-RS pair of the invention is capable of mediating incorporation of the α-hydroxy acid into a protein that is encoded by a polynucleotide, where the polynucleotide comprises a selector codon that is recognized by the O-tRNA. The anticodon loop of the O-tRNA recognizes the selector codon on an mRNA and incorporates the α-hydroxy acid at this site in the polypeptide. Generally, an orthogonal aminoacyl-tRNA synthetase of the invention preferentially charges (or aminoacylates) its O-tRNA with only one specific unnatural amino acid.

[0085] Applications of the novel O-RS/O-tRNA are also described. Site-specific incorporation of ester bonds facilitates studies of protein structure and function, as noted above. In addition, in one aspect the invention provides techniques for conveniently removing purification tags from proteins by hydrolysis of the incorporated ester bonds. In other aspects, site-specific modification of proteins at the ester bond is described.

[0086] In some aspects, to demonstrate (but not to limit) the present invention, the disclosure herein demonstrates that the α-hydroxy acid moiety can be incorporated into various model proteins. It is not intended that the incorporation of the unnatural amino acid be limited to any particular protein. From the present disclosure, it will be clear that the incorporation of the unnatural amino acid/?-hydroxy-L-phenyllactic acid and other α- hydroxy acids into particular proteins of interest is advantageous for a wide variety of purposes.

ORTHOGONAL tRNA/AMINOACYL-tRNA SYNTHETASE TECHNOLOGY [0087] 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 the pair functions independently of the synthetases and tRNAs endogenous to the translation system. Desired characteristics of the orthogonal pair include a tRNA that decodes or recognizes only a specific codon, e.g., a selector codon, that is not decoded by any endogenous tRNA, and an aminoacyl-tRNA synthetase that preferentially aminoacylates (or "charges") 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 tRNAs, e.g., of 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.

[0088] 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 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 July 7, 2004; WO 2005/007870, filed July 7, 2004; WO 2005/007624, filed July 7, 2004; WO 2006/110182, filed October 27, 2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS" and WO 2007/103490, filed March 7, 2007, entitled "SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS." 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(l):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.

Orthogonal Translation Systems [0089] Orthogonal translation systems generally comprise cells (which can be prokaryotic cells such as E. coli or eukaryotic 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, e.g.,p-hydroxy- L-phenyllactic 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 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 novel individual components, for example, novel orthogonal aminoacyl-tRNA synthetase polypeptides (e.g., SEQ ID NO:1), and polynucleotides that encode those polypeptides (e.g., SEQ ID NO:2).

[0090] 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.

[0091] 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.

[0092] 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 listing herein.

[0093] The host cell (or other translation system) 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 (e.g., with a different α-hydroxy 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.

[0094] As noted, in some embodiments, there exist multiple 0-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.

[0095] In certain embodiments, systems comprise a cell such as an E. coli 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. The translation system can also be a cell-free system, e.g., any of a variety of commercially available "m vitro" transcription/translation systems, in combination with an O-tRNA/O-RS pair and an unnatural amino acid as described herein.

[0096] 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.

Orthogonal tRNA (O-tRNA) [0097] 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.

[0098] Suppression efficiency can be determined by any of a number of assays known in the art. For example, a /?-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 OD₆₀₀ of about 0.5, and β-galactosidase assays are performed, e.g., using the BetaFluor™ β-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.

[0099] An exemplary O-tRNA of the invention is set forth in the sequence listing herein, for example, see SEQ ID NO:3. The disclosure herein also provides guidance for the design of additional equivalent O-tRNA species, and other suitable O-tRNAs are known in the art (see, e.g., references herein). 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. [0100] 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, and desirably, are other than wild-type tRNA molecules.

[0101] 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 translation system (e.g., a cell).

[0102] Methods of producing an orthogonal tRNA (O-tRNA) have been described; see, e.g., references herein. For example and in brief, O-tRNAs can be produced by generating a library of mutant tRNAs 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.

[0103] 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. [0104] 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 E. coli, a synthetase and/or a tRNA is chosen that does not display close sequence similarity to eubacterial organisms.

[0105] 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 OtRNAs. 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.

[0106] 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., β-lactamase, an enzyme that confers a detectable product, e.g., β-galactosidase, chloramphenicol acetyltransferase (CAT), or 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.

[0107] 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 β-lactamase (bla). For example, polynucleotide variants, e.g., bla variants, with a selector codon at a certain position (e.g., Al 84), 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.

[0108] 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.

[0109] 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 β-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.

[0110] 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.

[0111] 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.

[0112] 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 U S A. 90: 10444-8.

[0113] 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 July 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. Orthogonal Aminoacyl-tRNA Synthetase (Q-RS) [0114] An O-RS of the invention preferentially charges 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 as set forth in SEQ ID NO: 1, or a conservative variant 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 polynucleotide of SEQ ID NO:2. In one exemplary embodiment, the O-RS is derived from or homologous to a Methanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase such as that of SEQ ID NO:5, and optionally comprises an Arg residue at position 155, a GIy residue at position 173, a VaI residue at position 176, or a combination thereof, wherein amino acid position numbering corresponds to amino acid position numbering of the wild-type tyrosyl tRNA synthetase. The O-RS optionally also includes one or more of a GIu residue at position 36, an He residue at position 137, and a Tyr residue at position 151.

[0115] 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 (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.

[0116] 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, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE."

[0117] 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 similar to that described above for selection of O- tRNA, based on a combination of positive selection and negative selection, is typically used. 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. [0118] 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 organisms (e.g., microorganisms such as eubacteria or archaebacteria), such as libraries that comprise natural diversity (see, e.g., U.S. Patent No. 6,238,884 to Short et al; U.S. Patent No. 5,756,316 to Schallenberger et al; U.S. Patent No. 5,783,431 to Petersen et al; U.S. Patent No. 5,824,485 to Thompson et al; U.S. Patent No. 5,958,672 to Short et al), are optionally constructed and screened for orthogonal pairs.

[0119] 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.

[0120] 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.

[0121] Additional general details for producing O-RS and altering the substrate specificity of the synthetase can be found in Internal Publication Numbers 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. See also Example 1 hereinbelow, which describes production of an O-RS derived from the Methanococcus jannaschii tyrosyl-tRNA synthetase that specifically charges a cognate O- tRNA with the α-hydroxy acidp-hydroxy-L-phenyllactic acid.

SOURCE AND HOST ORGANISMS

[0122] 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 Archaea genes (i.e., archaebacteria) for use in a eubacterial host system.

[0123] For example, the O-tRNA and/or the O-RS 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-I , 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, or a combination of such organisms. 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. 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. See also, International Application Publication Number WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE," filed April 16, 2004, for screening O-tRNA and/or O-RS in one species for use in another species.

[0124] The O-tRNA, O-RS or 0-tRNA/O-RS pair can be selected or screened in vivo or in vitro and/or used in a cell, e.g., a non-eukaryotic cell or a eukaryotic cell, to produce a polypeptide with an unnatural amino acid. The cell used is not limited; any of the wide variety known in the art can be employed, as convenient. A non-eukaryotic cell can be from any of a variety of sources, e.g., a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus, or the like, or an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-I, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maήpaludis, Methanopyrus kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the like. A eukaryotic cell can be from any of a variety of sources, e.g., a plant (e.g., complex plants such as monocots or dicots), an algae, a protist, a fungus, a yeast (e.g., Saccharomyces cerevisiae), an animal (e.g., a mammal, an insect, an arthropod, etc.), or the like. For example, suitable insect host cells include, but are not limited to, Lepidopteran, Spodoptera frugiperda, Bombyx mori, Heliothis virescens, Heliothis zea, Mamestra brassicas, Estigmene acrea, and Trichoplusia ni insect cells; exemplary insect cell lines include BT1-TN-5B1-4 (High Five), BTI-TN-MGl, Sf9, Sf21, TN-368, D.Mel-2, and Schneider S-2 cells, among many others. To express a protein incorporating an unnatural amino acid, such insect cells are optionally infected with a recombinant baculovirus vector encoding the protein and a selector codon. A variety of baculovirus expression systems are known in the art and/or are commercially available, e.g., BaculoDirect™ (Invitrogen, Carlsbad, CA) and BD BaculoGold™ Baculovirus Expression Vector System (BD Biosciences, San Jose, CA). Compositions of eubacterial or other cells comprising translational components of the invention are also a feature of the invention.

[0125] 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

[0126] 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 p-hydroxy-L-phenyllactic acid residue, using these different selector codons.

[0127] 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, e.g.,/?-hydroxy-L-phenyllactic 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. 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 selector codon used is a stop codon, e.g., 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.

[0128] 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 (RFl) (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 RFl 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).

[0129] 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^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). Thus, 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. Components of the invention can be generated to use these rare codons in vivo.

[0130] 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) "xploring 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. MoI. Biol., 307: 755-769.

[0131] 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^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^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. MoI. 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(l):34-66 (2005), the content of which is incorporated by reference in its entirety. While the example below utilizes 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.

[0132] 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 et al, (2002) J. Am. Chem. Soc, 124:14626-14630. Other relevant publications are listed below.

[0133] 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 α/., (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, DipicPy, 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.

[0134] 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

[0135] As used herein, an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analog 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, and valine. The generic structure of an alpha-amino acid is illustrated by Formula I:

I

[0136] An unnatural amino acid can include 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^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.

[0137] Because unnatural amino acids such as those illustrated by Formula I typically differ from the natural amino acids in side chain, they 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, such unnatural amino acids have side chain groups that distinguish them from the natural amino acids.

[0138] 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-analog 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.

[0139] In another aspect, the invention provides unnatural amino acids having the general structure illustrated by Formula II below:

II

[0140] An unnatural amino acid having this structure is typically any structure where Ri is a substituent used in one of the twenty natural amino acids (e.g., tyrosine or phenylalanine) and R₂ is a substituent. Thus, this type of unnatural amino acid can be viewed as a natural amino acid derivative.

[0141] In addition to unnatural amino acids differing from natural amino acids in their side chains, unnatural amino acids can also optionally comprise modified backbone structures, e.g., as illustrated by the structures of Formulas III and IV:

III

IV

wherein Z typically comprises OH, NH₂, 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 Formulas I and II 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 III and IV. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids (i.e., where Z is OH, X is O, and Y is O in Formula III), α-thioacids, and α-aminothiocarboxylates, e.g., with side chains corresponding to the common twenty natural amino acids or unnatural side chains. In addition, substitutions at the α-carbon optionally include L, D, or α-α-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 analogs as well as 3,4,6,7,8, and 9 membered ring proline analogs, β and γ amino acids such as substituted β- alanine and γ-amino butyric acid.

[0142] 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.

[0143] As noted previously, α-hydroxy acids are of particular interest in the present invention, including p-hydroxy-L-phenyllactic acid (shown in Figure 1). Although p- hydroxy-L-phenyllactic acid is of primary interest in the Examples described herein, it is not intended that the invention necessarily be strictly limited to that structure. Indeed, a variety of easily-derived, structurally related analogs can be readily produced that retain the principle characteristic of/j-hydroxy-L-phenyllactic acid, and also are optionally specifically recognized by the aminoacyl-tRNA synthetases of the invention (e.g., the O-RS of SEQ ID NO: 1). It is intended that these related amino acid analogs are within the scope of the invention. One of skill in the art will recognize that a wide variety of unnatural analogs are easily derived, e.g., analogs in which the ring is para-substituted, ortho- substituted, or meta substituted, wherein the substituted ring 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 C₆ - C₂₀ 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.

[0144] In addition to /j-hydroxy-L-phenyllactic acid (and/or other α-hydroxy acids), other unnatural amino acids can be simultaneously incorporated into a polypeptide of interest, e.g., using an appropriate second 0-RS/O-tRNA pair in conjunction with an orthogonal pair provided by the present invention. Many such additional unnatural amino acids and suitable orthogonal pairs are known. See the present disclosure and the references cited herein. For example, see Wang and Schultz "Expanding the Genetic Code," Angewandte Chemie Int. Ed., 44(l):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.

[0145] Examples of α-hydroxy acids include, but are not limited to, p-hydroxy-L- phenyllactic acid (the α-hydroxy analog of tyrosine), leucic acid (the α-hydroxy analog of leucine), lactic acid (the α-hydroxy analog of alanine), 2-hydroxy-3-methylbutyric acid (the α-hydroxy analog of valine), 2-hydroxy-3-phenylpropionic acid, and α-hydroxy analogs of other natural and unnatural amino acids. Examples of additional unnatural amino acids include, but are not limited to, sulfotyrosine,/?-ethylthiocarbonyl-L-phenylalanine,/>-(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- carboxyrnethyl-L-phenylalanine,/?-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine, bipyridyl alanine, p-(2-amino-l-hy droxyethyl)-L- phenylalanine, /7-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine and/?-nitro-L- phenylalanine. Also, a/?-propargyloxyphenylalanine, a 3, 4-dihydroxy-L-pheny alanine (DHP), a 3, 4, 6-trihydroxy-L-phenylalanine, a 3,4,5-trihydroxy-L-phenylalanine, 4-nitro- phenylalanine, a/?-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-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, ap- acyl-L-phenylalanine, a/?-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a^-iodo-phenylalanine, a/»-bromophenylalanine, a/7-amino-L- phenylalanine, and an isopropyl-L-phenylalanine, and the like. Glutamine analogs include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Example phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanines, 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. The structures of a variety of unnatural amino acids are disclosed in the references cited herein. See also, WO 2006/110182, filed October 27, 2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS."

Chemical Synthesis of Unnatural Amino Acids [0146] Many of the unnatural amino acids provided above are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, WI, 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 γ-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)-l- methylbutyl]amino]quinoline (Chloroquine)." J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991) "Glutamine analogues as Potential Antimalarials" Eur. J. Med. Chem. 26, 201-5; Koskinen, A.M.P. & Rapoport, H. (1989) "Synthesis of 4- Substituted Prolines as Conformationally Constrained Amino Acid Analogues" J. Org. Chem. 54, 1859-1866; Christie, B.D. & Rapoport, H. (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 December 22, 2003.

Cellular Uptake of Unnatural Amino Acids [0147] 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 α-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 December 22, 2003; and Liu and Schultz (1999) "Progress toward the evolution of an organism with an expanded genetic code" PNAS 96:4780-4785.

[0148] Derivitization of amino acids (e.g., esterification or acylation of highly charged or hydrophilic amino acids with groups that hydrolyze in the cytoplasm) is optionally employed to improve uptake.

[0149] In some instances, unnatural amino acids are taken up by cells but are then metabolized. Incorporation of metabolically labile unnatural amino acids in in vivo translation systems can be facilitated by employing host cells in which specific metabolic enzymes are deleted; the growth medium of the strain is supplemented as necessary. See, e.g., Example 1 hereinbelow, in which an E. coli strain lacking tyrosine aminotransferase (tyrB) and aspartate aminotransferase (aspC) was used for incorporation of the α-hydroxy acidp-hydroxy-L-phenyllactic acid. See also Wang et al. (2006) "Expanding the Genetic Code" Annu. Rev. Biophys. Biomol. Struct. 35:225^9.

[0150] 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.

Biosynthesis of Unnatural Amino Acids [0151] 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 cells 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 j9-aminophenylalanine (as presented in an example in WO 2002/085923) 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.

[0152] 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 & 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.

[0153] 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.

[0154] 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.

[0155] 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.

Orthogonal Components for Incorporating Unnatural Amino Acids [0156] The invention provides compositions and methods for producing orthogonal components for incorporating the unnatural amino acidp-hydroxy-L-phenyllactic acid (see Figure 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.

[0157] In one aspect, a composition of the invention includes an orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially charges an O-tRNA withp-hydroxy-L-phenyllactic acid. In certain embodiments, the O-RS comprises an amino acid sequence comprising SEQ ID NO: 1 or a conservative variant thereof. In some embodiments, the O-RS preferentially charges the O-tRNA with/p-hydroxy-L-phenyllactic acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising that same O-tRNA, thep-hydroxy-L-phenyllactic acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID NO: 1 (e.g., at least 60%, 70%, 75%, 80%, 90%, 95%, or 99% or more efficiency). In certain embodiments of the invention, the O-RS preferentially aminoacylates the O-tRNA over any endogenous tRNA with/?-hydroxy-L-phenyllactic 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.

[0158] 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 listing (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% (e.g., at least 50%, 60%, 75%, 80%, or 90%) of the suppression efficiency of an orthogonal tyrosyl- tRNA synthetase pair derived from Methanococcus jannaschii (e.g., of SEQ ID NOs: 1 and

3).

[0159] A composition that includes an O-RS can optionally include a cell (e.g., a eubacterial cell, such as an E. coli cell and the like, or a eukaryotic cell such as a yeast cell) and/or a translation system. The composition optionally includes the unnatural amino acid p-hydroxy-L-phenyllactic acid.

[0160] A translation system is also an aspect of the invention, where the translation system includes an orthogonal-tRNA (O-tRNA), an orthogonal aminoacyl-tRNA synthetase (O-RS), and ap-hydroxy-L-phenyllactic acid unnatural amino acid. Typically, the O-RS preferentially charges 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 as set forth in SEQ ID NO:1 or a conservative variant thereof. Optionally, a cell (e.g., a eubacterial cell or a yeast cell) comprises the translation system.

[0161] A cell or other translation system of the invention can optionally further comprise an additional different 0-tRNA/O-RS pair and a second α-hydroxy or other unnatural amino acid, e.g., where this O-tRNA recognizes a second selector codon and this O-RS preferentially charges 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 first selector codon that is recognized by the first O-tRNA and a second selector codon that is recognized by the second O-tRNA. [0162] In certain embodiments, a cell of the invention is a eubacterial cell (such as

E. coll), 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.

[0163] 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 listing (e.g., SEQ ID NO:3 or 4) 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 listing, or a conservative variation thereof, or a sequence that is at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more identical thereto. 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 listing or examples herein, or a complementary polynucleotide sequence thereof.

[0164] 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).

[0165] Polynucleotides are also a feature of the invention. A polynucleotide of the invention (e.g., SEQ ID NO:2) 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 listing herein, and/or is complementary to 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.

[0166] 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.

[0167] Methods of producing components of an 0-tRNA/O-RS pair are also features of the invention, as are components produced by these methods. For example, methods for identifying an O-RS that charges an O-tRNA with/j-hydroxy-L-phenyllactic acid are also provided. In one class of embodiments, the O-RS is identified by mutating an amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase (e.g., a Methanococcus jannaschii tyrosyl-tRNA synthetase, see SEQ ID NOs:5 and 6) by site- directed mutagenesis, and selecting a resulting O-RS that preferentially charges the O-tRNA with/»-hydroxy-L-phenyllactic acid. The selecting step typically comprises positively selecting and negatively selecting for the O-RS from a pool comprising a plurality of mutant aminoacyl-tRNA synthetase molecules produced following the site-directed mutagenesis, e.g., as described elsewhere herein. An orthogonal aminoacyl-tRNA synthetase identified by the method is also a feature of the invention.

NUCLEIC ACID AND POLYPEPTIDE SEQUENCES AND VARIANTS [0168] As described herein, in one aspect 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 the polynucleotide or polypeptide sequences. Examples of said sequences, e.g., O-tRNA and O-RS amino acid and nucleotide sequences are disclosed herein (see Table 3, e.g., SEQ ID NOs: 1-4). 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 and polynucleotides and polypeptides substantially identical to a polynucleotide or polypeptide described herein. [0169] The construction and analysis of orthogonal synthetase species (O-RS) that are able to charge an O-tRNA with p-hydroxy-L-phenyllactic acid is described in Example 1. This Example describes the construction and analysis of an O-RS/O-tRNA pair that is able to incorporate the α-hydroxy acidp-hydroxy-L-phenyllactic acid; see SEQ ID NOs: 1 and 3.

[0170] One aspect of 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 as set forth in SEQ ID NO:2, 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 comprising SEQ ID NO:1 or a conservative variant thereof. Similarly, an artificial nucleic acid (a polynucleotide that is man made and is not naturally occurring) 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.

[0171] 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.

[0172] 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. Conservative Variations [0173] 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.

[0174] "Conservative variations" or "conservative variants" 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" or "conservative variants" 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. Conservative variants of SEQ ID NOs: 1 and 2 are thus a feature of the invention. Optionally, conservative variants of SEQ ID NOs: 1 or 2 retain one or more of (e.g., all three of) the RS residues selected after randomization in Example 1 below, i.e., Argl55, GIy 173, and Vall76; they optionally also retain GIu 36, He 137, and/or Tyr 151.

[0175] 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 1. Conservative Amino Acid Substitutions

Nucleic Acid Hybridization [0176] 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 NO:2, 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.

[0177] 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 5x- 10x as high as that observed for hybridization to any of the unmatched target nucleic acids.

[0178] 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, New York), 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 2007); 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.

[0179] 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°C, with the hybridization being carried out overnight. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (for a description of SSC buffer, see, Sambrook et al, Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

[0180] "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, e.g., 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, New York); 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 5x as high as that observed for hybridization of the probe to an unmatched target.

[0181] "Very stringent" conditions are selected to be equal to the thermal melting point (T_m) for a particular probe. The T_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 5⁰ C lower than the T_m for the specific sequence at a defined ionic strength and pH.

[0182] "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 10x 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 ¹A that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

[0183] 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 10x, 2OX, 50X, 10OX, or 500X 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 Vi that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.

[0184] 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. Unique Subsequences [0185] 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.

[0186] 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.

[0187] The invention also provides for target nucleic acids which hybridize 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.

Sequence Comparison, Identity, and Homology [0188] 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.

[0189] 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%, about 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 at least about 250 residues, or over the full length of the two sequences to be compared.

[0190] 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., identity), 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.

[0191] 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.

[0192] 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. MoI. Biol., 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. 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, WI), 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 2007).

[0193] 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. MoI. 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., J. MoI. Biol., 215:403-410 (1990)). 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).

[0194] 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, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). 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.

Mutagenesis and Other Molecular Biology Techniques [0195] 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, CA; Sambrook et al, Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 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 2008). 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.

[0196] 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.

[0197] 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, CA; Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 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 2007). 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., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), 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., Nature 327, 70-73 (1987)), and/or the like.

[0198] A highly efficient and versatile single plasmid system was developed for site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon (UAG) in E. coli. In this system, the pair of M. jannaschii suppressor tRNAtyr(CUA) and tyrosyl-tRNA synthetase are encoded in a single plasmid, which is compatible with most E. coli expression vectors. Monocistronic tRNA operon under control of proK promoter and terminator was constructed for optimal secondary structure and tRNA processing. Introduction of a mutated form of glnS promoter for the synthetase resulted in a significant increase in both suppression efficiency and fidelity. Increases in suppression efficiency were also obtained by multiple copies of tRNA gene as well as by a specific mutation (D286R) on the synthetase (Kobayashi et al, "Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion," Nat. Struct. Biol, 10(6):425-432 [2003]). The generality of the optimized system was also demonstrated by highly efficient and accurate incorporation of several different unnatural amino acids, whose unique utilities in studying protein function and structure were previously proven.

[0199] A catalogue of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., 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 e.g., Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001; 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 2008), and in Watson et al. (1992) Recombinant DNA, Second Ed., 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, The Great American Gene Company (Ramona, CA), ExpressGen Inc. (Chicago, IL), Operon Technologies Inc. (Alameda, CA) and many others.

[0200] 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, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

PROTEINS AND POLYPEPTIDES OF INTEREST

[0201] Methods of producing in a translation system a protein with an unnatural amino acid (e.g., p-hydroxy-L-phenyllactic acid) at a specified position are also a feature of the invention. Thus, one class of embodiments provides methods for producing a protein comprising a first α-hydroxy acid at a selected position, where the methods include (a) providing a translation system comprising a first α-hydroxy acid that is /?-hydroxy-L- phenyllactic acid, a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the/?- hydroxy-L-phenyllactic acid, and a nucleic acid encoding the protein, wherein the nucleic acid comprises at least one selector codon that is recognized by the first O-tRNA, and (b) incorporating the α-hydroxy acid at the selected position in the protein during translation of the protein in response to the selector codon, thereby producing the protein comprising the α-hydroxy acid at the selected position. The protein is optionally produced in a cell comprising the translation system, e.g., a eubacterial cell such as an E. coli cell or the like, a yeast cell, or another eukaryotic or prokaryotic cell. For example, the method can include 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, 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 selector codon, thereby producing the protein. As noted, the cell comprises an O-tRNA that functions in the cell and recognizes the selector codon, and an O-RS that preferentially charges the O-tRNA with the unnatural amino acid. A protein produced by any of the methods is also a feature of the invention.

[0202] 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.

[0203] The invention also provides compositions that include proteins, where the proteins comprise at least one p-hydroxy-L-phenyllactic acid residue. In certain embodiments, the protein comprises an amino acid sequence that is at least 75% identical to that of a known protein, a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof. Optionally, the composition comprises a pharmaceutically acceptable carrier.

[0204] The compositions of the invention and compositions made by the methods of the invention optionally are in a cell. The 0-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 April 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 0-tRNA/O-RS pair is introduced into a host, e.g., an Escherichia coli cell, the pair leads to the in vivo incorporation of an unnatural amino acid such as p-hydroxy-L-phenyllactic acid into a protein in response to a selector codon. Optionally, the compositions of the present invention can be in an in vitro translation system, or in an in vivo system(s).

[0205] A cell or other translation system 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 nL 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.

[0206] 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. In addition, of particular interest herein are methods that take advantage of the reactivity of the ester bond, for example, to facilitate removal of a peptide purification tag following protein purification or for covalent modification of the protein by transacylation, as described in greater detail below in the section entitled "Use of O-tRNA and O-RS and 0-tRNA/O-RS Pairs."

[0207] 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.

[0208] 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.

[0209] 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 April 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-I, 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, 1309, R83915, R91733, HCCl, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GROα/MGSA, GROβ, GROγ, MIP- lα, MIP- 16, MCP-I), 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-α, IFN-β, IFN-γ), interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, 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, 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-I, 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, SECl, SEC2, SEC3, SED, SEE), Superoxide dismutase (SOD), Toxic shock syndrome toxin (TSST-I), 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.

[0210] 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.

[0211] 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-I, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-I /LFA-I, 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, ReI, and steroid hormone receptors such as those for estrogen, progesterone, testosterone, aldosterone, the LDL receptor ligand and corticosterone.

[0212] 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.

[0213] Many of these proteins are commercially available, 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 LD₅₀ 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 relevanfenzymatic properties include shelf half-life, stability, enzymatic activity, production capability, or the like. [0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

Defining Polypeptides by Immunoreactivity [0219] 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.

[0220] 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."

[0221] 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.

USE OF O-tRNA AND Q-RS AND 0-tRNA/O-RS PAIRS

[0222] 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, Z-domain, or GFP 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.

[0223] As noted above, site-specific incorporation of an α-hydroxy acid - and concomitant introduction of an ester bond at a defined position in the polypeptide backbone - is of particular interest herein. In one aspect, since as noted above replacement of an amide bond with an ester bond alters hydrogen bonding patterns, site-specific incorporation of an α-hydroxy acid (e.g.,/?-hydroxy-L-phenyllactic acid) facilitates study of the roles of backbone hydrogen bonding in protein folding, thermodynamics, and kinetics, biomolecular interactions such as protein-protein and protein-substrate interactions, ion channel gating, and enzyme mechanisms, for example. Polyesters, e.g., folded polyesters and functional materials are optionally prepared.

[0224] In other aspects, reactivity of the ester bond is exploited, for example, for removal of C-terminal tags after affinity purification or to carry out a selective transacylation reaction to modify the C-terminus (or any region of a protein containing the ester linkage) with synthetic moieties such as reporters, therapeutic agents, oligonucleotides, etc.

[0225] In brief, the ester bond is subject to attack by nucleophiles. A protein in which an α-hydroxy acid has been incorporated can thus be reacted with a nucleophilic compound (i.e., a compound that supplies the entering group and acts as a nucleophile). The nucleophilic compound can be water, resulting in hydrolysis of the protein (schematically illustrated in Figure 7 Panel A); the N-terminal fragment of the original polypeptide comprises a free C-terminal carboxyl group, while the C-terminal fragment is left with an "N-terminal" hydroxyl. Similarly, the nucleophilic compound can be ammonia, resulting in ammoniolysis of the protein (schematically illustrated in Figure 7 Panel B); the N-terminal fragment of the original polypeptide comprises a C-terminal amide, while the C- terminal fragment is left with an "N-terminal" hydroxyl. In general, reactions of interest are transacylation reactions (also called acyl transfer reactions). Such transacylation reactions can also be used, e.g., to selectively modify the polypeptide with the nucleophilic group or another moiety attached thereto. See, e.g., the reaction schematically illustrated in Figure 7 Panel C, where in the nucleophilic compound R-Nu Nu can be, e.g., a thiol, amine, hydroxyl, alkoxyamine, hydroxylamine, hydrazine, hydrazide, etc. and/or R can be, e.g., an oligonucleotide or derivative thereof, label, fluorophore, polyethylene glycol (PEG), toxin, drug moiety, synthetic peptide, metal chelator, carbohydrate, polypeptide, affinity tag, biotin moiety, etc. These exemplary uses are described in greater detail in the following sections.

Site-Specific Hydrolysis of Fusion Proteins [0226] In one exemplary application, the ester bond is introduced between two polypeptides from different parental polypeptides (e.g., two different proteins, two homologous proteins from different species, an affinity tag and a protein of interest, etc.). The resulting fusion protein can then be fragmented by hydrolysis of the ester bond.

[0227] Thus, one class of embodiments provides methods of producing a first polypeptide comprising a first polypeptide sequence. The methods include providing a translation system comprising an α-hydroxy acid, a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the α-hydroxy acid, and a nucleic acid encoding a fusion protein. The nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by the first O-tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon. The α-hydroxy acid is incorporated at a selected position in the fusion protein during translation of the fusion protein in response to the selector codon, thereby producing the fusion protein comprising the α-hydroxy acid at the selected position and an ester bond in the protein backbone (between the first and second polypeptide sequences). After production of the fusion protein, the first polypeptide sequence is then released from the second polypeptide sequence by hydrolysis of the ester bond, producing the first polypeptide.

[0228] The methods can be employed to remove polypeptide tags after affinity purification of the fusion protein. Accordingly, the methods can include isolating the fusion protein from the translation system, for example, by providing a solid support comprising a binding moiety, binding the second polypeptide sequence to the binding moiety, and separating materials not captured on the solid support from the solid support, e.g., by washing, prior to hydrolysis of the ester bond. The first polypeptide can then be isolated from the second polypeptide sequence. Isolating the fusion protein and/or first polypeptide can involve purifying it either partially (e.g., achieving a 5X, 1OX, 10OX, 500X, or IOOOX or greater purification) or even substantially to homogeneity (e.g., where the protein is the main component of a solution, typically excluding the solvent and buffer components (e.g., salts and stabilizers) that the polypeptide is suspended in), according to standard procedures known to and used by those of skill in the art.

[0229] Hydrolysis of the ester bond can be conveniently achieved, for example, by incubating the fusion protein in an alkaline aqueous solution. Typically, the fusion protein is incubated at a pH and temperature for a time sufficient to essentially completely cleave the first polypeptide from the second polypeptide sequence. Optionally, hydrolysis also frees the first polypeptide from the solid support, to which the second polypeptide can remain bound, facilitating separation of the first and second polypeptides. In other embodiments, the fusion protein is released from the solid support prior to hydrolysis.

[0230] The second polypeptide sequence can be N-terminal of the first polypeptide sequence in the fusion protein. However, preferably, the second polypeptide sequence is C- terminal of the first polypeptide sequence in the fusion protein, such that hydrolysis results in a first polypeptide having no additional residues and having normal free N- and C- termini, as schematically illustrated in Figure 7 Panel A. In such embodiments, the α- hydroxy residue is located on the second polypeptide (which has an "N-terminal" hydroxyl instead of an N-terminal amine).

[0231] The second polypeptide optionally is or includes an affinity tag such as those well known in the art (i.e., a moiety, typically a polypeptide sequence, that facilitates purification of an attached protein, e.g., by high affinity and/or specificity binding to a cognate binding moiety). For example, the second polypeptide sequence can comprise one or more of a polyhistidine tag (e.g., a HIS-6 tag), a polyarginine tag, a polycysteine tag, a polyphenyalanine tag, a polyaspartic acid tag, a glutathione-S-transferase (GST) sequence, an S tag, an epitope tag (e.g., HA or myc), a maltose binding protein (MBP) sequence, a galactose-binding protein sequence, a cellulose binding domain, a SNAP tag, a biotin attachment site (e.g., an AviTag™ sequence) or avidin-binding sequence (e.g., a Strep- Tag®), or the like. Cognate binding moieties for such tags are likewise well known. Examples include, but are not limited to, immobilized metal ions (e.g., nickel-NTA), glutathione, S-protein, antibodies, amylose, galactose, cellulose, biotin, streptavidin, etc. Useful solid supports are similarly well known in the art, e.g., multiwell plates whose surfaces can be modified with biotin or streptavidin or another binding moiety, microspheres, chromatography resins (e.g., cationic, anionic, thiopropyl-modified, phenyl- modified, and other affinity resins), etc. The binding moiety which specifically recognizes and/or has high affinity for the second polypeptide sequence is bound to the solid support (e.g., covalently or noncovalently, directly or through a linker).

[0232] A large number of affinity tags and cognate binding moieties are known in the art and can be adapted to the practice of the present invention. For example, see, e.g., Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins" Protein Expression and Purification 11: 1-16, Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523- 533, and references therein). A few examples of suitable second polypeptides and binding moieties include, but are not limited to, a polyhistidine tag (e.g., a His-6, His-8, or His- 10 tag) that binds immobilized divalent cations (e.g., Ni²⁺), a biotin moiety (e.g., on an in vivo biotinylated polypeptide sequence) that binds immobilized avidin, a GST (glutathione S- transferase) sequence that binds immobilized glutathione, an S tag that binds immobilized S protein, an antigen that binds an immobilized antibody or domain or fragment thereof (including, e.g., T7, myc, FLAG, and B tags that bind corresponding antibodies), a FLASH Tag (a high affinity tag that couples to specific arsenic based moieties), a receptor or receptor domain that binds an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds immobilized IgG, maltose-binding protein (MBP) that binds immobilized amylose, an albumin-binding protein that binds immobilized albumin, a chitin binding domain that binds immobilized chitin, a calmodulin binding peptide that binds immobilized calmodulin, and a cellulose binding domain that binds immobilized cellulose. Another exemplary tag that can be used to couple the second polypeptide to the solid support is a SNAP-tag, commercially available from Covalys (www (dot) covalys (dot) com). The SNAP-tag is an approximately 20 kDa version of a protein O⁶-alkylguanine- DNA alkyltransferase which has a single reactive cysteine with a very high affinity for guanines alkylated at the Opposition. The alkyl group, including any immobilization moiety attached to the alkyl group (e.g., a surface-immobilized alkyl group), is transferred covalently from the guanine to the cysteine in the alkyltransferase protein.

[0233] Exemplary α-hydroxy acids include, but are not limited to, /?-hydroxy-L- phenyllactic acid, leucic acid, lactic acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxy-3- phenylpropionic acid, and α-hydroxy analogs of other natural and unnatural amino acids. It will be evident the ester bond is optionally introduced between two polypeptides from the same parental polypeptide instead of different parental polypeptides, and the resulting protein can be fragmented as described for fusion proteins.

Site-Specific Protein Modification [0234] A related class of embodiments provides methods of covalently attaching a first moiety to the C-terminus of a first polypeptide sequence. The methods include providing a translation system comprising an α-hydroxy acid, a first orthogonal aminoacyl- tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), wherein the first O-RS preferentially charges the first O-tRNA with the α-hydroxy acid, and a nucleic acid encoding a precursor protein. The nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by the first O- tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon. The α-hydroxy acid is incorporated at a selected position in the precursor protein during translation of the precursor protein in response to the selector codon, thereby producing the precursor protein comprising an ester bond in the protein backbone (between the first and second polypeptide sequences) and the α-hydroxy acid at the selected position. The resulting precursor protein is contacted with a nucleophilic compound comprising the first moiety, and the nucleophilic compound reacts with the ester bond in the precursor protein to attach the first moiety to the C-terminus of the first polypeptide sequence and release the second polypeptide sequence from the first polypeptide sequence.

[0235] In a preferred aspect, the nucleophilic compound is a compound other than water. For example, in one embodiment, the nucleophilic compound is ammonia, the first moiety comprises a nitrogen atom, and reacting the ammonia with the ester bond comprises ammoniolysis of the ester bond. See, e.g., the schematic illustration in Figure 7 Panel B. The resulting modified first polypeptide comprises a C-terminal amide, making the method useful, e.g., in production of certain hormones. As other examples, the nucleophilic compound can comprise an alkoxyamine, a hydroxylamine, a hydrazine, a hydrazide, an amine, a thiol, or a hydroxyl. Suitable reaction conditions for a given compound can be determined by one of skill using techniques known in the art.

[0236] The first moiety that is transferred to the first polypeptide optionally comprises one or more of a label (e.g., a fluorophore, spin label, or other biophysical probe), an affinity tag, a biotin moiety, an oligonucleotide or derivative (e.g., a tag, an antisense nucleic acid, or an siRNA, e.g., a chemically synthesized polynucleotide), a carbohydrate or sugar, a toxin, a drug, a polyethylene glycol (PEG) or derivative, a polypeptide (e.g., a synthetic peptide), a metal ion chelator, a cross-linking agent, or a fatty acid.

[0237] A variety of useful nucleophilic compounds are commercially available, and others can be readily produced by one of skill. As just a few examples, fluorophore hydrazides, amines, and hydrazines, as well as hydrazine and amine derivatives of biotin, are commercially available from Molecular Probes/Invitrogen, Inc., and are described in The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition or Web Edition (2008) from Invitrogen (available on the world wide web at probes (dot) invitrogen (dot) com/handbook).

[0238] An exemplary acyl transfer reaction is schematically illustrated in Figure 7

Panel C. The precursor protein can be reacted with a hydroxy lamine-oligo, a hydrazine- fiuorophore, etc., to label or otherwise modify the first polypeptide. The methods also provide a delivery mechanism for molecules such as antisense, siRNA, toxins or synthetic drugs (e.g., ene-dynes, antiproliferatives, and phosphatase inhibitor), among many other uses. For example, a bioactive moiety, such as a toxin or siRNA, can be attached to cancer cell specific immunoglobulins or cancer receptor-binding peptides via the acyl transfer reaction. The immunoglobulin acts as a biomolecular vehicle that provides a targeting delivery mechanism.

[0239] The precursor protein is optionally a fusion protein, but it need not be. Thus, in some embodiments, the first and second polypeptides originate from the same parental polypeptide (e.g., they can be two domains of a protein, where one domain is C-terminally modified and the other is removed). In other embodiments, the first and second polypeptides originate from different parental polypeptides. As for the embodiments above, the second polypeptide optionally comprises an affinity tag that is employed in purification as described above prior to its removal during the transacylation reaction. Exemplary α- hydroxy acids include, but are not limited to, /j-hydroxy-L-phenyllactic acid, leucic acid, lactic acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxy-3-phenylpropionic acid, and α- hydroxy analogs of other natural and unnatural amino acids.

KITS

[0240] Kits are also a feature of the invention. For example, a kit for producing a protein that comprises at least one α-hydroxy acid in a cell or other translation system is provided, where the kit includes at least one container containing a polynucleotide sequence encoding an O-tRNA and/or an O-tRNA, and/or a polynucleotide sequence encoding an O- RS and/or an O-RS (e.g., the O-tRNA and/or O-RS of SEQ ID NOs: 1 and 3 or conservative variants thereof). In one embodiment, the kit further includes the α-hydroxy acid, e.g.,p- hydroxy-L-phenyllactic acid. The kit optionally also includes instructional materials for producing the protein and/or a host cell (e.g., an E. coli cell comprising polynucleotide sequences encoding the O-tRNA and O-RS and having deletions in tyrB and aspC) . In one class of embodiments, the kit also includes one or more of a vector comprising a polynucleotide sequence encoding a polypeptide purification tag, a selector codon, and optionally a polylinker cloning site for insertion of a polynucleotide sequence encoding a protein of interest; materials for affinity purification of a resulting fusion protein (e.g., an affinity resin or other solid support comprising a binding moiety bound by the polypeptide purification tag, wash buffers, etc.); instructions for purifying the fusion protein and hydrolyzing the ester bond; an alkaline buffer (in which hydrolysis can be performed); instructions for modifying the protein of interest via a transacylation reaction; and reagents for modifying the protein via transacylation (e.g., a nucleophilic compound).

EXAMPLES

[0241] The following sets forth a series of experiments that demonstrate that the α- hydroxy acid/j-hydroxy-L-phenyllactic acid has been genetically incorporated into proteins in E. coli with good yields and high fidelity in response to an amber nonsense codon. The ability to site-specifically introduce backbone ester mutations provides a powerful method to study the role of the polypeptide backbone in structure, folding, biomolecular recognition and catalysis, as well as to selectively cleave or derivatize the protein backbone. To this end, this method has been used to site-specifically hydrolyze an affinity tag, as well as to determine the energetic contributions of backbone hydrogen bonds to protein stability.

[0242] 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. Accordingly, the following examples are offered to illustrate, but not to limit, the claimed invention.

EXAMPLE 1: ADDITION OF AN ALPHA HYDROXY ACID TO THE GENETIC CODE OF BACTERIA

[0243] Recently, a large number of amino acid building blocks with novel physical, biological and chemical properties have been genetically encoded in both prokaryotic and eukaryotic organisms. ^{[1> 2]} However, to date, substitutions in the polypeptide backbone itself are largely limited to in vitro protein translation systems^[3"7] or semisynthetic chemical methods. ^{[8 1 I]} These methods generally place restrictions on the nature, size or amounts of mutant protein that can be produced. This example shows that the α-hydroxy acid/?- hydroxy-L-phenyllactic acid can be directly incorporated into proteins in E. coli with good yields and high fidelity in response to an amber nonsense codon by means of an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair^[1' ^2] from archae bacteria (which does not cross react with endogenous counterparts in the host cell). The ability to site- specifically introduce backbone ester mutations provides a powerful method to study the role of the polypeptide backbone in structure, folding, biomolecular recognition and catalysis, as well as to selectively cleave or derivatize the protein backbone. To illustrate the utility of this method, it was used to site-specifically hydrolyze a protein affinity tag, as well as determine the energetic contribution of backbone hydrogen bonds to protein stability.

[0244] To demonstrate genetic introduction of α-hydroxy acids into proteins in bacteria, initial experiments focused on p-hydroxy-L-phenyllactic acid 1 (Figure 1), an isostere of tyrosine. However, initial feeding studies revealed that this and other α-hydroxy acids are metabolized by E. co//.^[12] When 1 was added to the growth media, the compound was undetectable in cell lysates and was depleted in the media. The metabolism of a related compound, p-methoxy-L-phenyllactic acid 2, whose products can be distinguished from endogenous metabolites, was then followed. When 2 was added to growth media, LC-MS analysis of E. coli lysates showed that it was transformed to the corresponding amino acid.^[12] The bacterial metabolism of α-hydroxy acids is likely to involve initial oxidation to the α-keto acid by an α-hydroxy acid dehydrogenase, followed by transamination to the corresponding amino acid by keto-acid transaminases (Figure 2 Panel A).^[12] Although there are many dehydrogenases in E. coli capable of oxidizing α-hydroxy acids, it is not clear which enzymes oxidize 1. For example, deletion of lactate dehydrogenase (JIdD) did not lead to accumulation of 1 in the cell lysate.^[12] In contrast, transamination ofp- hydroxyphenylpyruvate to tyrosine is known to involve tyrosine aminotransferase (tyrB) and aspartate aminotransferase^ ^3] (aspC) in the last step of tyrosine biosynthesis (Figure 2 Panel A). Therefore, both of these genes were sequentially disrupted in E. coli strain DHlOB with a phage λ-red recombinase-based gene knockout method^[14] resulting in the double knockout strain GWAPOl. The growth of GWAPOl cells in minimal media required tyrosine and aspartic acid supplementation (Figure 2 Panel B); addition of 1 mM 1 to the growth media resulted in > 100 μM levels of 1 in cell lysates.

[0245] To alter the substrate specificity of M. jannaschii tyrosyl-tRNA synthetase

(Λ//TyrRS) to aminoacylate 1 and not tyrosine, two libraries of A//TyrRS mutants were generated based on an analysis of the x-ray crystal structures of the M. jannaschii tRNA ^yr- TyrRS and L-tyrosine complex.^¹⁵' ^16] Five residues near the amino group of the tyrosine substrate were randomized in each library: Glu36, Ilel37, Tyrl51, Glnl55 and Glnl73 in plasmid pBK-lib-jwla (3 x 10⁷ in size), and residues Ilel37, Tyrl51, Glnl55, Glnl73, and He 176 in plasmid pBK-lib-jw2a (3 x 10⁷ in size). To identify Λ//TyrRS mutants selective for 1, these libraries were subjected to rounds of positive and negative selection as previously reported } ¹^ Surprisingly, only one round of negative and positive selection of the combined libraries generated a clone that grows at 100 μg/mL chloramphenicol in the presence of 1, but only at 20 μg/mL chloramphenicol in the absence of 1. This clone, PIaRS (SΕQ ID NO: 1), has the mutations Glnl55Arg, Glnl73Gly, and Ilel76Val. Without limitation to any particular mechanism, on the basis of the MjlyrRS structure/¹⁵' ^16] the Glnl55Arg and GIn 173GIy mutations likely render the synthetase inactive towards tyrosine by deleting two critical hydrogen bond acceptors for the α-amino group of the tyrosine substrate. Argl55 may still serve as a hydrogen bond donor to the α-hydroxyl group of 1. [0246] To determine the efficiency and fidelity for the incorporation of 1 into proteins, an amber stop codon was substituted for Lys99 in sperm whale myoglobin containing a C-terminal His₆ tag. Protein expression was carried out in the presence of the selected synthetase (PIaRS) and A^tRNA^ with 1 mM 1. As a negative control, protein expression was also carried out in the absence of 1. Analysis of the purified protein by SDS-PAGE showed that full-length protein was expressed only in the presence of 1 (Figure 3 Panel A), indicating that PIaRS does not utilize tyrosine or other endogenous amino acids to any significant degree. The yield of the mutant myoglobin was 2-3 mg/L. For comparison, the yield of myoglobin Lys99Tyr mutant in the presence of the wild type MfTyτRS and

under similar conditions was 5-10 mg/L. ESI-mass spectrometry analyses of the mutant myoglobin gave an observed average mass of 18390 Da, in close agreement with the calculated mass of 18391 Da for the Lys99 ->^• 1 myoglobin mutant. In comparison, the Lys99Tyr mutant has observed and calculated masses of 18389 Da and 18390 Da, respectively. The Lys99 — > 1 mutant consistently and reproducibly produced an observed mass 1 Da higher than that of the Lys99Tyr mutant, again indicating that 1 is selectively incorporated.

[0247] To confirm the selective incorporation of 1 and not tyrosine into protein, the ester backbone linkage was selectively hydrolyzed under alkaline conditions. Five myoglobin mutant proteins (Ser4 — > 1, Ala75 — » 1, Lys99 — > 1, TyrlO4 —> 1, and Lys99Tyr) were incubated in 0.67 M NaOH for 20 minutes at 4°C.^[17] As shown in Figure 3 Panels A-C, the Lys99Tyr mutant myoglobin was completely intact under these conditions, whereas the Lys99 — » 1 mutant was selectively and efficiently cleaved into two fragments. ESI-MS analysis showed that the two fragments have molecular masses of 11048 Da and 7360 Da, respectively (Figure 3 Panel C). For comparison, the predicted masses of the two fragments after hydrolysis are 11049 Da and 7360 Da, respectively. The other three mutants Ser4 — » 1, Ala75 — > 1, Tyrl04 — > 1 were also cleaved selectively and efficiently into two fragments with the correct masses, as shown by both SDS-PAGE (Figure 4) and LC-MS analysis (Table 2). Although some hydrolysis was observed in the SDS-PAGE for the mutants before base treatment, no hydrolysis was observed in LC^:MS experiments (Figure 4). This hydrolysis is therefore likely due to the basic SDS-PAGE buffer (pH 8.8). [0248] Table 2. ESI-MS data for Ser4 → 1, Ala75 → 1, Lys99 → 1, and TyrlO4 ->

1 mutants and wild type myoglobin (with N-terminal methionine), before and after base hydrolysis.

Before base hydrolysis After base hydrolysis

Protein

Expected mass Observed mass Expected mass Observed mass

WT 18355.1 18354 18355.1 18354

Ser4 → 1 18432.2 18431 361.5 + 18088.7 361 + 18087

Ala75 → 1 18448.2 18447 8393.7 + 10072.5 8393 + 10072

Lys99 → 1 18391.1 18390 11048.8 + 7360.3 1 1048 + 7360

TyrlO4 → l 18356.1 18355 1 1628.6 + 6745.5 11628 + 6745

[0249] Selective hydrolysis of the protein backbone can also be used to remove C- terminal fusion proteins and affinity tags to produce unmodified native proteins. Like the self-processing fusion tags derived from inteins,^¹⁹^ hydrolysis of an ester-linked C-terminal tag should afford an unaltered protein. In contrast, selective cleavage of C-terminal tags with proteases typically requires the addition of extra amino acids at the C-terminus and/or the absence of other cleavage sites in the protein. ^[19] To demonstrate the selective cleavage of a C-terminal His₆ tag, a Ser63 — » 1 mutant Z-domain protein containing a C-terminal His₆ tag immediately after the TAG63 site was expressed in the presence of the mutant synthetase (PIaRS), A// IRNA£[_A and 1 mM 1 in GWAPOl. After Ni-NTA affinity purification,

Z-domain protein was incubated at pH 9 at 4 ⁰C for 12 h, followed by dialysis against 20 mM phosphate buffer (pH 7.3). SDS-PAGE (Figure 5 Panel A) and ESI-MS (Figure 5 Panel B) indicated complete conversion to Z-domain protein without the His₆ tag. Circular dichroism measurements confirmed that the Z-domain protein was correctly folded (data not shown). The full length Z-domain protein has observed molecular mass of 6974 Da (without the N-terminal methionine, Figure 5 Panel B). For comparison, the predicted mass for the full length Z-domain protein is 6975 Da. Acyl transfer reactions of ester containing proteins to nucleophiles such as ammonia or alkoxyamine derivatives can also be carried out.

[0250] The amide-to-ester mutation can also be used to probe the role of backbone amide groups in catalysis, molecular recognition, and folding. In particular this substitution is a useful probe of backbone hydrogen bonding interactions in the formation of protein secondary structures. ^[9' ^10] The ester bond, like the amide bond, favors the trans conformation and has a significant cis-trans rotational barrier. ' * However, the ester substitution results in the loss of one hydrogen bond donor and a decrease in the basicity of the carbonyl oxygen. To this end, three mutants (Ala75 → 1, and TyrlO4 → 1, Ala75Tyr) and wild type myoglobin were used in unfolding studies to quantify the thermodynamic contribution of individual backbone H-bonds to protein stability. The myoglobin structure consist of eight helices (A to H) connected by short loops and turns (Figure 6 Panel A). The amide NH of Ala75 is located at the C-terminus of helix E and is hydrogen bonded to the carbonyl group of Thr71 (helix E); the carbonyl group of Ala75 is hydrogen bonded to water. The amide NH of TyrlO4 is located in helix G and is hydrogen bonded to the carbonyl group of ProlOl (helix G); the carbonyl group of TyrlO4 is hydrogen bonded to the amide NH of He 108 (helix G). To determine the difference in stabilities of the mutants, the chemical denaturation of myoglobin mutants was measured by circular dichroism (CD) in the presence of different concentrations of guanidinium hydrochloride (GuHCl); see Figure 6 Panel B. As shown in Figure 6 Panel C, substitution of Ala75 or TyrlO4 with 1 decreases the stability of the mutant protein by 1.66, or 2.45 kcal/mol, respectively, relative to the corresponding tyrosine mutants. In each case, the ester mutation is destabilizing, consistent with the fact that an ester is not a hydrogen bond donor and is a weaker hydrogen bond acceptor than an amide. The ΔΔG°(H₂O) values for the backbone amide-to-ester substitution determined here can be compared to those reported for T4 lysozyme.^[6] For T4 lysozyme, the ester substitution in the C-terminal position of an α-helix perturbed only one hydrogen bond and was destabilizing by 0.9 kcal/mol. Introduction of the ester linkage in the middle of a helix, which alters two hydrogen bonding interactions, destabilized the protein by 1.7 kcal/mol. Consistent with these results, ester substitution of TyrlO4, which perturbs two hydrogen bonding interactions, was more destabilizing than ester substitution of Ala75, which disrupts only one hydrogen bonding interaction.

[0251] In summary, the results described herein have shown that α-hydroxy acid 1 can be directly incorporated into proteins in E. coli with high fidelity and good efficiency in response to the amber codon TAG. This is a significant step in the expansion of the genetic code of living organisms beyond the realm of L-amino acids. ' Because of the versatility, low cost, and high yield of recombinant protein expression techniques, it facilitates systematic mutation of the protein backbone to investigate its role in protein folding/⁴' ^9] enzyme catalysis,^111] ion channel gating^[5] and molecular recognition both in vitro and in Other α-hydroxy acids can also be incorporated into proteins to ultimately address the question of whether a "polyester protein" can fold.^[17' ^l8]

Experimental Section [0252]

acid was purchased from Fluorochem. To generate the host strain, the tyrB and aspC genes were deleted by the methods described by Datsenko and Wanner^[14] and the resulting strain (GWAPOl) was confirmed by PCR and sequencing of genomic DNA. To characterize the biotransformation of α-hydroxy acids, DHlOB or GWAPOl cells were grown in 2YT media with 1 mM /j-methoxy-L-phenyllactic acid 2 overnight at 37 °C to OD 1.8, and the cells were lysed by sonication. The cell lysate and clarified media were then analyzed by LC-MS for the presence of 2 and its corresponding amino acid, /?-methoxy-L-phenylalanine 3; 2 is detected in the negative ion mode (mass = 195 Da), and 3 is detected in the positive ion mode (mass = 196 Da).

[0253] To selectively cleave the C-terminal His₆ tag of Z-domain protein, strain

GWAPOl was cotransformed with plasmids pLeiZ-TAG63 and pBK-PlaRS. Cells were grown in 5 mL 2YT media supplemented with kanamycin (50 μg/mL) and chloramphenicol (50 μg/mL). The 5 mL culture was transferred to 100 mL of 2YT with appropriate antibiotics and grown at 37 °C to an OD₆₀₀ of 0.6. Gene expression was then induced by the addition of 1 mM IPTG and 1 mM 1. After 10 hours, the cells were harvested by centrifugation and lysed by sonication. The Z-domain protein was purified by Ni-NTA affinity chromatography under native conditions. The purified protein was then incubated at pH 9 at 4 °C for 12 h to remove the His₆ tag. After dialysis against 20 mM phosphate buffer (pH 7.3), the wild type Z-domain protein was obtained.

[0254] To determine the stability of mutant and wild type myoglobin proteins, they were expressed with a C-terminal His₆ tag and purified to near homogeneity by Ni-NTA column chromatography under denaturing conditions. After dialysis against 20 mM phosphate buffer (pH 7.3), circular dichroism measurements confirmed that the apo myoglobin protein was correctly folded. Circular dichroism measurements were performed on an AVIV stopped flow circular dichroism spectrophotometer (Model 202SF) with 0.1- mm cuvettes for solutions containing 5 μM myoglobin protein. Samples were incubated with different concentrations of guanidinium hydrochloride in 10 mM phosphate buffer (pH 7.3) at 25 °C for 30 min before measurements. The free energy change, ΔG, between the fully folded (F) and unfolded (U) states was determined using the equation: ΔG = -RT InK = -RT In [(yF-y)/(y-yu)], where R is the gas constant, T is the absolute temperature, yF is the molar ellipticity of myoglobin in the fully folded state, yu is the molar ellipticity in the fully unfolded state, and y is the molar ellipticity at various concentrations of GuHCl. To determine the unfolding energy in the absence of guanidinium hydrochloride, ΔG°(H₂O), a linear least-squares analysis was performed using the equation: ΔG = ΔG°(H₂O) - m [D], where m is a constant, and [D] is the GuHCl concentration.

[0255] For mass spectroscopy analysis of fragmentation of Lys99 — > 1 mutant, the

Lys99 → 1 mutant was incubated in 0.67 M NaOH for 20 minutes at 4°C and then neutralized to pH 7.0 by the addition of IM HCl. The ESI-MS shows no mass peak for the full-length Lys99 — > 1 mutant after base treatment. Analysis of the ion envelops and deconvoluted masses indicate that the Lys99 — > 1 mutant was selectively and efficiently cleaved into two fragments. The observed masses were 11048 and 7360, respectively. For comparison, the predicted masses of the two fragments after hydrolysis are 11049 Da and 7360 Da, respectively. The fragment with molecular weight of 7360 is the C-terminal part of the myoglobin mutant that contains the polyhistidine tag.

References

[1] L. Wang, J. Xie, P. G. Schultz, Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 225.

[2] J. M. Xie, P. G. Schultz, Nat. Rev. MoI. Cell Bio. 2006, 7, 775.

[3] D. Mendel, V. W. Cornish, P. G. Schultz, Annu. Rev. Biophys. Biomol. Struct. 1995,

24, 435.

[4] J. A. Ellman, D. Mendel, P. G. Schultz, Science 1992, 255, 197. [5] T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz, J. Yang, Nat. Neurosci.

2001, 4, 239.

[6] J. T. Koh, V. W. Cornish, P. G. Schultz, Biochemistry 1997, 36, 11314. [7] H. H. Chung, D. R. Benson, P.G. Schultz, Science 1993, 259, 806. [8] P. E. Dawson, T. W. Muir, I. Clarklewis, S. B. H. Kent, Science 1994, 266, 776. [9] E. T. Powers, S. Deechongkit, J. W. Kelly, in Adv. Protein Chem. Vol. 72, 2006, pp.

39. [10] S. Deechongkit, H. Nguyen, E. T. Powers, P. E. Dawson, M. Gruebele, J. W. Kelly,

Nature 2004, 430, 101.

[11] X. Y. Yang, M. Wang, M. C. Fitzgerald, Bioorg. Chem. 2004, 32, 438. [12] J. C. Anderson, Towards polyester proteins: biotransformation of alpha-hydroxy acids, Ph. D. thesis, The Scripps Research Institute, 2003. [13] I. G. Fotheringham, S. A. Dacey, P. P. Taylor, T. J. Smith, M. G. Hunter, M. E.

Finlay, S. B. Primrose, D. M. Parker, R. M. Edwards, Biochem. J. 1986, 234, 593. [14] K. A. Datsenko, B. L. Wanner, Proc. Natl. Acad. Sci. 2000, 97, 6640. [15] T. Kobayashi, O. Nureki, R. Ishitani, A. Yaremchuk, M. Tukalo, S. Cusack, K.

Sakamoto, S. Yokoyama, Nat. Struct. MoI. Bio. 2003, 10, 425. [16] Y. Zhang, L. Wang, P. G. Schultz, I. A. Wilson, Protein Sci. 2005, 14, 1340. [17] Fahnesto.S, A. Rich, Science 1971, 173, 340. [18] A. L. Weber, S. L. Miller, J. MoI. Evolution 1981, 17, 273. [ 19] Waugh, D. S. Trends Biotechnol. 2005, 23, 316.

Table 3. Exemplary sequences.

[0256] 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. For example, all the techniques and apparatus described above can be used in various combinations. 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.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A translation system comprising:

(a) a first α-hydroxy acid that is p-hydroxy-L-phenyllactic acid;

(b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and

(c) a first orthogonal tRNA (O-tRNA); wherein said first O-RS preferentially charges said first O-tRNA with said/J-hydroxy-L- phenyllactic acid.

2. The translation system of claim 1, wherein said first O-RS preferentially charges said first O-tRNA with said/?-hydroxy-L-phenyllactic acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising said O-tRNA, said/?- hydroxy-L-phenyllactic acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID NO:1.

3. The translation system of claim 1, wherein said first O-RS comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and conservative variants thereof.

4. The translation system of claim 1, wherein said first O-RS is homologous to a wild- type tyrosyl tRNA synthetase from Methanococcus jannaschii.

5. The translation system of claim 4, wherein said first O-RS comprises an Arg residue at position 155, a GIy residue at position 173, a VaI residue at position 176, or a combination thereof, wherein amino acid position numbering corresponds to amino acid position numbering of the wild-type tyrosyl tRNA synthetase.

6. The translation system of claim 1, wherein said first O-tRNA is an amber suppressor tRNA.

7. The translation system of claim 1, wherein said first O-tRNA comprises or is encoded by a polynucleotide sequence set forth in SEQ ID NO:3.

8. The translation system of claim 1, comprising 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.

9. The translation system of claim 8, further comprising a second O-RS and a second O-tRNA, wherein the second O-RS preferentially charges the second O-tRNA with a second α-hydroxy or unnatural amino acid that is different from the first α-hydroxy acid, and wherein the second O-tRNA recognizes a selector codon that is different from the selector codon recognized by the first O-tRNA.

10. The translation system of claim 1, wherein said system comprises a host cell comprising said first α-hydroxy acid, said first O-RS and said first O-tRNA.

11. The translation system of claim 10, wherein said host cell is a eubacterial cell.

12. The translation system of claim 11, wherein said eubacterial cell is an E. coli cell.

13. The translation system of claim 12, wherein said E. coli cell comprises disruptions in tyrB and aspC.

14. The translation system of claim 10, wherein said host cell comprises a polynucleotide encoding said first O-RS.

15. The translation system of claim 14, wherein said polynucleotide comprises a polynucleotide sequence set forth in SEQ ID NO:2.

16. The translation system of claim 10, wherein said host cell comprises a polynucleotide encoding said first O-tRNA.

17. A method for producing in a translation system a protein comprising a first α- hydroxy acid at a selected position, the method comprising:

(a) providing a translation system comprising:

(i) a first α-hydroxy acid that is /j-hydroxy-L-phenyllactic acid; (ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), wherein said first O-RS preferentially charges said first O-tRNA with said/>-hydroxy-L- phenyllactic acid; and (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 (b) incorporating said α-hydroxy acid at said selected position in said protein during translation of said protein in response to said selector codon, thereby producing said protein comprising said α-hydroxy acid at the selected position.

18. The method of claim 17, wherein said first O-RS preferentially charges said first O- tRNA with said/j-hydroxy-L-phenyllactic acid with an efficiency that is at least 50% of the efficiency observed for a translation system comprising said O-tRNA, saidp-hydroxy-L- phenyllactic acid, and an aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQ ID NO:l

19. The method of claim 17, wherein said translation system comprises a polynucleotide encoding said first O-RS.

20. The method of claim 17, wherein said first O-RS comprises an amino acid sequence selected from the group consisting SEQ ID NO:1 and conservative variants thereof.

21. The method of claim 17, wherein said first O-RS is homologous to a wild- type tyrosyl tRNA synthetase from Methanococcus jannaschii.

22. The method of claim 21, wherein said first O-RS comprises an Arg residue at position 155, a GIy residue at position 173, a VaI residue at position 176, or a combination thereof, wherein amino acid position numbering corresponds to amino acid position numbering of the wild-type tyrosyl tRNA synthetase.

23. The method of claim 17, wherein said providing a first orthogonal aminoacyl-tRNA synthetase 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 charges said O-tRNA with said α-hydroxy acid, thereby providing said first orthogonal aminoacyl-tRNA synthetase.

24. The method of claim 23, wherein said selecting step comprises positively selecting and negatively selecting for said O-RS from a pool comprising a plurality of mutant aminoacyl-tRNA synthetase molecules produced following said site-directed mutagenesis.

25. The method of claim 17, wherein said translation system comprises a polynucleotide encoding said first O-tRNA.

26. The method of claim 17, wherein said first O-tRNA is an amber suppressor tRNA.

27. The method of claim 17, wherein said providing a translation system comprises providing a nucleic acid that encodes or comprises said O-tRNA, wherein said nucleic acid comprises a polynucleotide sequence set forth in SEQ ID NO:3.

28. The method of claim 17, wherein said providing a translation system comprises providing a nucleic acid that encodes said O-RS, wherein said nucleic acid comprises a polynucleotide sequence set forth in SEQ ID NO:2.

29. The method of claim 17, wherein said nucleic acid encoding said protein comprises an amber selector codon.

30. The method of claim 17, further wherein said protein comprises a second α-hydroxy or unnatural amino acid that is different from said first α-hydroxy acid, and wherein said translation system further comprises a second O-RS and a second O-tRNA, wherein the second O-RS preferentially charges the second O-tRNA with the second α-hydroxy or unnatural amino acid, and wherein the second O-tRNA recognizes a selector codon in the nucleic acid encoding said protein that is different from the selector codon recognized by the first O-tRNA.

31. The method of claim 17, wherein said providing a translation system comprises providing a host cell, wherein said host cell comprises said first α-hydroxy acid, said first O-RS, said first O-tRNA and said nucleic acid, and wherein said incorporating step comprises culruring said host cell.

32. The method of claim 31, wherein said host cell is a eubacterial host cell.

33. The method of claim 32, wherein said eubacterial host cell is an E. coli host cell.

34. The method of claim 33, wherein said E. coli cell comprises disruptions in tyrB and asp C.

35. The method of claim 31, wherein said host cell comprises a nucleic acid encoding said O-RS.

36. The method of claim 35, wherein said nucleic acid encoding said O-RS comprises a polynucleotide sequence set forth in SΕQ ID NO:2.

37. The method of claim 17, wherein said method comprises step (c) optionally purifying said protein from the translation system and step (d) reacting said protein with a nucleophilic compound.

38. The method of claim 37, wherein said nucleophilic compound is water and said reacting comprises hydrolysis of the protein or wherein said nucleophilic compound is ammonia and said reacting comprises ammoniolysis of the protein.

39. The method of claim 37, wherein said reacting is by transacylation.

40. The method of claim 37, wherein said nucleophilic compound comprises one or more of a label, a fluorophore, an affinity tag, a biotin moiety, an oligonucleotide, a carbohydrate, a toxin, a drug, a polyethylene glycol, a synthetic peptide, or a metal ion chelator.

41. The method of claim 37, wherein said nucleophilic compound comprises an alkoxyamine, a hydroxylamine, a hydrazine, a hydrazide, an amine, a thiol, or a hydroxyl.

42. A composition comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SΕQ ID NO: 1 and conservative variants thereof.

43. The composition of claim 42, wherein said conservative variant polypeptide charges a cognate orthogonal tRNA (O-tRNA) with an 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 the amino acid sequence of SΕQ ID NO: 1.

44. The composition of claim 42, wherein said composition comprises a cell comprising the polypeptide.

45. A polynucleotide encoding the polypeptide of claim 42.

46. The polynucleotide of claim 45, wherein said polynucleotide comprises the polynucleotide sequence of SEQ ID NO:2.

47. A vector comprising a polynucleotide of claim 45.

48. An expression vector comprising a polynucleotide of claim 45.

49. A cell comprising a vector, the vector comprising a polynucleotide of claim 45.

50. A composition comprising a polynucleotide comprising a polynucleotide sequence set forth in SEQ ID NO:2 or the complement thereof.

51. A method of producing a first polypeptide comprising a first polypeptide sequence, the method comprising:

(a) providing a translation system comprising:

(i) an α-hydroxy acid;

(ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS);

(iii) a first orthogonal tRNA (O-tRNA), wherein said first O-RS preferentially charges said first O-tRNA with said α-hydroxy acid; and

(iv) a nucleic acid encoding a fusion protein, wherein said nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by said first O-tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon;

(b) incorporating said α-hydroxy acid at a selected position in said fusion protein during translation of said fusion protein in response to said selector codon, thereby producing said fusion protein comprising an ester bond in the protein backbone and said α- hydroxy acid at the selected position; and (c) hydrolyzing the ester bond, thereby releasing the first polypeptide sequence from the second polypeptide sequence and producing the first polypeptide.

52. The method of claim 51, comprising, after step (b) and before step (c), isolating the fusion protein from the translation system.

53. The method of claim 52, wherein isolating the fusion protein from the translation system comprises providing a solid support comprising a binding moiety, binding the second polypeptide sequence to the binding moiety, and separating materials not captured on the solid support from the solid support.

54. The method of claim 51, comprising, after step (c), isolating the first polypeptide from the second polypeptide sequence.

55. The method of claim 51, wherein hydrolyzing the ester bond comprises incubating the fusion protein in an alkaline aqueous solution.

56. The method of claim 51, wherein in the fusion protein the second polypeptide sequence is C-terminal of the first polypeptide sequence.

57. The method of claim 51, wherein the second polypeptide sequence comprises one or more of a polyhistidine tag, a polyarginine tag, a polycysteine tag, a polyphenyalanine tag, a polyaspartic acid tag, a GST sequence, an S tag, an epitope tag, a maltose binding protein sequence, a galactose-binding protein sequence, or a cellulose binding domain.

58. The method of claim 51, wherein the α-hydroxy acid is /7-hydroxy-L-phenyllactic acid.

59. A method of covalently attaching a first moiety to the C-terminus of a first polypeptide sequence, the method comprising:

(a) providing a translation system comprising: (i) an α-hydroxy acid;

(ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), wherein said first O-RS preferentially charges said first O-tRNA with said α-hydroxy acid; and (iv) a nucleic acid encoding a precursor protein, wherein said nucleic acid comprises a first polynucleotide sequence encoding the first polypeptide sequence, a selector codon that is recognized by said first O-tRNA, and a second polynucleotide sequence encoding a second polypeptide sequence, wherein the first and second polynucleotide sequences are fused in frame with each other and separated by the selector codon;

(b) incorporating said α-hydroxy acid at a selected position in said precursor protein during translation of said precursor protein in response to said selector codon, thereby producing said precursor protein comprising an ester bond in the protein backbone and said α-hydroxy acid at the selected position; and

(c) contacting the precursor protein with a nucleophilic compound comprising the first moiety, wherein the nucleophilic compound is a compound other than water, and wherein the nucleophilic compound reacts with the ester bond in the precursor protein to attach the first moiety to the C-terminus of the first polypeptide sequence and release the second polypeptide sequence from the first polypeptide sequence.

60. The method of claim 59, wherein the first moiety comprises a nitrogen atom, wherein the nucleophilic compound is ammonia, and wherein reacting said ammonia with the ester bond comprises ammoniolysis of the ester bond.

61. The method of claim 59, wherein the nucleophilic compound comprises an alkoxyamine, a hydroxylamine, a hydrazine, a hydrazide, an amine, a thiol, or a hydroxyl.

62. The method of claim 59, wherein the first moiety comprises one or more of a label, a fluorophore, an affinity tag, a biotin moiety, an oligonucleotide, a carbohydrate, a toxin, a drug, a polyethylene glycol, a synthetic peptide, or a metal ion chelator.

63. The method of claim 59, wherein the α-hydroxy acid is p-hydroxy-L-phenyllactic acid.