WO2021165968A1 - Mutant aminoacyl-trna synthetases - Google Patents

Mutant aminoacyl-trna synthetases Download PDF

Info

Publication number
WO2021165968A1
WO2021165968A1 PCT/IL2021/050194 IL2021050194W WO2021165968A1 WO 2021165968 A1 WO2021165968 A1 WO 2021165968A1 IL 2021050194 W IL2021050194 W IL 2021050194W WO 2021165968 A1 WO2021165968 A1 WO 2021165968A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutated
leucine
tyrosine
mutant
seq
Prior art date
Application number
PCT/IL2021/050194
Other languages
French (fr)
Inventor
Miriam Amiram
Sigal GELKOP
Bar ISRAELI
Daniela STRUGACH
Original Assignee
B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University filed Critical B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
Priority to EP21757370.8A priority Critical patent/EP4107258A4/en
Publication of WO2021165968A1 publication Critical patent/WO2021165968A1/en
Priority to US17/892,163 priority patent/US20230313168A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02006—Leucyltransferase (2.3.2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)

Definitions

  • the present invention is in the field of artificial amino acid incorporation.
  • Site-specific modification of proteins is a powerful means for investigation and manipulation of the properties of proteins, and has been utilized for a variety of applications, such as fluorescent labeling, analysis of structure and functions, and manipulation of the chemical, biological, and pharmacological properties of target molecules. Beyond single-site modifications, multi-site modifications have been demonstrated to extend and further exploit the potential of such applications, for example for direct polymerization of target proteins, site- specific conjugation of single protein to multiple ligands, and increased performance in analytical chemistry assays.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • an alkyne or azide group must be site-specifically incorporated into the protein. This can be achieved using several methodologies including enzymatic or chemical modification of selected residues (typically post- protein purification), or by incorporation of unnatural amino acids (uAAs) that bear an alkyne or an azide group. Several studies describe the incorporation of such uAAs by substitution of a natural amino acid with a close synthetic analog in auxotrophic strain, which has been used for labeling in various organisms.
  • uAAs can be incorporated site specifically via codon reassignment or frameshift codons by using orthogonal translation systems (OTSs) consisting of an aminoacyl tRNA synthetase (aaRS), which is able to charge only a cognate tRNA that is not aminoacylated by endogenous aaRSs.
  • OTSs orthogonal translation systems
  • aaRS aminoacyl tRNA synthetase
  • aaRS aminoacyl tRNA synthetase
  • a TAG stop codon is assigned to the uAA.
  • the azobenzene molecule Upon irradiation with light of the appropriate wavelength (Atrans cis), the azobenzene molecule undergoes a dramatic switch from the trans to the cis configuration (shortening by at least ⁇ 3.5 A), with a concomitant change from a hydrophobic to a hydrophilic (polar) molecule ( ⁇ 3 Debyes). Importantly, this process is reversible, and with time or upon irradiation with a second, different, wavelength within the blue light range (Acis— Trans), the azobenzene molecule relaxes back to the trans configuration.
  • incorporation of azobenzene into a polypeptide chain can be mediated by incorporation of azobenzene-containing non-standard amino acid (nsAA), using expanded genetic code method as is used for the alkyne or azide groups.
  • nsAA non-standard amino acid
  • This expansion has enabled template -based incorporation of >100 nsAAs containing diverse chemical groups including post- translational modifications, photocaged amino acids, bio-orthogonal reactive groups, and spectroscopic labels.
  • light-responsive nsAA only incorporation of a single nsAA into a single protein has ever been successfully achieved.
  • the present invention provides mutant aminoacyl-tRNA synthetase (aaRS) proteins.
  • Nucleic acid molecules encoding the mutant aaRSs are provided.
  • Orthogonal translation systems comprising the mutant aaRSs or the nucleic acid molecules are provided.
  • Cells comprising the orthogonal translation systems, mutant aaRSs or nucleic acid molecules are provided. Methods of using the mutant aaRSs, nucleic acid molecules, orthogonal translation systems and cells are also provided.
  • a mutant aminoacyl-tRNA synthetase comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; leucine 65 mutated to valine; glutamic acid 107 mutated to alanine; phenylalanine 108 mutated to tyrosine; glutamine 109 mutated to methionine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid;
  • the mutant is selected from the group consisting of: a) a mutant comprising tyrosine 32 mutated to leucine, aspartic acid 158 mutated to serine, isoleucine 159 mutated to methionine, leucine 162 mutated to lysine, and alanine 167 mutated to histidine; b) a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to alanine, leucine 162 mutated to glutamic acid, and alanine 167 mutated to histidine; c) a mutant comprising alanine 32 mutated to threonine, leucine 65 mutated to valine, glutamic acid 107 mutated to alanine, phenylalanine 108 mutated to
  • the mutant aaRS comprises an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • aaRS mutant aminoacyl-tRNA synthetase
  • aaRS comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine
  • the mutant aaRS of the invention comprises: a) aspartic acid 158 mutated to glycine; b) isoleucine 159 mutated to tyrosine; and c) leucine 162 mutated to serine or leucine 162 mutated to arginine.
  • the mutant aaRS of the invention further comprises alanine 167 mutated to phenylalanine.
  • the mutant aaRS of the invention further comprises tyrosine 32 mutated to leucine or tyrosine 32 mutated to glycine.
  • the mutant aaRS of the invention further comprises leucine 65 mutated to valine or leucine 65 mutated to glycine.
  • the mutant is selected from the group consisting of: a) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; b) a mutant comprising tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; c) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107
  • the mutant comprises an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
  • the amino acid sequence of an aaRS is SEQ ID NO: 1.
  • mutant aaRS of the invention further comprises a mutation of arginine 257 to glycine, a mutation of aspartic acid 286 to arginine or both.
  • nucleic acid molecule comprising a coding region encoding a mutant aaRS of the invention.
  • the coding region comprises a nucleic acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
  • the coding region is operably linked to at least one regulatory element configured to express the coding region in a target cell.
  • an orthogonal translation system comprising, a) a mutant aaRS of the invention, or a nucleic acid molecule of the invention, and b) an orthogonal tRNA compatible with the mutant aaRS and comprising an anticodon that corresponds to a stop codon.
  • orthogonal translation system of the invention further comprises a non-standard amino acid (nsAA) recognized by the mutant aaRS.
  • nsAA non-standard amino acid
  • the nsAA is an unnatural amino acid (uAA).
  • the uAA comprises a biorthogonal chemical moiety.
  • the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azide or an alkyne group.
  • the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azobenzene group.
  • the nsAA is a modified phenylalanine.
  • the modified phenylalanine is 4-propargyloxy-L- phenylalanine (pPR).
  • the uAA comprising an azobenzene group is selected from phenylalanine-4’ -azobenzene (AzoPhe). tri-fluorinated azobenzene (Azo3F), and tetra- ortho-fluorinated azobenzene (Azo4F) amino acids.
  • the stop codon is a TAG stop codon.
  • a cell comprising an orthogonal translation system of the invention.
  • the cell of the invention further comprises an expression vector comprising an open reading frame (ORF) comprising at least one of the stop codons within the open reading frame.
  • ORF open reading frame
  • the ORF comprises a plurality of stop codons.
  • the ORF comprises at least 10 stop codons.
  • the ORF is operatively linked to at least one regulatory element capable of inducing expression of the ORF within the cell.
  • the cell is devoid of native TAG stop codons and does not express release factor 1 (RF1).
  • the cell comprises RF1 and at least one native TAG stop codon.
  • a method of producing a protein comprising a nsAA comprising introducing into a cell an expression vector comprising an open reading frame encoding the protein wherein the open reading frame comprises a stop codon, wherein the cell comprises an orthogonal translation system of the invention.
  • the method of the invention is for labeling the protein, and the method further comprises converting the nsAA into a detectably labeled amino acid and wherein the mutant aaRS is the mutant aaRS of the invention.
  • the converting comprises addition of a detectable moiety by Click chemistry.
  • the method of the invention is for producing a light- responsive protein, wherein the mutant aaRS is the mutant aaRS of the invention.
  • a protein comprising a nsAA produced by a method of the invention.
  • Figures 1A-C (1A) A table depicting amino acid substitutions present in mutant aminoacyl tRNA synthetases capable of incorporating alkyne-containing non-standard amino acids. The mutation sites are with respect to a M. janaschii tyrosyl-tRNA synthetase. (IB) A table depicting amino acid substitutions present in mutant aminoacyl tRNA synthetases capable of incorporating azobenzene-containing non-standard amino acids. The mutation sites are with respect to a wild-type M. janaschii tyrosyl-tRNA synthetase. (1C) Production of GFP(3TAG) by chromosomally integrated parent and evolved aaRS variants in E. coli strain C321.ARF1.
  • FIGS 2A-Z Multi-site incorporation of pPR by the parent translation systems and evolution of chromosomally integrated pPR-RS variants.
  • (2C-E) Incorporation of (2C) 3, (2D) 10 and (2E) 30 pPRs in a single protein by evolved aaRS variants, expressed on a plasmid in C321.ARF1.
  • * L R ⁇ 0.05, *** L R ⁇ 0.0005, and **** L R ⁇ 0.0001 indicate comparison of each evolved variant with the parent pPR-RS (2C-D) or with the wild-type protein (2E).
  • n 3; error bars indicate S.D.
  • (2G) production by Mutl-RS in C321.ARF1 and 2xYT media (2H) production by Mut2-RS in C321.ARF1 and 2xYT media, (21) production by Mutl-RS in C321.ARF1 and minimal media (MM), (2J) production by Mut2-RS in C321.ARF1 and minimal media, (2K) production by Mutl-RS in BF21 and 2xYT media, (2L) production by Mut2-RS in BF21 and 2xYT media, (2M) production by Mutl-RS in BF21 and minimal media, (2N) production by Mut2-RS in BF21 and minimal media.
  • (2W-Z) Time-course kinetic analysis of EFP(30TAG)-GFP production by Mutl-RS and Mut2-RS expressed from multi copy plasmids.
  • Figures 3A-D MALDI-TOF analysis of WT ELP(10Tyrosine)-GFP protein expressed in (3A) BL21 and (3B) C321.ARF1, and ELP(10pPR)-GFP protein expressed by Mutl-RS in (3C) BL21 and (3D) C321.ARF1, respectively.
  • Figures 5A-E (5A) In-gel fluorescence analysis of purified ELPs containing 1 or 10 instances of pPR conjugated to TAMRA-azide at various protein concentrations, namely: (1)
  • 5B-E TAMRA labeling of C321.ARF1 cells expressing (5B) ELP(lpPR) by the parentpPR-RS; (5C) ELP(lpPR) by Mutl-RS; (5D) ELP(lOpPR) b the parent pPR-RS and (5E) ELP(lOpPR) by the Mutl-RS. Percentage of labeled cells was calculated using ImageJ and is given for each image.
  • FIG. 6A-F Conjugation of multiple fluorophores to ELPs in bacteria.
  • 1 parent-pPR-RS, BL21; 2: Mutl-RS, BL21; 3: parent pPR-RS, C321.ARF1; 4: Mutl-RS, C321.ARF1.
  • FIGS 8A-D Incorporation of phenylalanine-4'-azobenzene (AzoPhe) in expressed proteins.
  • *P ⁇ 0.01 indicates comparison of literary aaRS with the evolved.
  • #P ⁇ 0.01 indicates comparison of evolved aaRS (lOAzo) with the endogenous (lOTyr).
  • Figures 9A-G (9A) Illustration of the reversible trans-to-cis isomerization of an azobenzene molecule. (9B) Illustrations and properties of azobenzene -uAAs 1, 2, and 3. (9C) Illustration of the mechanism for altering the Tt of the ELP by azobenzene isomerization. A change in the transition temperature by cis/trans isomerization generates a “window” in which isothermal (e.g., at T*), light-mediated change in ELP solubility can be achieved. (9D) Schematic illustration of reporter proteins for the incorporation of either 2 (GFP) or 1, 5, or 10 (ELP-GFP) uAAs at TAG codons.
  • GFP 2
  • ELP-GFP ELP-GFP
  • Figures 10A-D (10A) Production of GFP(2TAG) by the previously described AzoRS and four evolved variants, expressed from a single chromosomal copy. (10B-D) Production of
  • ELP-GFP fusion proteins containing either (10B) 1, (IOC) 5, or (10D) 10 instances of the azobenzene-uAAs depicted in 10B and expressed by episomal versions of the previously described AzoRS, our evolved variants (AzoRS 1-4), or MjTyrRS (producing tyrosine- containing control ELPs) in the C321.ARF l strain.
  • the level of GFP fluorescence indicates the production of the ELP-GFP fusion and, therefore, the efficiency of sAA incorporation.
  • FIG 11 MALDI-TOF analysis of ELP60(WT) [expected: 22,760.4, found: 22726.03], ELP60(2xl) [expected: 23,148.87, found: 23083.17], ELP60(6xl) [expected: 23,841.65, found: 23793.87], and ELP60(10xl) [expected: 24,562.47 found: 24519.49].
  • Figure 12 Turbidity profile, as a function of temperature and light irradiation for ELP6o(tyrosinexlO), 25 mM solution in water.
  • Figure 13A-R Characterization of the light-responsive properties of ELPs containing multiple instances of azobenzene -uAA 1.
  • 13A-C Turbidity profiles as a function of temperature and light irradiation for ELPs (25 mM solutions in water) containing either (13A) 2 (supplemented with 1 M NaCl), (13B) 6, or (13C) 10 instances of 1.
  • 13D-F CD spectra of light-irradiated ELPs (7.5 pM solutions in water) containing either (13D) 2, (13E) 6, or (13F)
  • Figures 14A-L Characterization of the light-responsive properties of ELP containing multiple instances of azobenzene-uAA 2 (25 pM solutions in water, unless otherwise indicated).
  • Figure 15 Turbidity profile as a function of temperature and light irradiation for ELP60(3X10) at concentration of 12.5 pM.
  • FIGS. 16A-16V (16A-B) Cryo-TEM images of self-assembled molecules of 1 isomerized to the (16A) trans or (16B) cis conformations.
  • (16C-J Dynamic light scattering analysis of ELPs containing (16C) 10 instances of tyrosine, (16D) 10 instances of a benzophenone -bearing uAA, (16E) 2 instances of 1, irradiated with blue light, (16F) 2 instances of 1, irradiated with UV light, (16G) 6 instances of 1, irradiated with blue light, (16H) 6 instances of 1, irradiated with UV light, (161) 10 instances of 1, irradiated with blue light, (16J) 10 instances of 1, irradiated with UV light.
  • Figures 17A-F Cryo-TEM images of the self-assembly of ELPs containing 10 instances of either 1 irradiated with (17A) blue or (17B) uv light, 2 irradiated with (17C) blue or (17D) green light, or 3 irradiated with (17E) blue or (17F) green light.
  • Figures 18A-N Characterization of the self-assembly of diblock ELPs as a function of temperature and azobenzene isomerization.
  • FIG. 20 Post-purification fluorescent labeling of ELPs.
  • ELP(lOpPR) (right) shows improved signals and reduced limit of detection for proteins as compared with only a single pPR residue (ELP(lpPR), right).
  • Figure 21 In vitro TAMRA labeling of ELP(lOpPR) in non-recoded BL21 strain and in the GRO. Proteins were expressed in either BL21 by the (1) parent or (2) Mutl-RS, or in the GRO by (3) parent pPR-RS or (4) Mutl-RS. Typhoon imaging at 532nm.
  • Figures 22A-B Staining of the OTS through conjugation of pPR to TAMRA.
  • FIG. 24A-J Sequence and signal intensities of peptides identified LC-MS of tryptic fragments.
  • 24A ELP(10TAG)-GFP MS, expressed by parent pPR-RS in the C321.ARF1 strain.
  • 24B ELP(10TAG)-GFP MS, expressed by parent pPR-RS in the BL21 strain.
  • 24C ELP( 10TAG)-GFP MS, expressed by Mutl-RS in the C321.ARF1 using ImM pPR.
  • 24D Sequence and signal intensities of peptides identified LC-MS of tryptic fragments.
  • ELP( 10TAG)-GFP MS expressed by Mutl-RS in the C321.ARF1 using 0.25mM pPR.
  • 24E ELP(10TAG)-GFP MS, expressed by Mutl-RS in the BL21 E. cob strain using ImM pPR.
  • 24F ELP(10TAG)-GFP MS, expressed by Mutl-RS in the BL21 E. cob strain using 0.25 mM pPR.
  • 24G ELP(10TAG)-GFP MS, expressed by Mut2-RS in the C321.ARF1 using ImM pPR.
  • 24H ELP(30TAG) MS, expressed in the C321.ARF1 by Mutl-RS, using different pPR concentrations.
  • Figure 25 Fluorescent quantification of microscopy images.
  • the present invention provides, in some embodiments, mutant aminoacyl-tRNA synthetase (aaRS) proteins.
  • Nucleic acid molecules encoding the mutant aaRSs are also provided, as are orthogonal translation systems comprising the mutant aaRSs or nucleic acid molecules and cells comprising the orthogonal translation system. Methods of use are also provided.
  • the present invention is based on the surprising development of highly efficient aaRS variants capable of multi-site incorporation of uAAs in a genomically recoded organism (GRO) that lacks all native TAG codons as web as the associated release factor (RF1). Surprisingly some new aaRS variants were even functional in wild-type cells.
  • the toolbox for multi-site and site-selective protein labeling has thus been greatly expanded via evolution of efficient aaRS variants for the multi-site incorporation of the alkyne -bearing uAA, 4-propargyloxy-L- phenylalanine (pPR), azobenzene-bearing phenylalanine-4 ’-azobenzene (AzoPhe), tri- fluorinated azobenzene (Azo3F) and tetra-ortho-fluorinated azobenzene (Azo4F). While OTSs have been previously developed, they are suitable for single-site pPR incorporation per-protein generally.
  • the present invention provides a mutant aminoacyl-tRNA synthetase (aaRS).
  • the mutant aaRS comprises an amino acid sequence of an aaRS comprising at least one amino acid mutation. In some embodiments, the mutant aaRS comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations. In some embodiments, the mutant aaRS comprises 2 mutations. In some embodiments, the mutant aaRS comprises 5 mutations.
  • the mutant aaRS comprises 6 mutations. In some embodiments, the mutant aaRS comprises 7 mutations. In some embodiments, the mutant aaRS comprises 8 mutations. In some embodiments, the mutant aaRS comprises 9 mutations. In some embodiments, the mutant aaRS comprises 11 mutations.
  • mutation refers to any mutation such as can be introduced into an amino acid sequence or into a nucleic acid sequence by any method known in the art.
  • a mutation is a deletion.
  • a mutation is an insertion.
  • a mutation is a substitution.
  • a mutation is a conversion of one amino acid to another.
  • a mutation is a conversion of one nucleotide to another.
  • a mutation is a conversion of a plurality of nucleotides to other nucleotides.
  • a mutation introduced into a nucleic acid sequence when translated, results in a mutant amino acid sequence.
  • the mutation is not a silent mutation.
  • the mutation increases the incorporation rate of a non-standard amino acid (nsAA) into a protein. In some embodiments, the mutation increases the rate of recognition of the aaRS of its cognate tRNA. In some embodiments, the mutation increases the rate of recognition of the aaRS of an orthogonal tRNA. In some embodiments, the mutation increases the rate of recognition of an amino acid. In some embodiments, the mutation increases the rate of recognition of the aaRS of its cognate amino acid. In some embodiments, the mutation increases the rate of recognition of the aaRS of an orthogonal amino acid.
  • nsAA non-standard amino acid
  • the amino acid is a non-standard amino acid (nsAA). In some embodiments, the nsAA is an unnatural amino acid (uAA). In some embodiments, a nsAA is a uAA. In some embodiments, the amino acid is an orthogonal amino acid. In some embodiments, the amino acid is a non-naturally occurring amino acid. In some embodiments, the amino acid is a man-made amino acid.
  • the term “unnatural amino acid” as used herein refers to any amino acid that is not genetically encoded for in an organism. The term “unnatural amino acid” as used herein refers to an amino acid that that is not inherently present within the organism.
  • Methods of generating mutations include, but are not limited to, site-directed mutagenesis, nucleotide excision, nucleotide addition, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), multiplexed automated genome engineering (MAGE) and polymerase chain reaction (PCR) with mutation generating primers or probes.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • MAGE multiplexed automated genome engineering
  • PCR polymerase chain reaction
  • Aminoacyl-tRNA synthetase is a well-known protein that catalyzes the attachment of amino acids to the 3’ end of their cognate tRNAs.
  • the aaRS is an archaeal aaRS.
  • the aaRS is a Methanocaldococcus jannaschii (Mj) protein. In some embodiments, the aaRS is a Mj aaRS. Mj is also known as Methanococcus jannaschii. In some embodiments, the aaRS recognizes a tRNA molecule. In some embodiments, the aaRS transfers an amino acid to the tRNA molecule. In some embodiments, the aaRS transfers an amino acid to the tRNA molecule. In some embodiments, the aaRS transfers an amino acid derived molecule to the tRNA molecule. In some embodiments, the aaRS is an orthogonal aaRS (o-aaRS).
  • o-aaRS orthogonal aaRS
  • the aaRS is a uAA-specific o- aaRS.
  • uAA-specific o-aaRS refers to an orthogonal amino-acyl - tRNA synthetase that recognizes only the uAA and the tRNA of the system or cell of the invention.
  • the amino acid derived molecule is a non-standard amino acid (nsAA).
  • the nsAA is an unnatural amino acid (uAA).
  • the uAA is a D amino acid or an L amino acid.
  • the uAA is a D amino acid.
  • the uAA is an L amino acid.
  • the uAA is an azide- or an alkyne-containing amino acid.
  • the uAA is an azide containing amino acid.
  • the uAA is an alkyne containing amino acid.
  • the uAA is an azobenzene-containing amino acid.
  • the uAA is a modified phenylalanine.
  • the modified phenylalanine is 4-propargyloxy-L-phenylalanine (pPR).
  • the modified phenylalanine is phenylalanine-4 ’-azobenzene (AzoPhe).
  • the azobenzene-containing amino acid is AzoPhe or tri-fluorinated azobenzene (Azo3F).
  • the azobenzene-containing amino acid is AzoPhe, Azo3F or tetra-ortho- fluorinated azobenzene (Azo4F).
  • the azobenzene-containing amino acid is AzoPhe. In some embodiments, the azobenzene-containing amino acid is Azo3F. In some embodiments, the azobenzene-containing amino acid is Azo4F. In some embodiments, the aaRS transfers 4-propargyloxy-L-phenylalanine (pPR) to the tRNA molecule. In some embodiments, the aaRS transfers phenylalanine-4’ -azobenzene (AzoPhe), tri-fluorinated azobenzene (Azo3F) or tetra-ortho-fluorinated azobenzene (Azo4F) to the tRNA molecule.
  • pPR 4-propargyloxy-L-phenylalanine
  • the aaRS transfers phenylalanine-4’ -azobenzene (AzoPhe), tri-fluorinated azobenzene (Azo3F) or tetra-ortho
  • the aaRS transfers phenylalanine-4 ’-azobenzene (AzoPhe) to the tRNA molecule. In some embodiments, the aaRS transfers tri-fluorinated azobenzene (Azo3F) to the tRNA molecule. In some embodiments, the aaRS transfers tetra-ortho-fluorinated azobenzene (Azo4F) to the tRNA molecule.
  • the tRNA molecule is an orthogonal tRNA (o-tRNA). In some embodiments, the tRNA molecule comprises a stop anticodon. In some embodiments the tRNA molecule comprises an amber anticodon. In some embodiments, the aaRS does not recognize a canonical tRNA in a cell. In some embodiments, the canonical tRNA comprises an anticodon with complementarity to a tyrosine codon. In some embodiments, the cell is a target cell. In some embodiments, the cell is a cell comprising the mutant aaRs. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is an Escherichia coli cell. In some embodiments, the cell is selected from a bacterium, an Escherichia coli cell, a eukaryotic cell, a yeast cell a fungal cell, a plant cell, an animal cell.
  • orthogonal refers to molecules (e.g.,
  • orthogonal tRNA synthetase and “orthogonal tRNA” pairs) that can process information in parallel with wild-type molecules (e.g., tRNA synthetases and tRNAs), but that do not engage in crosstalk with the wild-type molecules of a cell.
  • wild-type molecules e.g., tRNA synthetases and tRNAs
  • the orthogonal tRNA synthetase preferentially aminoacylates a complementary orthogonal tRNA (O-tRNA), but no other cellular tRNAs, with a non-canonical amino acid (e.g., Propargyl-l-Lysine), and the orthogonal tRNA is a substrate for the orthogonal synthetase but is not substantially aminoacylated by any endogenous tRNA synthetases.
  • orthogonal is with respect to a target cell.
  • the target cell is a cell of the invention.
  • orthogonal refers to an inability or reduced efficiency, e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency, of an O-tRNA to function with an endogenous tRNA synthetase (RS) compared to an endogenous tRNA to function with the endogenous tRNA synthetase, or of O-tRNA synthetase (O-RS) to function with an endogenous tRNA compared to an endogenous tRNA synthetase to function with the endogenous tRNA.
  • RS endogenous tRNA synthetase
  • O-RS O-tRNA synthetase
  • an O-tRNA in a cell is aminoacylated by any endogenous RS of the cell with reduced or even zero efficiency, when compared to aminoacylation of an endogenous tRNA by the endogenous RS.
  • an O-tRNA synthetase aminoacylates any endogenous tRNA a cell of interest with reduced or even zero efficiency, as compared to aminoacylation of the endogenous tRNA by an endogenous RS.
  • the O-tRNA anticodon loop recognizes a codon, which is not recognized by endogenous tRNAs, on the mRNA and incorporates the UAA at this site in the polypeptide, details of which are further described, for example, in U.S. Pat. No.
  • the unique codon may include nonsense codons, such as, stop codons, four or more base codons, rare codons, codons derived from natural or unnatural base pairs and/or the like.
  • the unique codon is the TAG stop codon.
  • aaRS recognition of a tRNA molecule refers to the association of an aaRS with a specific tRNA molecule including but not limited to contact at the anticodon or the acceptor stem of the tRNA molecule.
  • transfer to a tRNA molecule refers to the process by which an amino acid or an amino acid derived molecule is associated with an aaRS or a mutant aaRS and moved onto the 3 ’-hydroxyl group on the CCA tail of the tRNA molecule. The process is also referred to in the art as “charging the tRNA molecule”.
  • cancer describes an endogenous molecule that is present in a cell without any transgenic manipulation to the cell or to the progenitors of the cell.
  • the aaRS into which the mutation is introduced comprises or consists of the amino acid sequence
  • an amino acid sequence of Mj aaRS consists of SEQ ID NO: 1.
  • an amino acid sequence of wild-type aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto.
  • an amino acid sequence of aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto.
  • the an amino acid sequence of a non-mutant aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto.
  • the amino acid numbering provided herein is with respect to the sequence of SEQ ID NO: 1.
  • SEQ ID NO: 1 comprises a wildtype sequence for an aaRS and the isolated peptide is a mutant aaRS.
  • the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; leucine 65 mutated to valine; glutamic acid 107 mutated to alanine; phenylalanine 108 mutated to tyrosine; glutamine 109 mutated to methionine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid; leucine 162 mutated to lysine; leucine 162 mutated to valine; leucine 162 mutated to arginine; leucine 162 mutated to
  • the mutation is tyrosine 32 mutated to leucine, or threonine. In some embodiments the mutation is tyrosine 32 mutated to leucine. In some embodiments the mutation is tyrosine 32 mutated to threonine. In some embodiments the mutation is leucine 65 mutated to valine. In some embodiments, the mutation is glutamic acid 107 mutated to alanine. In some embodiments, the mutation is phenylalanine 108 mutated to tyrosine. In some embodiments, the mutation is glutamine 109 mutated to methionine. In some embodiments, the mutation is aspartic acid 158 mutated to serine, or glycine.
  • the mutation is aspartic acid 158 mutated to serine. In some embodiments, the mutation is aspartic acid 158 mutated to glycine. In some embodiments, the mutation is isoleucine 159 mutated to alanine, methionine, cysteine, or tyrosine. In some embodiments, the mutation is isoleucine 159 mutated to alanine. In some embodiments, the mutation is isoleucine 159 mutated to methionine. In some embodiments, the mutation is isoleucine 159 mutated to cysteine. In some embodiments, the mutation is isoleucine 159 mutated to tyrosine.
  • the mutation is leucine 162 mutated to glutamic acid, lysine, valine, arginine, serine or cysteine. In some embodiments, the mutation is leucine 162 mutated to glutamic acid. In some embodiments, the mutation is leucine 162 mutated to lysine. In some embodiments, the mutation is leucine 162 mutated to valine. In some embodiments, the mutation is leucine 162 mutated to arginine. In some embodiments, the mutation is leucine 162 mutated to serine. In some embodiments, the mutation is leucine 162 mutated to cysteine.
  • the mutation is alanine 167 mutated to histidine, aspartic acid or tyrosine. In some embodiments, the mutation is alanine 167 mutated to histidine. In some embodiments, the mutation is alanine 167 mutated to aspartic acid. In some embodiments, the mutation is alanine 167 mutated to tyrosine. It will be understood by a skilled artisan that any combination of the above recited mutations is envisioned and may be present in the mutant aaRS of the invention.
  • the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; leucine 162 mutated to arginine;
  • the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to alanine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to tyrosine; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated
  • the mutation is tyrosine 32 mutated to leucine or glycine. In some embodiments, the mutation is tyrosine 32 mutated to leucine. In some embodiments, the mutation is tyrosine 32 mutated to glycine. In some embodiments, the mutation is leucine 65 mutated to valine or glycine. In some embodiments, the mutation is leucine 65 mutated to valine. In some embodiments, the mutation is leucine 65 mutated to glycine. In some embodiments, the mutation is glutamic acid 107 mutated to serine, asparagine or aspartic acid. In some embodiments, the mutation is glutamic acid 107 mutated to serine.
  • the mutation is glutamic acid 107 mutated to asparagine. In some embodiments, the mutation is glutamic acid 107 mutated to aspartic acid. In some embodiments, the mutation is phenylalanine 108 mutated to arginine. In some embodiments, the mutation is glutamine 109 mutated to methionine, serine, leucine or cysteine. In some embodiments, the mutation is glutamine 109 mutated to methionine. In some embodiments, the mutation is glutamine 109 mutated to serine. In some embodiments, the mutation is glutamine 109 mutated to leucine. In some embodiments, the mutation is glutamine 109 mutated to cysteine.
  • the mutation is aspartic acid 158 mutated to glycine. In some embodiments, the mutation is isoleucine 159 mutated to tyrosine. In some embodiments, the mutation is leucine 162 mutated to serine or arginine. In some embodiments, the mutation is leucine 162 mutated to serine. In some embodiments, the mutation is leucine 162 mutated to arginine. In some embodiments, the mutation is alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, and isoleucine 159 mutated to tyrosine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to serine or arginine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to serine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to arginine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to leucine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to leucine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to valine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to valine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to leucine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to leucine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to valine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to valine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to valine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to valine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to valine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to valine or glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to valine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to glycine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to valine.
  • the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to glycine.
  • the aaRS further comprises mutation of arginine 257 to glycine, mutation of aspartic acid 286 to arginine, or both. In some embodiments, the aaRS further comprises mutation of arginine 257 to glycine. In some embodiments, the aaRS further comprises mutation of aspartic acid 286 to arginine. In some embodiments, the aaRS further comprises mutation of both arginine 257 to glycine and aspartic acid 286 to arginine. In some embodiments, SEQ ID NO: 1 further comprises these two known mutations. In some embodiments, the sequence into which the mutations of the invention are introduced comprises or consists of
  • LKNAVAEELIKILEPIRKRL (SEQ ID NO: 28) or a sequence with 95% identity thereto.
  • the sequence into which the mutations of the invention are introduced consists of SEQ ID NO: 28.
  • the mutant aaRS comprises tyrosine 32 mutated to leucine, aspartic acid 158 mutated to serine, isoleucine 159 mutated to methionine, leucine 162 mutated to lysine, alanine 167 mutated to histidine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 2.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to alanine, leucine 162 mutated to glutamic acid, alanine 167 mutated to histidine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the mutant aaRS comprises: tyrosine 32 mutated to threonine, leucine 65 mutated to valine, glutamic acid 107 mutated to alanine, phenylalanine 108 mutated to tyrosine, glutamine 109 mutated to methionine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to cysteine, leucine 162 mutated to arginine, alanine 167 mutated to aspartic acid, arginine 257 mutated to glycine and aspartic acid 286 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSAYMLDKDYTLN VYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 4), or a fragment, a derivative or analog thereof.
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 4.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to methionine; leucine 162 mutated to serine, alanine 167 mutated to histidine, arginine 257 mutated to glycine and aspartic acid 286 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 5.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to cysteine, alanine 167 mutated to tyrosine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 6.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 12) or a fragment, a derivative or an analog thereof.
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 12.
  • the mutant aaRS comprises: tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRL ALKTTLKR ARRSMELI AREDENPKV AE VI YPIMQ VN G YH Y S G VD VF V GGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 13) or a fragment, a derivative or an analog thereof.
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 13.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEKLREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSVSLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 14) or a fragment, a derivative or an analog thereof.
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 14.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to asparagine, phenylalanine 108 mutated to valine, glutamine 109 mutated to leucine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 15.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to aspartic acid, aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 16.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSVCLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 17) or a fragment, a derivative or an analog thereof.
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 17.
  • the mutant aaRS comprises: tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; and leucine 162 mutated to arginine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 18.
  • the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to glycine; glutamic acid 107 mutated to aspartic acid, phenylalanine 108 mutated to arginine, glutamine 109 mutated to methionine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
  • the mutant aaRS comprises or consists of the amino acid sequence:
  • the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 19.
  • the fragment, derivative or analog comprises at least one of the recited mutations.
  • the fragment, derivative or analog is an active fragment, derivative or analog.
  • active refers to possessing an aaRS activity.
  • the aaRS activity is the ability to catalyzes the attachment of an amino acid to its cognate tRNA.
  • the aaRS activity is the ability to recognize an amino acid.
  • the aaRS activity is the ability to recognize a tRNA.
  • the aaRS activity is the ability to transfer an amino acid to a tRNA.
  • a derivative refers to any polypeptide that is based off the polypeptide of the invention and still comprises the recited mutations.
  • a derivative is not merely a fragment of the polypeptide, nor does it need to have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification.
  • a derivative may be a derivative of a fragment of the polypeptide of the invention.
  • a derivative of a sequence comprises at least 70, 75, 80, 85, 90, 92, 93, 95, 97, 99 or 100% identity to that sequence. Each possibility represents a separate embodiment of the invention.
  • a derivative of a sequence comprises at least 90% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 95% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 97% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 99% identity to that sequence.
  • a fragment comprises at least 50, 100, 150, 200, or 250 amino acids of the aaRS. Each possibility represents a separate embodiment of the invention.
  • a fragment is a functional fragment.
  • a fragment comprises at least 50 amino acids of the aaRS.
  • a fragment comprises at least 100 amino acids of the aaRS.
  • the fragment is a portion of the polypeptide comprises any one of a leucine at position 32, a threonine at position 32, a valine at position 65, an alanine at position 107, a tyrosine at position 108, a methionine at position 109, a serine at position 158, a glycine at position 158, an alanine at position 159, a methionine at position 159, a cysteine at position 159, a tyrosine at position 159, a glutamic acid at position 162, a lysine at position 162, a valine at position 162, an arginine at position 162, a serine at position 162, a cysteine at position 162, a histidine at position 167, an aspartic acid at position 167, and a tyrosine at position 167.
  • any fragment of the isolated polypeptide of the invention will still comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 80, or at least 100 amino acids surrounding position 32, position 65, position 107, position 108, position 109, position 158, position 159, position 162, or position 167 of the polypeptide.
  • Each possibility represents a separate embodiment of the present invention.
  • the fragment is a portion of the polypeptide comprises any one of a leucine at position 32, a glycine at position 32, a valine at position 65, a glycine at position
  • a serine at position 107 an asparagine at position 107, a aspartic acid at position 107, a valine at position 108, a arginine at position 108, a methionine at position 109, a serine at position 109, a leucine at position 109, a cysteine at position 109, a glycine at position 158, a tyrosine at position 159, a an alanine at position 162, a serine at position 162, and a phenylalanine at position 167.
  • Such a fragment will still be recognizable as being from the polypeptide of the invention, and as such will be at least 10 amino acids in length.
  • any fragment of the isolated polypeptide of the invention will still comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 80, or at least 100 amino acids surrounding position 32, position 65, position 107, position 108, position 109, position 158, position 159, position 162, or position 167 of the polypeptide.
  • Each possibility represents a separate embodiment of the present invention.
  • analog includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
  • one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine
  • substitution of one basic residue such as lysine, arginine or histidine for another
  • substitution of one acidic residue such as aspartic acid or glutamic acid for another
  • the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a fragment, analog or derivative thereof. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a fragment, analog or derivative thereof.
  • the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
  • the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO:
  • the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 or a fragment, analog or derivative thereof.
  • the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
  • the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19or a fragment, analog or derivative thereof.
  • the present invention provides an isolated polypeptide, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
  • the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the peptides, polypeptides and proteins described herein have modifications rendering them more stable while in the body, more capable of penetrating into cells or capable of eliciting a more potent effect than previously described.
  • the terms “peptide”, “polypeptide” and “protein” apply to naturally occurring amino acid polymers.
  • the terms “peptide”, “polypeptide” and “protein” apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • isolated polypeptide refers to a peptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature.
  • a preparation of isolated peptide contains the peptide in a highly-purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • a highly-purified form i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • nucleic acid molecule encoding a mutant aaRS of the invention, or a fragment, a derivative or an analog thereof.
  • nucleic acid molecule comprising a coding region encoding a mutant aaRS of the invention, or a fragment, a derivative or an analog thereof.
  • the nucleic acid molecule encodes a mutant aaRS of the invention. In some embodiments, the nucleic acid molecule comprises a coding region encoding a mutant aaRS of the invention.
  • the nucleic acid molecule is selected from DNA, RNA, cDNA, genomic DNA (gDNA), vector DNA, vector RNA, LNA, PNA and a combination thereof.
  • the nucleic acid molecule is DNA.
  • the nucleic acid molecule is RNA.
  • the nucleic acid molecule is cDNA.
  • the nucleic acid molecule is gDNA.
  • the nucleic acid molecule is LNA.
  • the nucleic acid molecule is PNA.
  • the nucleic acid molecule is a hybrid molecule comprising more than one type of nucleic acid.
  • the phrases "coding sequence” and “coding region” are interchangeable and refer to the region that when translated results in the production of an expression product, such as a polypeptide, protein, or enzyme, and specifically the mutant aaRS.
  • the coding region is operably linked to at least one regulatory element.
  • the regulatory element is configured to express the coding region in a target cell.
  • the regulatory element is configured to express a protein encoded by the coding region in a target cell.
  • the regulatory element is a promoter.
  • the regulatory element is an enhancer.
  • the regulatory element is a silencer.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • expression of the coding region refers to a state in which mRNA is transcribed from the coding region acting as a template.
  • expression of the coding region refers to a state in which polypeptide is translated from the mRNA transcribed from the coding region.
  • promoter refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
  • the nucleic acid molecule is a vector.
  • the vector is a DNA vector.
  • the vector is an RNA vector.
  • the vector is an expression vector.
  • the expression vector is configured for expression in a bacterial cell.
  • the expression vector is configured for expression in a mammalian cell.
  • the expression vector is configured for expression in a target cell.
  • a gene or protein within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome.
  • the gene is in an expression vector such as plasmid or viral vector.
  • the vector is introduced into a cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82,
  • a vector of the invention may be introduced into a target cell by any method known in the art, including but not limited to those provided herein. In some embodiments, the introducing produces a cell of the invention.
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • additional elements such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • the vector may be a DNA plasmid delivered via non-viral methods or via viral methods.
  • the viral vector may be a retroviral vector, a herpes viral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector.
  • the promoters may be active in mammalian cells.
  • the promoters may be a viral promoter.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 ( ⁇ ), pGL3, pZeoSV2( ⁇ ), pSecTag2, pDisplay, pEF/myc/cyto, pCM V /myc/cy to , pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDS VE, and any other vector allowing expression of proteins under the direction of the S V -40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • recombinant viral vectors which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression.
  • lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • plant expression vectors are used.
  • the expression of a polypeptide coding sequence is driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 3:17-311 (1987)] are used.
  • plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.
  • constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)].
  • Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
  • a gene or protein can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy).
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy).
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 7.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 7.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 7.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 8. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 8. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 8.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 9.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 9.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 9.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 10.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 10.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 10.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 11.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 11.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 11.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 20.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 20.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 20.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • sequence of the nucleic acid molecule comprises SEQ ID NO: 21.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 21.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 21. [0150]
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 22.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 22.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 22.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 23.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 23. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 23.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 24.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 24.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 24.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 25.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 25.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 25.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 26.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 26. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 26.
  • a sequence of the nucleic acid molecule comprises or consists of the sequence:
  • the sequence of the nucleic acid molecule comprises SEQ ID NO: 27.
  • the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 27.
  • the coding region of the nucleic acid molecule consists of SEQ ID NO: 27.
  • each of SEQ ID NO:7-l 1 comprises a coding sequence for a mutant aaRS.
  • each of 20-27 comprises a coding sequence for a mutant aaRS.
  • each of the nucleic acid molecules comprises a coding sequence coding for a mutant aaRS. It will be understood by a skilled artisan, that as the protein is the active molecule any substitution to the nucleic acid sequence that does not alter the protein encoded is also envisioned. As the codons for amino acids are degenerate, one codon may be switched for a synonymous codon.
  • the coding region encodes a recombinant protein.
  • the recombinant protein is a mutant aaRS.
  • the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring.
  • the polypeptide is a recombinant protein.
  • the term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector used to express the recombinant protein in a cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vectors e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses.
  • Viral vectors also include polynucleotides carried by a virus for transfecting into host cells.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid coding for the protein of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • additional elements such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • an orthogonal translation system comprising: a. a mutant aaRS of the invention or a nucleic acid molecule of the invention; and b. a tRNA compatible with the aaRS.
  • the orthogonal translation system is configured for translation in a target cell. In some embodiments, the orthogonal translation system is configured for in vitro translation. In some embodiments, the orthogonal translation system is configured for administration to a subject. In some embodiments, the orthogonal translation system is configured for administration to a cell. In some embodiments, the orthogonal translation system is configured for transfection to a cell. In some embodiments, the orthogonal translation system comprises a mutant aaRS of the invention. In some embodiments, the orthogonal translation system comprises a nucleic acid molecule of the invention.
  • the tRNA is an orthogonal tRNA. In some embodiments, the tRNA is a non-naturally occurring tRNA. In some embodiments, the tRNA is a Mj tRNA. In some embodiments, the Mj tRNA is the tRNA corresponding to a stop codon. In some embodiments, the tRNA corresponds to a stop codon. In some embodiments, the stop codon is a stop codon that is absent in a target cell. In some embodiments, the stop codon is a stop codon that is depleted in a target cell. In some embodiments, the tRNA is recognized by the aaRS. In some embodiments, the tRNA is compatible with the mutant aaRS.
  • the tRNA is recognized by the mutant aaRS. In some embodiments, the mutation does not affect the aaRS’s recognition of the tRNA. In some embodiments, the mutation enhances the aaRS’s recognition of the tRNA. In some embodiments, the tRNA comprises an anticodon. In some embodiments, the anticodon corresponds to a stop codon. In some embodiments, the anticodon recognizes a stop codon. In some embodiments, the anticodon anneals to a stop codon. In some embodiments, the stop codon is a TAG stop codon. In some embodiments, the stop codon is a TGA stop codon. In some embodiments, the stop codon is a TAA stop codon. In some embodiments, the stop codon is not a TGA stop codon. In some embodiments, the stop codon is not a TAA stop codon.
  • the orthogonal translation system further comprises an nsAA.
  • the nsAA is a uAA.
  • the uAA comprises a chemical moiety.
  • the chemical moiety is a biorthogonal chemical moiety.
  • the uAA is not naturally found in a target cell.
  • the biorthogonal chemical moiety is not naturally found in a target cell.
  • the chemical moiety is an azide or an alkyne group.
  • the chemical moiety comprises an azide or an alkyne group.
  • Unnatural amino acids comprising azide and/or alkyne groups are well known in the art and non-limiting example include 3-Azido-D alanine, 3-azido- L-alanine, 4-azido-D-homoalanine, 4-azido-L-homoalanaine, 5-azido-D-ornithine, 5-azido-L- ornithine, 6-azido-D lysine, 6-azido-L-lysine, Boc-(R)-4-(2-propynyl)-L-proline, Boc- propargyl-Glycine-OH, Fmoc-(S)-propargyl-alanine-OH, Fmoc-(R)-propargyl-alanine-OH, and pPR.
  • the chemical moiety is an azide group. In some embodiments, the chemical moiety is an alkyne group. In some embodiments, the chemical moiety is an azobenzene group. Unnatural amino acids comprising azobenzene groups are well known in the art and non limiting example include 4,4’-AMPB, 3,3’-AMPB, 3,4’-AMPB, 3,3’-APB, AzoPhe, Azo3F and Azo4F. In some embodiments, the uAA is a modified phenylalanine.
  • the modified phenylalanine is selected from 4-propargyloxy-L-phenylalanine (pPR), and phenylalanine-4’ -azobenzene (AzoPhe). In some embodiments, the modified phenylalanine is pPR. In some embodiments, the modified phenylalanine is AzoPhe. In some embodiments, a uAA comprising an azobenezene group is selected from AzoPhe, Azo3F and Azo4F. In some embodiments, Azo3F is 2,4,6-tri-fluorinated azobenzene. In some embodiments, a uAA comprising an azobenezene group is AzoPhe. In some embodiments, a uAA comprising an azobenezene group is Azo3F. In some embodiments, a uAA comprising an azobenezene group is Azo4F.
  • the mutant aaRS comprises a mutation found in SEQ ID NO: 2-6 and the uAA comprises an azide or an alkyne group. In some embodiments, the mutant aaRS comprises a sequence of SEQ ID NO: 2-6 and the uAA comprises an azide or an alkyne group. In some embodiments, the mutant aaRS comprises a mutation found in SEQ ID NO: 12-19 and the uAA comprises an azobenzene group. In some embodiments, the mutant aaRS comprises a sequence of SEQ ID NO: 12-19 and the uAA comprises an azobenzene group.
  • a cell comprising a mutant aaRS of the invention.
  • a cell comprising a nucleic acid molecule of the invention.
  • a cell comprising an orthogonal translation system of the invention.
  • the cell is a target cell.
  • the cell is a mammalian cell.
  • the cell is a bacterial cell.
  • the bacterium is E. coli.
  • the cell is not an archaeal cell.
  • the cell is an unmodified cell.
  • the cell is unmodified with the exception of the presence of a protein, nucleic acid or system of the invention.
  • the cell is a genetically modified cell.
  • the genome of the cell is unmodified. In some embodiments, the genome of the cell is modified. In some embodiments, the cell is devoid of TAG stop codons. In some embodiments, the TAG stop codons are endogenous TAG stop codons. In some embodiments, the TAG stop codons are native TAG stop codons. In some embodiments, the cell is depleted of TAG stop codons. In some embodiments, depleted comprises at least 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% of the stop codons of the cell having been removed. Each possibility represents a separate embodiment of the invention.
  • the TAG stop codons are mutated to TGA or TAA stop codons. In some embodiments, the TAG stop codons are mutated to TGA stop codons. In some embodiments, the TAG stop codons are mutated to TAA stop codons. In some embodiments, the stop codon that is depleted or absent from the cell is the stop codon that corresponds to the anticodon loop of the tRNA.
  • the cell is devoid of release factor 1 (RF1). In some embodiments, the cell does not express RF1. In some embodiments, the cell has decreased expression of RF1. In some embodiments, decreased is with respect to a wild-type cell. In some embodiments, decreased is with respect to a non-modified cell. In some embodiments, decreased is at least a 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% reduction in expression. Each possibility represents a separate embodiment of the invention. In some embodiments, the RF1 gene has been genomically ablated from the cell. In some embodiments, the cell is an RF1 knockout cell.
  • the cell is a wild-type cell. In some embodiments, the cell expresses RF1. In some embodiments, the cell expresses RF1 at normal levels. In some embodiments, the cell comprises at least one TAG stop codon. In some embodiments, the cell comprises its natural content of TAG stop codons. In some embodiments, the cell does not comprise a TAG stop codon mutated to a TGA or TAA stop codon. [0173] In some embodiments, the cell further comprises a vector comprising an open reading frame (ORF). In some embodiments, the ORF is a coding region. In some embodiments, the ORF comprises at least one stop codon within the open reading frame.
  • ORF open reading frame
  • the stop codon is a stop codon that corresponds to the anticodon of the tRNA of the orthogonal translation system. In some embodiments, the at least one stop codon is not the last codon of the ORF. In some embodiments, at least one codon coding for an amino acid is present after the stop codon in the ORF. In some embodiments, the amino acid encoded after the stop codon is a natural amino acid. In some embodiments, the last codon of the ORF is a stop codon that does not correspond to the anticodon of the tRNA of the orthogonal translation system.
  • the vector is an expression vector.
  • the vector is configured to express a protein encoded by the ORF in the cell.
  • the ORF is operatively linked to at least one regulatory element.
  • the regulatory element is configured to induce expression of the protein encoded by the ORF in the cell.
  • the regulatory element is capable of induce expression of the protein encoded by the ORF in the cell.
  • the ORF comprises at least one stop codon. In some embodiments, the ORF comprises at least two stop codons. In some embodiments, the OFR comprises a plurality of stop codons. In some embodiments, the ORF comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 stop codons. Each possibility represents a separate embodiment of the invention. In some embodiments, the ORF comprises at least 10 stop codons. In some embodiments, the ORF comprises at least 30 stop codons. It will be understood by a skilled artisan that the number of stop codons recited herein does not refer to the stop codon at the end of the ORF that is responsible for stopping translation. The stop codon at the end of the ORF that stops translation will not correspond to the anticodon of the tRNA of the orthogonal translation system.
  • the ORF encodes a protein of interest. In some embodiments, the ORF encodes a protein to comprise an nsAA. In some embodiments, the protein of interest is a protein to be tagged. In some embodiments, the protein or interest is a protein to be made light responsive. Methods of use
  • a method of producing a protein comprising an nsAA comprising introducing into a cell an expression vector comprising an ORF encoding the protein, wherein the ORF comprises at least one stop codon, and wherein the cell comprises an orthogonal translation system of the invention, thereby producing a protein comprising an nsAA.
  • the protein is a target protein.
  • the expression vector comprising an ORF encoding the protein is an expression vector as described herein above.
  • the orthogonal translation system is an orthogonal translation system comprising a nsAA.
  • the cell comprises the nsAA.
  • the orthogonal translation system is compatible with the nsAA.
  • the tRNA of the orthogonal translation system is compatible with the nsAA.
  • the mutant aaRS of the orthogonal translation system is compatible with the nsAA.
  • the method further comprises introducing the orthogonal translation system into the cell. In some embodiments, the method further comprises introducing the nsAA into the cell. In some embodiments, introducing comprises transfection. In some embodiments, introducing comprises nucleofection. In some embodiments, introducing comprises genomic alteration. In some embodiments, introducing comprises genome editing.
  • the method is for labeling a protein.
  • the method is for labeling and the nsAA is an azide or alkyne group containing nsAA.
  • the method is for labeling and the mutant aaRS comprises a mutation found in SEQ ID NO: 2-6.
  • the method is for labeling and the mutant aaRS comprises a sequence of SEQ ID NO: 2-6.
  • the method is for labeling and further comprises converting the nsAA into a detectably labeled amino acid.
  • converting comprises addition of a detectable moiety by Click chemistry.
  • the Click chemistry is copper- catalyzed Click chemistry.
  • the Click chemistry is not copper-catalyzed Click chemistry.
  • the Click chemistry comprises azide and/or alkene cycloaddition.
  • a “detectable moiety” is any molecule or portion of a molecule that can be specifically detected by a method known in the art.
  • detectable moieties include, but are not limited to fluorescent moieties, radioactive moieties, bulky groups, dyes, and a tag.
  • the term "moiety”, as used herein, relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures.
  • the term “moiety” further means part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule.
  • the detectable moiety is a fluorescent moiety.
  • method is for producing a light-responsive protein.
  • a light-responsive protein is a light-sensitive protein.
  • the method is for producing a light-responsive protein and the nsAA comprises an azobenzene group.
  • the method is for producing a light-responsive protein and the mutant aaRS of the orthogonal translation system comprises a mutation found in SEQ ID NO: 12-19.
  • the method is for producing a light-responsive protein and the mutant aaRS of the orthogonal translation system comprises a sequence of SEQ ID NO: 12-19.
  • the method further comprises irradiating the produced protein with light.
  • a protein comprising a nsAA.
  • the protein is a protein comprising a nsAA. In some embodiments, the protein is a light-responsive protein. In some embodiments, the protein is a light-sensitive protein. In some embodiments, the protein is an ELP. In some embodiments, the protein is a self assembling protein. In some embodiments, the protein is a diblock. In some embodiments, the protein is a ELP diblock copolymer.
  • the protein comprises at least one nsAA. In some embodiments, the protein comprises a plurality of nsAA. In some embodiments, the protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 nsAA. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises at least 5 nsAA. In some embodiments, the protein comprises at least 10 nsAA. In some embodiments, the protein comprises at least 15 nsAA. In some embodiments, the protein comprises at least 20 nsAA. In some embodiments, the protein comprises at least 30 nsAA. In some embodiments, the protein comprises at least 50 nsAA. In some embodiments, the protein comprises at least 100 nsAA.
  • all the nsAA in the protein are the same nsAA. In some embodiments, the nsAA comprise at least two different nsAA. In some embodiments, the nsAA are present at predetermined positions in the protein. In some embodiments, at least one nsAA is inserted in a hydrophobic segment of an ELP diblock co-polymer. In some embodiments, all the nsAA are inserted in a hydrophobic segment of an ELP diblock co-polymer.
  • each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • the azobenzene-uAAs 1 and 2 were purchased from Giotto Biotech and the azobenzene- uAA 3 was purchased from Chiroblock. Restriction endonucleases and ligation enzymes were purchased from New England Biolabs. DNA amplification was performed using The KAPA2G Fast HotStart ReadyMix or the KAPA HiFi PCR kit (Roche). Plasmid purification was conducted with Plasmid HiYield mini-prep (RBC Bioscience) and the PCR/restriction product was purified using a HiYield gel/PCR extraction kit (RBC Bioscience). Ligation was performed using the Quick LigationTM Kit or with the T4 DNA Ligase, both purchased from New England Biolabs.
  • Ligation products were transformed into 5 -alpha Competent E. coli (High Efficiency) or Stbl2 Competent E. coli (High Efficiency), purchased from New England Biolabs. SDS solution was purchased from Bio-Rad. Anhydrotetracycline hydrochloride was purchased from Sigma- Aldrich. C321.AA (Isaacs lab) and pEvol-pAzFRS.l.tl were a gift from Farren Isaacs (Addgene plasmids # 73581 and # 73547).
  • AARS libraries were generated by MAGE-based diversification of previously isolated genomically integrated mutants, pAcF-RS.tl, pAcFRS.2.tl and pAzFRS.2.tl.
  • cultures Prior to MAGE cycling, cultures were established by inoculating the liquid medium with a single bacterial colony or by adding 30 m ⁇ of a confluent liquid culture (1: 100 dilution) at 34 °C to mid-logarithmic growth (OD at 600 nm of 0.6 - 0.7) in a shaking incubator.
  • MAGE oligos are known in the art, and are provided for example in Amiram et al., 2015, “Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids”, Nature Biotechnology, 22, 1272-1279, herein incorporated by reference in its entirety.
  • the oligo-cell mixture was transferred to a pre-chilled 1 mm gap electroporation cuvette (Bio- Rad) and electroporated under the following parameters: 1.8 kV, 200 V and 25 mF.
  • LB media (3 ml) was immediately added to the electroporated cells. The cells were recovered from electroporation and grown at 34 °C for 3-3.5 h. Once the cells reached mid-log stage, they were used in additional MAGE cycles, subjected to negative and positive selection cycles, or frozen for further use.
  • Plasmid construction Plasmids bearing GFP -based reporter genes were known in the art. Plasmids bearing the OTS variants for pPR incorporation were constmcted by insertion of aaRS genes to a previously described plasmid harboring a pl5A origin of replication and a chloramphenicol resistance marker. The gene encoding for the parent-pPR-RS OTS was chemically synthetized (IDT), and aaRS genes were PCR-amplified from chromosomal templates. All variants were inserted sequentially using the flanking restriction sites restriction sites Bglll and Sail, to produce inducible expression under the control of araBAD promoter and the rrnB terminator. The second constitutive copy of the aaRS, typically found in the pEvol system was removed.
  • the GFP(2TAG) reporter gene was chemically synthesized (IDT), restricted with Xhol and Hindlll restriction enzymes, and ligated to a similarly cut reporter plasmid.
  • the ELP 6 o genes were chemically synthesized as half-proteins, ELP30 genes (GeneArt, Thermo Fisher), restricted with BseRI, and ligated sequentially using PreRDL, under the control of the pTac promoter in a pet24 modified vector (GeneScript).
  • Plasmids bearing the OTS variants for azobenzene -u A A incorporation were constmcted by inserting aaRS genes into a previously described plasmid (pEvol) harboring a pi 5 A origin of replication and a chloramphenicol resistance marker.
  • the gene encoding for the AzoRS OTS was synthetized (IDT), and the evolved genomic aaRS genes were PCR-amplified from chromosomal templates. All variants were inserted sequentially by using the flanking restriction sites Bglll and Sail to obtain inducible expression under the control of the araBAD promoter and the rrnB terminator.
  • the second constitutive copy of the aaRS typically found in the pEvol system was removed.
  • Ligation was conducted with the Quick LigationTM Kit (NEB ® ) and the ligation products were transformed into NEB® 5- alpha Competent E. coli (High Efficiency), later plated on LB-agar plates supplemented with chloramphenicol (25 pg ml 1 ) and analyzed by Sanger sequencing.
  • aaRS expression was then induced by the addition of 0.2% arabinose
  • GFP expression was induced by the addition of 60 ng/m ⁇ anhydrotetracy cline
  • the uAA was added at a concentration of 1 mM.
  • inducers for aaRS and GFP expression were added immediately after inoculation in the plate. Cultures and inducers were added individually to each well. Cells were incubated at 34 °C overnight. Following expression, cells were centrifuged at 4,000 g for 5 min. Supernatant medium was removed and cells were resuspended in PBS.
  • GFP fluorescence was measured on a Biotek spectrophotometric plate reader using excitation and emission wavelengths of 485 and 528 nm, respectively. Fluorescence signals were normalized by dividing the fluorescence counts by the OD600 reading.
  • aaRS was then induced by adding arabinose (0.2%); GFP expression was induced by adding anhydrotetracycline (60 ng ml 1 ); and the uAA was added at a concentration of 0.25 mM.
  • the cells were centrifuged at 4,000 g for 5 min, the supernatant medium was removed, and the cells were resuspended in PBS.
  • GFP fluorescence was measured on a Biotek spectrophotometric plate reader by using excitation and emission wavelengths of 485 nm and 528 nm, respectively. Fluorescence signals were normalized by dividing the fluorescence counts by the ODeoo reading.
  • ELP expression and purification Before batch expression, starter cultures (1:25 v/v of final expression volume) of 2xYT media supplemented with 30 pg/ml kanamycin and 25 pg/ml chloramphenicol were inoculated with transformed cells from a fresh agar plate or from stocks stored at -80 °C, and incubated overnight at 34 °C while shaking at 220 r.p.m. Cells were centrifuged at 4,000 g for 10 min, supernatant medium was removed and cells were resuspended in remaining media, and transferred to expression flasks (containing 2xYT media, antibiotics, 0.2% arabinose and the uAA).
  • ELP(10TAG)-GFP For the expression of ELP(10TAG)-GFP by Mutl-RS in the genomically recoded organism, cells were supplemented with 0.25 mM of the uAA. For expression of ELP(30TAG)- GFP or for expression in BL21, cells were supplemented with 1 irM uAA. Cells were incubated at 34 °C for 4-5 h and then reporter protein expression was induced with 60 pg/ml anhydrotetracycline. Cells were harvested 24 h after inoculation by centrifugation at 4,000 g for 30 min at 4 °C. The cell pellet was resuspended by vortex in ⁇ 2 ml PBS buffer and stored at -80 °C or immediately purified.
  • resuspended pellets were lysed by ultrasonic disruption (18 cycles of 10 s sonication separated by 40 s intervals).
  • Poly(ethyleneimine) (0.2 ml of 10% solution) was added to each lysed suspension before centrifugation at 4,000 g for 15 min at 4 °C to separate cell debris from the soluble cell lysate.
  • All ELP constructs were purified by a modified inverse transition cycling (ITC) protocol consisting of multiple “hot” and “cold” spins using sodium citrate to trigger the phase transition.
  • ITC inverse transition cycling
  • the soluble cell lysate was incubated for 1-2 min at 75 °C to denature native E. coli proteins.
  • the cell lysate was then cooled on ice, centrifuged for 2 min at -14,000 r.p.m and the pellet was discarded.
  • the ELP phase transition was triggered by adding sodium citrate to the cell lysate or the product of a previous cycle of ITC at a final concentration of -0.5 M.
  • the solutions were then centrifuged at -14,000 r.p.m for 2 min and the pellets were resuspended in PBS, followed by a 2 min “cold” spin performed without addition of sodium citrate to remove denatured contaminant. Additional rounds of ITC were carried out as needed, using a saturated solution of sodium citrate until sufficient purification was achieved.
  • Protein concentration was calculated by measuring the OD280 of purified protein according to the following extinction coefficients: Tyr (WT protein): 33,935, ELP(lpPR)-GFP: 33,645, ELP(5pPR)-GFP: 32,485, ELP(10pPR)-GFP: 31,035, based on extinction coefficient of pPR (1200 M-cm-1).
  • starter cultures (1:40 v/v of final expression volume) of 2xYT media, supplemented with kanamycin (30 pg ml 1 ) and chloramphenicol (25 pg ml 1 ), were inoculated with transformed cells from either a fresh agar plate or from stocks stored at -80 °C, incubated overnight at 34 °C while shaking at 220 rpm, and transferred to expression flasks containing 2xYT media, antibiotics, arabinose (0.2%), and azobenzene -u A A (0.25 mM).
  • ELP 6 o(10TAG), ELP 6 o(6TAG), and ELP 6 o(2TAG) by AzoRS- 4 the C321.ARF1 strain 1401 , supplemented with azobenzene-uAA (0.25 mM) and arabinose (0.2%), was incubated at 34 °C for 4-5 h and then protein expression was induced with isopropyl b-d-l-thiogalactopyranoside (IPTG, 1 mM). The cells were harvested 24 h after inoculation by centrifugation at 4,000 g for 30 min at 4 °C.
  • IPTG isopropyl b-d-l-thiogalactopyranoside
  • the cell pellet was then resuspended by vortex in milli-Q water ( ⁇ 4 ml) and either stored at -80 °C or purified immediately.
  • resuspended pellets were lysed by ultrasonic disruption (18 cycles of 10 s sonication, separated by 40 s intervals of rest).
  • Poly(ethyleneimine) was added (0.2 ml of a 10% solution) to each lysed suspension before centrifugation at 4,000 g for 15 min at 4 °C to separate cell debris from the soluble cell lysate.
  • All ELP constructs were purified by a modified inverse transition cycling (ITC) protocol [20b] consisting of multiple “hot” and “cold” spins by using sodium chloride to trigger the phase transition.
  • ITC inverse transition cycling
  • the soluble cell lysate was incubated for 1-2 min at 42-55 °C to denature the native E.coli proteins. The cell lysate was then cooled on ice, centrifuged for 2 min at -14,000 rpm, and the pellet was discarded.
  • the ELP phase transition was triggered by adding sodium chloride to the cell lysate or to the product of a previous cycle of ITC at a final concentration of -5 M. The solutions were then centrifuged at -14,000 rpm for 10 min and the pellets were resuspended in milli-Q water, after which a 2 min “cold” spin was performed without sodium chloride to remove denatured contaminant. Additional rounds of ITC were conducted as needed using a saturated solution of sodium chloride until sufficient purification was achieved.
  • Protein concentrations were calculated by measuring the OD280 of the purified protein according to the following extinction coefficients: ELP 6 o(tyrosinexlO): 16,390, ELP 6 o(lxlO): 26,900, ELP6O(1X6): 16,736, and ELP 6 o(2xlO): 6,572, based on the extinction coefficient of 1 (2,541 M cm 1 ); ELP 6 o(2xlO): 41590, ELP 60 (2x6): 25550, and ELP 60 (2xlO): 9510, based on the extinction coefficient of 2 (4010 M cm 1 ); and ELP 6 o(3xlO): 79122 , ELP 6 o(3x6): 74546, and ELP6O(3X 10): 25482, based on the extinction coefficient of 3 (123250 M cm 1 ).
  • Intact mass measurements Intact mass measurements of the proteins were performed using the MALDI-TOF instrument (MALDI-TOF/TOF autoflex speed), at the Ilse Katz Institute for Nanoscale Science and Technology (Ben-Gurion University of the Negev). Spectrum analysis was performed by the Flexanalysis software.
  • ELP(ITAG) and ELP(IOTAG), both without GFP were expressed in the genomically recoded organism or in the BL21 strain, by the parent - pPR-RS or evolved Mutl-RS.
  • Starter cultures of 2xYT media supplemented with 30 pg/ml kanamycin and 25 pg/ml chloramphenicol were inoculated with transformed cells from a fresh agar plate or from stocks stored at -80°C, and incubated overnight at 34 °C while shaking at 220 r.p.m.
  • Concentrations of other reagents in the reaction were as following: 2% v ⁇ v DMSO, 0.1 mM TAMRA, 0.5 mM THPTA premixed with 0.1 mM CuS04 for 20 min, 2.5 mM sodium ascorbate.
  • DPBS solution was added up to desired volume. Reaction was performed for 1 hour at 25 °C, in a shaking incubator at 400 r.p.m in the dark. Cells were washed by cycles of 3 min centrifugation at -14,000 r.p.m followed by pellet resuspension in PBS, until the supernatant was colorless.
  • Phase transition analysis To characterize the inverse transition temperature of EFP variants, the ODeoo of the EFP solution (in milli-Q water, unless otherwise noted) was monitored as a function of temperature, with heating and cooling performed at a rate of 1 °C min 1 on a UV- vis spectrophotometer equipped with a multicell thermoelectric temperature controller (Thermo Scientific).
  • DLS Dynamic light scattering
  • Circular Dichroism (CD) analysis The secondary structure of ELPs was studied using an Jasco J-715 spectropolarimeter (Tokyo) equipped with a PTC-348WI temperature controler, using a 1 -mm quartz cuvette instrument by scanning from 280 nm to 180 nm at either 10 °C or 30 °C. Purified constructs were diluted to 7.5 mM in water. Data were considered for analysis whenever the Dynode voltage was below 800 V.
  • the samples were studied using a FEI Talos F200C TEM, at 200kV maintained at - 180 °C; and images are recorded on a FEI Ceta 16M camera (4k x 4k CMOS sensor) at low dose conditions, to minimize electron beam radiation damage.
  • the measurements were done at the Ilse Katz Institute for Nanoscale Science and Technology (Ben-Gurion University of the Negev).
  • EXAMPLE 1 Evolution and performance of chromosomally integrated nsAA-RS variants
  • MjTyrRS M. janaschii tyrosyl-tRNA synthetase
  • mutants of the MjTyrRS were subjected to 5 or 10 rounds of MAGE -based diversification followed by tolC- mediated (1) negative, (2) positive, and (3) negative selections (colicin El (ColEl) -mediated negative selection, or SDS-mediated positive selections cycles).
  • a GFP fluorescence assay indicated that multi-site pPR incorporation by parent-pPR-RS, expressed from a multi-copy plasmid, in the GRO produced ⁇ 5%, ⁇ 2% and -24.5% of pPR-containing GFP(3TAG), ELP(IOTAG)- GFP and ELP(30TAG)- GFP, respectively, as compared to WT proteins (Fig. 2B).
  • the inventors also compared the efficacy of the parent-pPR-RS, which was integrated into a permissive region in the GRO genome so that the aaRS is expressed from only a single chromosomal copy.
  • Mutl-RS The best-performing variant, Mutl-RS (Fig. 1C), was further evaluated in the production of proteins with three to 30 instances of the uAA in the presence of twofold or fourfold reduced concentrations of pPR (l M mM is typically added to the growth medium; Fig. 4A).
  • ELP(30TAG)-GFP In contrast, production of ELP(30TAG)-GFP resulted in protein losses of ⁇ 40 L % and ⁇ 70 L % in the presence of two- or fourfold reduced pPR concentrations, respectively.
  • our evolved aaRS outperformed the parent synthetase by 20- to 200-fold improved protein yields at all pPR concentrations [except for ELP(30TAG)- GFP, which could not be produced by the parent in these conditions].
  • Detected protein yields were 24.52 ⁇ 1.9 and 54.42 ⁇ 5.7 AA mg/L for ELP(IOTAG)- GFP and ELP(30TAG)-GFP, respectively, when expressed with 1 AA mM pPR in the growth medium (compared with 8.98 ⁇ 0.88 AA mg/L and 14.97 ⁇ 0.85 AA mg/L, respectively, of the equivalent WT proteins).
  • EXAMPLE 3 Evolved pPR-RSs enable rapid and non-toxic protein labeling in vivo
  • Commonly used fluorescent labeling methods include fusion to GFP variants or to self labeling enzymes (e.g., SNAP- and CLIP-tag and self-labeling tags (e.g., tetracysteine tag).
  • self labeling enzymes e.g., SNAP- and CLIP-tag
  • self-labeling tags e.g., tetracysteine tag.
  • these methods are limited, as the large size of the fused proteins (-20-27 kDa) may perturb the cellular localization, structure, or function of the fused protein, while the utilization of small, genetically encoded labeling tags often results in nonspecific staining of the membrane and hydrophobic pockets and thiols in off-target proteins.
  • site- specific pPR incorporation in ELP-fusion proteins enables labeling at multiple, precise positions with minimal changes to the target protein sequence.
  • ELP fusion proteins As scaffolds for fluorophore conjugation sites. ELPs have already been successfully fused to a variety of proteins and typically do not reduce (and can even enhance) protein yields. Herein is shown that they can also enable the conjugation of multiple fluorophore labels while preventing or minimizing perturbation of proper protein folding or function, which can be caused by internal labeling.
  • every third pentapeptide contained an X-guest residue that encoded for pPR, which resulted in a ⁇ 12 kPa ELP protein.
  • ELPs containing natural amino acids or uAAs have previously been designed and utilized for various applications, such as protein purification, hydrogel formation, drug delivery, tumor targeting and tissue engineering.
  • a GFP fluorescence assay indicated that the multi-site incorporation of 1, 2, or 3 by AzoRS, when expressed from a multi-copy plasmid in C321.ARF1, produced up to -96%, 14%, and -4% of EFP(1 TAG)-GFP, EFP(5TAG)- GFP, and EFP(10TAG)-GFP, respectively, as compared with control GFP and EFP proteins, which contained tyrosines incorporated by the wild-type MjTyrRS system (Fig. 9E-G).
  • a modified protein-evolution strategy was used that was previously developed to identify improved MjTyrRS mutants, which can efficiently charge an amber suppressor tRNA with azobenzenes 1, 2, or 3 in C321.ARF1.
  • genomically integrated aaRS variants were subjected to 5-10 rounds of multiplex automated genome engineering (MAGE)-based diversification, using degenerate ssDNA oligonucleotides (Table 2), followed by successive to/C-mediated negative-positive-negative selection cycles (ColEl -mediated negative selection or SDS-mediated positive selection).
  • MAGE multiplex automated genome engineering
  • the first (negative) selection cycle was used to eliminate non-orthogonal variants generated in the diversification process, which, even if rare, would otherwise be enriched in the subsequent positive selection cycle; the second (positive) selection cycle was used to enrich the efficient aaRS variants; and the third (negative) selection cycle was used to eliminate “cheater” non- orthogonal clones generated in response to the stress applied in the positive selection step.
  • the production of GFP(2TAG) in the presence of 1 was used to evaluate activity in genomically integrated individual clones.
  • Several improved variants were identified that, when expressed from a single chromosomal copy, were capable of 14-56-fold higher GFP(2TAG) production compared with the parent enzyme (Fig. 10A).
  • Table 1 GFP and ELP sequences
  • Table 2 Degenerate ssDNA MAGE oligonucleotides
  • Single-stranded DNA oligonucleotides with two phosphorothioate bonds at the 5' end were purchased from Integrated DNA Technologies.
  • the degenerate base n represents all four bases, and k represents G/T.
  • Table 3 Annotations of specific mutations in evolved aaRS variants, as compared with the WT Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS) sequence. In addition to the indicated mutations, all mutants also harbor the R257G and D286R mutations, which have been shown to improve tRNA binding.
  • EXAMPLE 5 ELPs with a UV-light-responsive phase-separation behavior
  • ELPs were selected as hosts for azobenzene incorporation since the hydrophobic azobenzene molecule was expected to dramatically reduce the Tt when incorporated in multiple sites in the ELPs.
  • ELPeoWT ELP6O(2TAG)
  • ELP6O(6TAG) ELP6O(lOTAG)
  • ELPeo(lOTAG) ELPeo(lOTAG)
  • ELP 6 o(lxlO) is the protein product of the ELP6O( 10TAG) gene, wherein 1 was incorporated in 10 encoded TAG codons.
  • the ELP60 protein series was first produced in the C321.ARF1 strain by using AzoRS-4 and azobenzene-uAA 1. To determine protein yields, small batches of ELP 6 o(lx2), ELP 6 o(lx6), and ELP6O(1X10) were purified, and the protein yields were 35.69 ⁇ 3.69, 22.9 ⁇ 1.27, and 24.34 ⁇ 1.69 mg L 1 , respectively, as compared with 39.72 ⁇ 0.68 mg L 1 of ELP 6 o(WT). The accuracy of incorporating 1 was evaluated by intact mass-spectrometry (MS) (Fig.
  • Tables 4-7 Sequence and signal intensities of peptides identified LC-MS of tryptic fragments.
  • X denoted the azobenzene-uAA.
  • Table 4 ELP6O(10X1)MS, expressed by AzoRS in the C321.ARF1 strain.
  • Table 6 ELP6O(10X2)MS, expressed by AzoRS-4 in the C321.ARF1 strain.
  • ELP6O(10X3)MS expressed by AzoRS-4 in the C321.ARF1 strain.
  • the ELPs were irradiated at 365 nm or 405 nm to induce isomerization to the cis (more hydrophilic) or trans (more hydrophobic) configuration, respectively.
  • ELPs bearing mostly the cis isomers exhibited a higher Tt than ELPs bearing mostly the trans isomers.
  • the ATtds/trans induced by the isomerization process increased with the number of incorporated instances of 1, from zero [for the control protein ELP 6 o(tyrosinexlO); Fig. 12] to ⁇ 12 °C for ELPeo(lxlO) (Fig. 13A-C). It is also evident that the Tt of the ELPs decreases with increasing azobenzene content.
  • the secondary structure of light-irradiated ELPs was examined by using circular dichroism (CD) spectroscopy at various temperatures. All ELPs — including the control ELPs — showed the characteristic disordered negative peak at around -190 nm, which decreased in magnitude as the temperature increased (Fig. 13D-F).
  • the magnitude of the negative peak was greater in the control ELP 6 o(tyrosinexlO) than in ELPs containing 1, and it was similar in the control ELP 6 o(benzophenonexlO), which contains an uAA with two aromatic rings, and in ELPeo(lxlO) (Fig. 13J-K).
  • the effect of isomerization was also evident in the CD spectra and increased with increasing numbers of 1 incorporated per ELP chain.
  • ELP polymers were chemically synthesized and polymerized with the sequence [fv(VPGVG), fx(VPGXG)] n , where X represents an azobenzene -bearing amino acid and fv and fx represent the mole-fraction of each pentapeptide.
  • ELP Tt can be manipulated by triggering the cis-trans isomerization of the azobenzene groups, with a ATtcis/u-ans 0.5 or 5 °C for ELPs with a 5% or 15% mole fraction of azobenzene incorporated in the X position, respectively.
  • the molecular weight distribution of these ELPs was not reported and only the VPGVG motif was explored.
  • the incorporation of 1 by the evolved aaRSs disclosed herein enabled the precise production of ELPs bearing various numbers of 1, allowing us to evaluate the effect of increasing azobenzene incorporation on the Tt of the ELP by comparing it across ELPs comprising exactly 60 pentapeptides for each construct.
  • Example 6 Producing ELPs with a visible-light-responsive phase-separation behavior
  • the CD signature of ELP 6 o(2xlO) confirmed its disordered conformation, albeit with smaller variations in the magnitude of the negative peak (around 190 nm) between ELPs bearing mostly cis or mostly trans isomers, as compared with ELP 60 (1X10) (Fig. 14J-L).
  • Azobenzene molecules are known to self-assemble and stimulate the self-assembly of various azobenzene conjugates. Therefore, it was hypothesized that azobenzene molecules also engender ELP self-assembly, which, in turn, may affect the local ELP concentration and, therefore, its Tt. Indeed, even when present as an amino acid side-chain, molecule 1 clearly self- assembled, in both the cis and trans configurations, and in different geometries depending on the isomerization state (Fig. 16A-B).
  • ELPs bearing only two instances of 3 did appear to self-assemble (Fig. 16A-V).
  • ELP 6 o(lxlO), ELP 6 o(2xlO), and ELP 6O (3X10) were imaged by cryo-transmission electron microscopy (cryo-TEM). All azobenzene-ELPs self- assembled into thin sheets, but ELP 6 o(2xlO) and ELP 6 o(3xlO) also formed clusters of aggregates.
  • Example 7 Producing ELP diblock copolymers with a light-responsive self-assembly behavior
  • ELP 6 o(WT)-ELP 6 o(10TAG) An ELP fusion protein, termed ELP 6 o(WT)-ELP 6 o(10TAG), consisting of the gene for ELP 6 o(WT) (the hydrophilic block) fused at the genetic level to the gene for ELP 6 o(10TAG) (the hydrophobic block) was generating, thus setting a 1:1 hydrophilic :hydrophobic block ratio.
  • ELP 6 o(WT)- ELP 6O (1X10) and ELP 6 o(WT)-ELP 6 o(2xlO) were then expressed and their light-responsive self- assembly behavior characterized using UV-vis spectrometry and DLS.
  • the DLS confirmed the formation of self- assembled nanostructures for all proteins and the light-dependent assembly of these structures, with a ATSELF-ASSEMBLY of ⁇ 5 for ELP 6 o(WT)-ELP 6 o(lxlO) and of ⁇ 1 °C for ELP 6 o(WT)- ELP 6O (2X10) (Fig. 18A, colored dots, Fig. 18G).
  • identical azobenzene bearing ELPs i.e., ELP 6 o(lxlO) and ELP 6 o(2xlO)
  • nanostructures of -25 ⁇ 15 nm were the predominant species observed in all proteins, small amounts (-2% by volume at the onset of micelle formation) of larger nanostructures (a several hundred nm) appeared to form as well, and their proportion increased with increasing temperatures (up to -15% or 25% for ELP 6 o(WT)-ELP 6 o(lxlO) and of ⁇ 1 °C for ELP 6 o(WT)- ELP 6O (2X10), respectively, at 40 °C).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Mutant aminoacyl-tRNA synthetase (aaRS) proteins are provided. Nucleic acid molecules encoding the mutant aaRSs, orthogonal translation systems comprising the mutant aaRSs or nucleic acid molecules, cells comprising the orthogonal translation systems, as well as methods of using same are also provided.

Description

MUTANT AMINOACYL-TRNA SYNTHETASES
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of priority to U.S. Provisional Application No. 62/978,895 titled “MUTANT AMINOACYL-TRNA SYNTHETASES” filed February 20, 2020, the contents of which are incorporate herein by reference in their entirety.
FIELD OF INVENTION
[002] The present invention is in the field of artificial amino acid incorporation.
BACKGROUND OF THE INVENTION
[003] Site-specific modification of proteins is a powerful means for investigation and manipulation of the properties of proteins, and has been utilized for a variety of applications, such as fluorescent labeling, analysis of structure and functions, and manipulation of the chemical, biological, and pharmacological properties of target molecules. Beyond single-site modifications, multi-site modifications have been demonstrated to extend and further exploit the potential of such applications, for example for direct polymerization of target proteins, site- specific conjugation of single protein to multiple ligands, and increased performance in analytical chemistry assays.
[004] Traditional methods for protein modification via the chemically reactive cysteine and lysine residues often result in heterogenous product, may disturb the proper folding and function of the modified protein, or may require additional mutations to the protein to achieve single-site modification. In contrast, site-specific introduction of small bio-orthogonal groups to proteins followed by a chemo- selective reaction, does not require the introduction of additional mutations, enables site-selection to minimize interference with the folding and function of the protein, and results in homogenously modified product.
[005] One of the most commonly utilized bio-orthogonal reactions is the copper catalyzed azide-alkyne cycloaddition (CuAAC), which has been employed in a variety of applications, such as modifying and labeling nucleic acids, viruses and proteins both in-vitro and in-vivo. In addition to bio-orthogonality, reactions of this family are rapid, regioselective, and result in high yields of the conjugated product. However, CuAAC bioconjugation application in living systems is hindered due to the Cu(I)-induced generation of reactive oxygen species (ROS) with consequent cellular toxicity. Several methodologies have addressed this limitation by using different ligands to reduce the Cu-mediated toxicity to cells, by utilizing an azide probe that contains an internal copper-chelating moiety, or by using strained alkynes which obviate the need for copper catalysts. However, unlike azides and alkynes, the azide probes with internal copper chelating moiety and strained alkynes are large structures, therefore they may interfere with the function of the labeled protein. In addition, the strain promoted azide-alkyne cycloaddition (SPAAC) is a much slower reaction than the CuAAC and is not strictly bio-orthogonal and regioselective.
[006] As a pre-requisite for site-specific conjugation via CuAAC, an alkyne or azide group must be site-specifically incorporated into the protein. This can be achieved using several methodologies including enzymatic or chemical modification of selected residues (typically post- protein purification), or by incorporation of unnatural amino acids (uAAs) that bear an alkyne or an azide group. Several studies describe the incorporation of such uAAs by substitution of a natural amino acid with a close synthetic analog in auxotrophic strain, which has been used for labeling in various organisms. However, while this method has been applied in various organisms, growth defects due to amino acid substitution, relatively low aminoacylation of the endogenous tRNA with the uAA, compared to the natural amino acid limit the yield of the target protein. In addition, this method replaces all instances of the relevant natural amino acid with the uAA, and therefore is not site specific. Alternatively, uAAs can be incorporated site specifically via codon reassignment or frameshift codons by using orthogonal translation systems (OTSs) consisting of an aminoacyl tRNA synthetase (aaRS), which is able to charge only a cognate tRNA that is not aminoacylated by endogenous aaRSs. Typically, a TAG stop codon (transcribed to UAG during mRNA synthesis) is assigned to the uAA.
[007] Genetic code expansion to integrate light sensitive moieties into proteins. Generally, there are three common classes of light-responsive moieties that are used to impart light -responsive behavior for biopolymers: (1) fusion to a genetically encoded light-responsive protein, however the main drawback is lack of generality, (2) incorporation of an irreversible photolabile group, and (3) incorporation of a reversible photoswitchable group. Among the third group molecules, azobenzenes, a class of molecules known to undergo reversible light-based isomerization, are the most robust and commonly used photoswitches. Upon irradiation with light of the appropriate wavelength (Atrans cis), the azobenzene molecule undergoes a dramatic switch from the trans to the cis configuration (shortening by at least ~3.5 A), with a concomitant change from a hydrophobic to a hydrophilic (polar) molecule (~3 Debyes). Importantly, this process is reversible, and with time or upon irradiation with a second, different, wavelength within the blue light range (Acis— Trans), the azobenzene molecule relaxes back to the trans configuration.
[008] Incorporation of azobenzene into a polypeptide chain can be mediated by incorporation of azobenzene-containing non-standard amino acid (nsAA), using expanded genetic code method as is used for the alkyne or azide groups. This expansion has enabled template -based incorporation of >100 nsAAs containing diverse chemical groups including post- translational modifications, photocaged amino acids, bio-orthogonal reactive groups, and spectroscopic labels. However, with respect to light-responsive nsAA only incorporation of a single nsAA into a single protein has ever been successfully achieved.
[009] Several challenges have limited genetic code modification-based integration of nsAA technology to in some applications only one instance, or in others a few instances, of site-specific uAA incorporation per protein. Improved translation systems that can circumvent these limitations and improved nsAA integration technology are greatly needed.
SUMMARY OF THE INVENTION
[010] The present invention provides mutant aminoacyl-tRNA synthetase (aaRS) proteins. Nucleic acid molecules encoding the mutant aaRSs are provided. Orthogonal translation systems comprising the mutant aaRSs or the nucleic acid molecules are provided. Cells comprising the orthogonal translation systems, mutant aaRSs or nucleic acid molecules are provided. Methods of using the mutant aaRSs, nucleic acid molecules, orthogonal translation systems and cells are also provided.
[Oil] According to a first aspect, there is provided a mutant aminoacyl-tRNA synthetase (aaRS) comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; leucine 65 mutated to valine; glutamic acid 107 mutated to alanine; phenylalanine 108 mutated to tyrosine; glutamine 109 mutated to methionine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid; leucine 162 mutated to lysine; leucine 162 mutated to valine; leucine 162 mutated to arginine; leucine 162 mutated to serine; leucine 162 mutated to cysteine; alanine 167 mutated to histidine, alanine 167 mutated to aspartic acid and alanine 167 mutated to tyrosine.
[012] According to some embodiments, the mutant is selected from the group consisting of: a) a mutant comprising tyrosine 32 mutated to leucine, aspartic acid 158 mutated to serine, isoleucine 159 mutated to methionine, leucine 162 mutated to lysine, and alanine 167 mutated to histidine; b) a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to alanine, leucine 162 mutated to glutamic acid, and alanine 167 mutated to histidine; c) a mutant comprising alanine 32 mutated to threonine, leucine 65 mutated to valine, glutamic acid 107 mutated to alanine, phenylalanine 108 mutated to tyrosine, glutamine 109 mutated to methionine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to cysteine, leucine 162 mutated to arginine, and alanine 167 mutated to aspartic acid; d) a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to methionine, leucine 162 mutated to serine, and alanine 167 mutated to histidine; and e) a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, alanine 162 mutated to cysteine, and alanine 167 mutated to tyrosine.
[013] According to some embodiments, the mutant aaRS comprises an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. [014] According to another aspect, there is provided a mutant aminoacyl-tRNA synthetase (aaRS) comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; leucine 162 mutated to arginine; and alanine 167 mutated to phenylalanine.
[015] According to some embodiments, the mutant aaRS of the invention comprises: a) aspartic acid 158 mutated to glycine; b) isoleucine 159 mutated to tyrosine; and c) leucine 162 mutated to serine or leucine 162 mutated to arginine.
[016] According to some embodiments, the mutant aaRS of the invention further comprises alanine 167 mutated to phenylalanine.
[017] According to some embodiments, the mutant aaRS of the invention further comprises tyrosine 32 mutated to leucine or tyrosine 32 mutated to glycine.
[018] According to some embodiments, the mutant aaRS of the invention further comprises leucine 65 mutated to valine or leucine 65 mutated to glycine.
[019] According to some embodiments, the mutant is selected from the group consisting of: a) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; b) a mutant comprising tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; c) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; d) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to asparagine, phenylalanine 108 mutated to valine, glutamine 109 mutated to leucine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; e) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to aspartic acid, aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; f) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; g) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; and leucine 162 mutated to arginine; and h) a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to glycine; glutamic acid 107 mutated to aspartic acid, phenylalanine 108 mutated to arginine, glutamine 109 mutated to methionine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
[020] According to some embodiments, the mutant comprises an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. [021] According to some embodiments, the amino acid sequence of an aaRS is SEQ ID NO: 1.
[022] According to some embodiments, mutant aaRS of the invention further comprises a mutation of arginine 257 to glycine, a mutation of aspartic acid 286 to arginine or both.
[023] According to another aspect, there is provided a nucleic acid molecule comprising a coding region encoding a mutant aaRS of the invention.
[024] According to some embodiments, the coding region comprises a nucleic acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
[025] According to some embodiments, the coding region is operably linked to at least one regulatory element configured to express the coding region in a target cell.
[026] According to another aspect, there is provided an orthogonal translation system, comprising, a) a mutant aaRS of the invention, or a nucleic acid molecule of the invention, and b) an orthogonal tRNA compatible with the mutant aaRS and comprising an anticodon that corresponds to a stop codon.
[027] According to some embodiments, orthogonal translation system of the invention further comprises a non-standard amino acid (nsAA) recognized by the mutant aaRS.
[028] According to some embodiments, the nsAA is an unnatural amino acid (uAA).
[029] According to some embodiments, the uAA comprises a biorthogonal chemical moiety.
[030] According to some embodiments, the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azide or an alkyne group.
[031] According to some embodiments, the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azobenzene group.
[032] According to some embodiments, the nsAA is a modified phenylalanine. [033] According to some embodiments, the modified phenylalanine is 4-propargyloxy-L- phenylalanine (pPR).
[034] According to some embodiments, the uAA comprising an azobenzene group is selected from phenylalanine-4’ -azobenzene (AzoPhe). tri-fluorinated azobenzene (Azo3F), and tetra- ortho-fluorinated azobenzene (Azo4F) amino acids.
[035] According to some embodiments, the stop codon is a TAG stop codon.
[036] According to another aspect, there is provided a cell comprising an orthogonal translation system of the invention.
[037] According to some embodiments, the cell of the invention further comprises an expression vector comprising an open reading frame (ORF) comprising at least one of the stop codons within the open reading frame.
[038] According to some embodiments, the ORF comprises a plurality of stop codons.
[039] According to some embodiments, the ORF comprises at least 10 stop codons.
[040] According to some embodiments, the ORF is operatively linked to at least one regulatory element capable of inducing expression of the ORF within the cell.
[041] According to some embodiments, the cell is devoid of native TAG stop codons and does not express release factor 1 (RF1).
[042] According to some embodiments, the cell comprises RF1 and at least one native TAG stop codon.
[043] According to another aspect, there is provided a method of producing a protein comprising a nsAA, the method comprising introducing into a cell an expression vector comprising an open reading frame encoding the protein wherein the open reading frame comprises a stop codon, wherein the cell comprises an orthogonal translation system of the invention. [044] According to some embodiments, the method of the invention is for labeling the protein, and the method further comprises converting the nsAA into a detectably labeled amino acid and wherein the mutant aaRS is the mutant aaRS of the invention.
[045] According to some embodiments, the converting comprises addition of a detectable moiety by Click chemistry.
[046] According to some embodiments, the method of the invention is for producing a light- responsive protein, wherein the mutant aaRS is the mutant aaRS of the invention.
[047] According to another aspect, there is provided a protein comprising a nsAA produced by a method of the invention.
[048] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
[049] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[050] Figures 1A-C: (1A) A table depicting amino acid substitutions present in mutant aminoacyl tRNA synthetases capable of incorporating alkyne-containing non-standard amino acids. The mutation sites are with respect to a M. janaschii tyrosyl-tRNA synthetase. (IB) A table depicting amino acid substitutions present in mutant aminoacyl tRNA synthetases capable of incorporating azobenzene-containing non-standard amino acids. The mutation sites are with respect to a wild-type M. janaschii tyrosyl-tRNA synthetase. (1C) Production of GFP(3TAG) by chromosomally integrated parent and evolved aaRS variants in E. coli strain C321.ARF1.
[051] Figures 2A-Z: Multi-site incorporation of pPR by the parent translation systems and evolution of chromosomally integrated pPR-RS variants. (2A) Schematic illustration of reporter proteins for incorporation of 3, 10 and 30 unnatural amino acids (uAAs) and equivalent control wild-type (WT) proteins. (2B) GFP expression from WT GFP reporter, or from GFP(3TAG) EFP(10TAG)-GFP or EFP(30TAG)-GFP reporters produced by the parent pPR-RS, expressed by plasmid (P) or genomic integration (G). Red bars indicate addition of uAA (pPR), blue bars indicate no addition of uAA (pPR). (2C-E) Incorporation of (2C) 3, (2D) 10 and (2E) 30 pPRs in a single protein by evolved aaRS variants, expressed on a plasmid in C321.ARF1. *LR<0.05, ***LR<0.0005, and ****LR<0.0001 indicate comparison of each evolved variant with the parent pPR-RS (2C-D) or with the wild-type protein (2E). n= 3; error bars indicate S.D. (2F) Production of EFP(10TAG)-GFP by Mutl-RS and previously evolved aaRS variants, in the absence (red bars) and the presence of the respective uAAs, using the C321.ARF1 strain. (pPR for Mutl-RS, pAcF for pAcF-RS.2.tl, pAzF for pAzFRS.2.tl). n=3; error bars indicate s.d. (2G-N) Time-course kinetic analysis of GFP(3TAG) production by Mutl-RS and Mut2-RS expressed from multi-copy plasmids. (2G) production by Mutl-RS in C321.ARF1 and 2xYT media, (2H) production by Mut2-RS in C321.ARF1 and 2xYT media, (21) production by Mutl-RS in C321.ARF1 and minimal media (MM), (2J) production by Mut2-RS in C321.ARF1 and minimal media, (2K) production by Mutl-RS in BF21 and 2xYT media, (2L) production by Mut2-RS in BF21 and 2xYT media, (2M) production by Mutl-RS in BF21 and minimal media, (2N) production by Mut2-RS in BF21 and minimal media. (20- V) Time-course kinetic analysis of EFP(10TAG)-GFP production by Mutl-RS and Mut2-RS expressed from multi-copy plasmids. (20) production by Mutl-RS in C321.ARF1 and 2xYT media, (2P) production by Mut2-RS in C321.ARF1 and 2xYT media, (2Q) production by Mutl-RS in C321.ARF1 and minimal media, (2R) production by Mut2-RS in C321.ARF1 and minimal media, (2S) production by Mutl-RS in BF21 and 2xYT media, (2T) production by Mut2-RS in BF21 and 2xYT media, (2U) production by Mutl-RS in BF21 and minimal media, (2V) production by Mut2-RS in BF21 and minimal media. (2W-Z) Time-course kinetic analysis of EFP(30TAG)-GFP production by Mutl-RS and Mut2-RS expressed from multi copy plasmids. (2W) production by Mutl-RS in C321.ARF1 and 2xYT media, (2X) production by Mut2-RS in C321 ARFl and 2xYT media, (2Y) production by Mutl-RS in C321 ARFl and minimal media, (2Z) production by Mut2-RS in C321 ARFl and minimal media. Fluorescence signals were normalized by dividing the fluorescence counts by the final OD600 reading n = 3. Note: error bars indicating s.d. may be too small to be visible.
[052] Figures 3A-D: MALDI-TOF analysis of WT ELP(10Tyrosine)-GFP protein expressed in (3A) BL21 and (3B) C321.ARF1, and ELP(10pPR)-GFP protein expressed by Mutl-RS in (3C) BL21 and (3D) C321.ARF1, respectively.
[053] Figures 4A-B: (4A) Evaluation of pPR incorporation in the presence of standard (1 mM), twofold (0.5 mM) and fourfold (0.25 mM) reduced pPR concentrations. (4B) Multisite incorporation of pPR by Mutl-RS compared with the parent pPR-RS, in the BL21 and C321.ARF1 strains. n= 3; error bars indicate S.D.
[054] Figures 5A-E: (5A) In-gel fluorescence analysis of purified ELPs containing 1 or 10 instances of pPR conjugated to TAMRA-azide at various protein concentrations, namely: (1)
30 mM; (2) 6 pM; (3) 3 pM; (4) 1.5 pM; (5) 0.6 pM; (6) 0.33 pM; (7) 0.165 pM. (5B-E) TAMRA labeling of C321.ARF1 cells expressing (5B) ELP(lpPR) by the parentpPR-RS; (5C) ELP(lpPR) by Mutl-RS; (5D) ELP(lOpPR) b the parent pPR-RS and (5E) ELP(lOpPR) by the Mutl-RS. Percentage of labeled cells was calculated using ImageJ and is given for each image.
[055] Figures 6A-F : Conjugation of multiple fluorophores to ELPs in bacteria. (6A) Conjugation of ELP(ITAG) and ELP(IOTAG), produced by either the parent pPR-RS (P) or evolved Mutl-RS (E) in C321.ARF1. Conjugation of (6B) ELP(ITAG) and (6C) ELP(IOTAG) produced by either the parent pPR-RS or Mutl-RS in BL21 compared to C321.ARF1. 1 : parent-pPR-RS, BL21; 2: Mutl-RS, BL21; 3: parent pPR-RS, C321.ARF1; 4: Mutl-RS, C321.ARF1. (6D) In-vitro TAMRA labeling of cell lysates containing ELP(lOpPR) expressed in BL21 strain by the (1) the parent pPR-RS or (2) Mutl-RS, or expressed in the C321.ARF1 strain by (3) parent pPR-RS or (4) Mutl-RS. (6E-F) Labeling of the OTS by conjugation of pPR to TAMRA. (6E) In-vivo and (6F) in-vitro fluorescent labeling of C321. (l)ARFl cells harboring the Mutl-RS plasmid, (2) ARFl cells harboring the Mutl-RS plasmid, with induction of the aaRS, and (3) ARFlcells harboring both the Mutl-RS and ELP(lOpPR) plasmids. A double-band (indicated by a red arrow) was detected when OTS was induced. [056] Figures 7A-B: (7A) Labeling efficiency of cells harboring ELP(lOpPR) expressed by Mutl-RS in the presence of reduced copper concentrations. n=3; values are means ± s.d. (7B) Growth curves of C321.ARF1 cells following an in-vivo click reaction n = 3; values are means ± s.d.
[057] Figures 8A-D: Incorporation of phenylalanine-4'-azobenzene (AzoPhe) in expressed proteins. (8A) GFP expression in GRO from ELP(10Tyr or 30Tyr)-GFP reporters, or from ELP(1TAG, 5TAG, 10TAG or 30TAG)-GFP reporters produced by literary (L) or Mut7 aaRS. Red bars indicate addition of uAA (AzoPhe), grey bars indicate no addition of uAA. error bars; mean ± standard error. *P< 0.01 indicates comparison of literary aaRS with the evolved. #P< 0.01 indicates comparison of evolved aaRS (lOAzo) with the endogenous (lOTyr). (8B) Multi-site-specific incorporation of AzoPhe into E2(10TAG). Mass-spectrometry results (MALDI-TOF) of (top) E2 with 10 TAG, expressed with episomal evolved aaRS, in presence of AzoPhe, compared with (bottom) ELP (lOTyr). (8C). MALDI (z=2, z=3) spectra of purified proteins expressed in the GRO. ELP(Tyr)-GFP (Left), ELP(10pPR)-GFP (Right) The deviation from the theoretical MW is indicated. (8D). MALDI (z=2, z=3) spectra of purified proteins expressed in BL21 ELP(Tyr)-GFP (Left), ELP(10pPR)-GFP (Right) The deviation from the theoretical MW is indicated.
[058] Figures 9A-G: (9A) Illustration of the reversible trans-to-cis isomerization of an azobenzene molecule. (9B) Illustrations and properties of azobenzene -uAAs 1, 2, and 3. (9C) Illustration of the mechanism for altering the Tt of the ELP by azobenzene isomerization. A change in the transition temperature by cis/trans isomerization generates a “window” in which isothermal (e.g., at T*), light-mediated change in ELP solubility can be achieved. (9D) Schematic illustration of reporter proteins for the incorporation of either 2 (GFP) or 1, 5, or 10 (ELP-GFP) uAAs at TAG codons. (9E-G) Incorporation of either (9E) 1, (9F) 5, or (9G) 10 instances of azobenzene-uAAs 1, 2, or 3 in a single ELP by the previously described AzoRS, expressed from a multi-copy plasmid. Error bars indicate SD ( n = 3).
[059] Figures 10A-D: (10A) Production of GFP(2TAG) by the previously described AzoRS and four evolved variants, expressed from a single chromosomal copy. (10B-D) Production of
ELP-GFP fusion proteins containing either (10B) 1, (IOC) 5, or (10D) 10 instances of the azobenzene-uAAs depicted in 10B and expressed by episomal versions of the previously described AzoRS, our evolved variants (AzoRS 1-4), or MjTyrRS (producing tyrosine- containing control ELPs) in the C321.ARF l strain. The level of GFP fluorescence indicates the production of the ELP-GFP fusion and, therefore, the efficiency of sAA incorporation.
*P<0.05, **P<0.001, ***P<0.0005, ****P<0.0001 indicate comparison of each evolved variant with the AzoRS. n = 3; error bars indicate s.d.
[060] Figure 11: MALDI-TOF analysis of ELP60(WT) [expected: 22,760.4, found: 22726.03], ELP60(2xl) [expected: 23,148.87, found: 23083.17], ELP60(6xl) [expected: 23,841.65, found: 23793.87], and ELP60(10xl) [expected: 24,562.47 found: 24519.49].
[061] Figure 12: Turbidity profile, as a function of temperature and light irradiation for ELP6o(tyrosinexlO), 25 mM solution in water.
[062] Figure 13A-R: Characterization of the light-responsive properties of ELPs containing multiple instances of azobenzene -uAA 1. (13A-C) Turbidity profiles as a function of temperature and light irradiation for ELPs (25 mM solutions in water) containing either (13A) 2 (supplemented with 1 M NaCl), (13B) 6, or (13C) 10 instances of 1. (13D-F) CD spectra of light-irradiated ELPs (7.5 pM solutions in water) containing either (13D) 2, (13E) 6, or (13F)
10 instances of 1 at 10 °C or 30 °C. (13G-H) Turbidity profiles as a function of the duration of irradiation with either (13G) UV or (13H) blue-light for ELPs containing 10 instances of 1 (25 pM solutions in water). (131) Reversibility of the light-mediated transition (600 nm), and of azobenzene isomerization (325 nm), over multiple cycles of 30 s illumination of ELPs (25 pM solutions in water) containing 10 instances of azobenzene-uAA 1 at 26 °C. (13J-K) CD spectra of (13J) ELP60(tyrosinexl0) and (13K) ELP60(benzophenonexl0), both as 7.5 pM solutions in water, tested at 10 °C or 30 °C. (13L-N) Turbidity profiles as a function of temperature and light irradiation for ELP60(lxl0) at concentrations of (13L) 12.5 pM, (13M) 25 pM, or (13N) 50 pM (C) in water. (130) Turbidity profiles (heating and cooling) of light-irradiated ELP60(lxlO), 25 pM in water. (13P-R) UV-vis spectra of light-irradiated ELPs containing 10 instances of azobenzene-uAA (13P) 1, (13Q) 2, or (13R) 3. Insets show the red-shifted band separation for azobenzene-uAAs 2 and 3.
[063] Figures 14A-L: Characterization of the light-responsive properties of ELP containing multiple instances of azobenzene-uAA 2 (25 pM solutions in water, unless otherwise indicated). (14A-C) Turbidity profiles as a function of temperature and light irradiation for ELPs containing either (14A) 2 (supplemented with 1 M NaCl), (14B) 6, or (14C) 10 instances of 2. (14D-E) Turbidity profiles as a function of the duration of irradiation with either (14D) blue or (14E) green light for ELPs containing 10 instances of 2. (14F) Reversibility of the light- mediated transition (600 nm), and azobenzene isomerization (340 nm), over (first) five cycles of 5 min and then 5 cycles of 30 s illumination of ELPs containing 10 instances of azobenzene - uAA 2 at 24 °C. (14G-I) Turbidity profiles as a function of temperature and light irradiation for ELP60(2X10) at concentrations of (14G) 12.5 mM, (14H) 25 pM, or (141) 50 pM in water. (14J- L) Comparison of the CD spectra of (14J) ELP60(lxlO), (14K) ELP60(2xl0), and (14L) ELP60(3xl0), all 7.5 pM solutions in water, as a function of light irradiation at 10°C or 30°C.
[064] Figure 15: Turbidity profile as a function of temperature and light irradiation for ELP60(3X10) at concentration of 12.5 pM.
[065] Figures 16A-16V: (16A-B) Cryo-TEM images of self-assembled molecules of 1 isomerized to the (16A) trans or (16B) cis conformations. (16C-J) Dynamic light scattering analysis of ELPs containing (16C) 10 instances of tyrosine, (16D) 10 instances of a benzophenone -bearing uAA, (16E) 2 instances of 1, irradiated with blue light, (16F) 2 instances of 1, irradiated with UV light, (16G) 6 instances of 1, irradiated with blue light, (16H) 6 instances of 1, irradiated with UV light, (161) 10 instances of 1, irradiated with blue light, (16J) 10 instances of 1, irradiated with UV light. (16K-P) Dynamic light scattering analysis of ELPs containing (16K) 2 instances of 2, irradiated with blue light, (16L) 2 instances of 2, irradiated with green light, (16M) 6 instances of 2, irradiated with blue light, (16N) 6 instances of 2, irradiated with green light, (160) 10 instances of 2, irradiated with blue light, (16P) 10 instances of 2, irradiated with green light. (16Q-V) Dynamic light scattering analysis of ELPs containing (16Q) 2 instances of 3, irradiated with blue light, (16R) 2 instances of 3, irradiated with green light, (16S) 6 instances of 3, irradiated with blue light, (16T) 6 instances of 3, irradiated with green light, (16U) 10 instances of 3, irradiated with blue light, (16V) 10 instances of 3, irradiated with green light.
[066] Figures 17A-F: Cryo-TEM images of the self-assembly of ELPs containing 10 instances of either 1 irradiated with (17A) blue or (17B) uv light, 2 irradiated with (17C) blue or (17D) green light, or 3 irradiated with (17E) blue or (17F) green light.
[067] Figures 18A-N: Characterization of the self-assembly of diblock ELPs as a function of temperature and azobenzene isomerization. (18A) Turbidity profiles (solid lines) as a function of temperature and light irradiation for ELP6o(WT)-ELP6o(lxlO); dots indicate particle size, as determined by DLS. (18B) Reversibility of the light-mediated self-assembly of ELP6o(WT)- ELP6O(1X10), over ten cycles of 30 s illumination (25 mM solutions in water) at 25 °C. (18C-F) Cryo-TEM images of (18C-D) blue- or (18E-F) UV- light irradiated ELP6o(WT)-ELP6o(lxlO). (18G) Turbidity profiles (solid lines) as a function of temperature and light irradiation for ELP60(WT)-ELP60(2xl0); dots indicate particle size, as determined by DLS. (18H) Turbidity profiles (solid lines) as a function of temperature and light irradiation for ELP60(WT)- ELP60(2xl0); dots indicate particle size, as determined by DLS. (18I-L) Cryo-TEM images of the self-assembly of ELP60(WT)-ELP60(2xl0 irradiated with (181- J) blue or (18K-L) green light. (18M-N) Dynamic light scattering analysis of (18M) ELP60(WT)-ELP60(lxl0) and (18N) ELP60(WT)-ELP60(2xl0) as a function of light-irradiation, analyzed at 38 °C.
[068] Figures 19A-D: Kinetic analysis of GFP production by aaRS variants expressed on plasmids. Time course analysis of (19A) GFP(3TAG) expression by Mutl-RS, (19B) GFP(3TAG) expression by Mut2-RS, (19C) ELP(10TAG)-GFP expression by Mutl-RS and (19D) ELP(10TAG)-GFP expression by Mut2-RS, expressed on multi-copy plasmids in the presence of pPR or with no uAA. n=3; Error bars, mean ± s.d.
[069] Figure 20: Post-purification fluorescent labeling of ELPs. In-gel fluorescence analysis of ELPs containing 1 or 10 instances of pPR, conjugated to TAMRA-azide, at varied protein concentrations. ELP(lOpPR) (right) shows improved signals and reduced limit of detection for proteins as compared with only a single pPR residue (ELP(lpPR), right).
[070] Figure 21: In vitro TAMRA labeling of ELP(lOpPR) in non-recoded BL21 strain and in the GRO. Proteins were expressed in either BL21 by the (1) parent or (2) Mutl-RS, or in the GRO by (3) parent pPR-RS or (4) Mutl-RS. Typhoon imaging at 532nm.
[071] Figures 22A-B: Staining of the OTS through conjugation of pPR to TAMRA.
(22A) In-vivo, or (22B) in-vitro fluorescent labeling of cells harboring Mutl-RS plasmid (1) without or (2) with induction of the OTS, or (3) cells harboring both Mutl-RS and ELP(lOpPR) plasmids. Double-band (marked by red arrow) is detected when OTS in induced, suggesting these bands represent the aminoacylated aaRS and aminoacylated aaRS-tRNA complex. [072] Figure 23: Expected and experimental molecular weights of ELP(10TAG)-GFP by MALDI-TOF mass spectrometry analysis. Molecular weights (Da) calculated based on doubly charged proteins. pPR-bearing proteins were expressed by Mutl-RS.
[073] Figures. 24A-J: Sequence and signal intensities of peptides identified LC-MS of tryptic fragments. (24A) ELP(10TAG)-GFP MS, expressed by parent pPR-RS in the C321.ARF1 strain. (24B) ELP(10TAG)-GFP MS, expressed by parent pPR-RS in the BL21 strain. (24C) ELP( 10TAG)-GFP MS, expressed by Mutl-RS in the C321.ARF1 using ImM pPR. (24D)
ELP( 10TAG)-GFP MS, expressed by Mutl-RS in the C321.ARF1 using 0.25mM pPR. (24E) ELP(10TAG)-GFP MS, expressed by Mutl-RS in the BL21 E. cob strain using ImM pPR. (24F) ELP(10TAG)-GFP MS, expressed by Mutl-RS in the BL21 E. cob strain using 0.25 mM pPR. (24G) ELP(10TAG)-GFP MS, expressed by Mut2-RS in the C321.ARF1 using ImM pPR. (24H) ELP(30TAG) MS, expressed in the C321.ARF1 by Mutl-RS, using different pPR concentrations. (241) ELP(30TAG)MS, expressed by Mut2-RS in the C321.ARF1, using ImM pPR. (24 J) Identification of Mutl-RS in the fluorescent band following in-vivo click reaction in C321.ARF1 and in-gel trypsin digestion.
[074] Figure 25: Fluorescent quantification of microscopy images.
DETAILED DESCRIPTION OF THE INVENTION
[075] The present invention provides, in some embodiments, mutant aminoacyl-tRNA synthetase (aaRS) proteins. Nucleic acid molecules encoding the mutant aaRSs are also provided, as are orthogonal translation systems comprising the mutant aaRSs or nucleic acid molecules and cells comprising the orthogonal translation system. Methods of use are also provided.
[076] The present invention is based on the surprising development of highly efficient aaRS variants capable of multi-site incorporation of uAAs in a genomically recoded organism (GRO) that lacks all native TAG codons as web as the associated release factor (RF1). Surprisingly some new aaRS variants were even functional in wild-type cells. The toolbox for multi-site and site-selective protein labeling has thus been greatly expanded via evolution of efficient aaRS variants for the multi-site incorporation of the alkyne -bearing uAA, 4-propargyloxy-L- phenylalanine (pPR), azobenzene-bearing phenylalanine-4 ’-azobenzene (AzoPhe), tri- fluorinated azobenzene (Azo3F) and tetra-ortho-fluorinated azobenzene (Azo4F). While OTSs have been previously developed, they are suitable for single-site pPR incorporation per-protein generally. For example previous attempts to incorporate pPR found proteins harboring a single pPR were expressed by the system in only moderate yields (<20% or -42% of wild-type proteins in E.coli cell free protein synthesis, depending on the position for uAA incorporation in the protein). The newly evolved aaRS variants are capable of incorporating up to 10 or 30 instances of the uAA in a single protein, in both the commonly used, non-recoded E. coli strain BL21 and the GRO, respectively. Further, it is shown herein that multi-site incorporation of uAAs in proteins allows rapid, robust, and non-toxic fluorescent labeling of these proteins or generation of light responsive polymers in vivo. Not only that, but there is shown herein the genetic encoding of light-responsive phase transition and self-assembly of a PBB using photo- switchable uAAs. In particular, azobenzene-containing ELPs with a predetermined number of azobenzenes, incorporated at specific positions, were generated. This allowed for manipulation of the transition temperature of the ELP and control of the ELP’s self-assembly and geometry. Finally, light-responsive nanostructures were engineered by incorporating azobenzene-uAAs in the hydrophobic segment of ELP diblock co-polymers.
Mutant aaRS
[077] By a first aspect, the present invention provides a mutant aminoacyl-tRNA synthetase (aaRS).
[078] In some embodiments, the mutant aaRS comprises an amino acid sequence of an aaRS comprising at least one amino acid mutation. In some embodiments, the mutant aaRS comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutations. In some embodiments, the mutant aaRS comprises 2 mutations. In some embodiments, the mutant aaRS comprises 5 mutations.
In some embodiments, the mutant aaRS comprises 6 mutations. In some embodiments, the mutant aaRS comprises 7 mutations. In some embodiments, the mutant aaRS comprises 8 mutations. In some embodiments, the mutant aaRS comprises 9 mutations. In some embodiments, the mutant aaRS comprises 11 mutations.
[079] As used herein, the term “mutation” refers to any mutation such as can be introduced into an amino acid sequence or into a nucleic acid sequence by any method known in the art. In some embodiments, a mutation is a deletion. In some embodiments, is an insertion. In some embodiments, a mutation is a substitution. In some embodiments, a mutation is a conversion of one amino acid to another. In some embodiments, a mutation is a conversion of one nucleotide to another. In some embodiments, a mutation is a conversion of a plurality of nucleotides to other nucleotides. In some embodiments, a mutation introduced into a nucleic acid sequence when translated, results in a mutant amino acid sequence. In some embodiments, the mutation is not a silent mutation.
[080] In some embodiments, the mutation increases the incorporation rate of a non-standard amino acid (nsAA) into a protein. In some embodiments, the mutation increases the rate of recognition of the aaRS of its cognate tRNA. In some embodiments, the mutation increases the rate of recognition of the aaRS of an orthogonal tRNA. In some embodiments, the mutation increases the rate of recognition of an amino acid. In some embodiments, the mutation increases the rate of recognition of the aaRS of its cognate amino acid. In some embodiments, the mutation increases the rate of recognition of the aaRS of an orthogonal amino acid.
[081] In some embodiments, the amino acid is a non-standard amino acid (nsAA). In some embodiments, the nsAA is an unnatural amino acid (uAA). In some embodiments, a nsAA is a uAA. In some embodiments, the amino acid is an orthogonal amino acid. In some embodiments, the amino acid is a non-naturally occurring amino acid. In some embodiments, the amino acid is a man-made amino acid. The term “unnatural amino acid” as used herein refers to any amino acid that is not genetically encoded for in an organism. The term “unnatural amino acid” as used herein refers to an amino acid that that is not inherently present within the organism. This refers to any amino acid other than 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. Examples of unnatural amino acids are common in the art.
[082] Methods of generating mutations are well-described in the art and include, but are not limited to, site-directed mutagenesis, nucleotide excision, nucleotide addition, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), multiplexed automated genome engineering (MAGE) and polymerase chain reaction (PCR) with mutation generating primers or probes. [083] Aminoacyl-tRNA synthetase is a well-known protein that catalyzes the attachment of amino acids to the 3’ end of their cognate tRNAs. In some embodiments, the aaRS is an archaeal aaRS. In some embodiments, the aaRS is a Methanocaldococcus jannaschii (Mj) protein. In some embodiments, the aaRS is a Mj aaRS. Mj is also known as Methanococcus jannaschii. In some embodiments, the aaRS recognizes a tRNA molecule. In some embodiments, the aaRS transfers an amino acid to the tRNA molecule. In some embodiments, the aaRS transfers an amino acid to the tRNA molecule. In some embodiments, the aaRS transfers an amino acid derived molecule to the tRNA molecule. In some embodiments, the aaRS is an orthogonal aaRS (o-aaRS). In some embodiments, the aaRS is a uAA-specific o- aaRS. As used herein the term “uAA-specific o-aaRS” refers to an orthogonal amino-acyl - tRNA synthetase that recognizes only the uAA and the tRNA of the system or cell of the invention.
[084] In some embodiments, the amino acid derived molecule is a non-standard amino acid (nsAA). In some embodiments, the nsAA is an unnatural amino acid (uAA). In some embodiments, the uAA is a D amino acid or an L amino acid. In some embodiments, the uAA is a D amino acid. In some embodiments, the uAA is an L amino acid. In some embodiments, the uAA is an azide- or an alkyne-containing amino acid. In some embodiments, the uAA is an azide containing amino acid. In some embodiments, the uAA is an alkyne containing amino acid. In some embodiments, the uAA is an azobenzene-containing amino acid. In some embodiments, the uAA is a modified phenylalanine. In some embodiments, the modified phenylalanine is 4-propargyloxy-L-phenylalanine (pPR). In some embodiments, the modified phenylalanine is phenylalanine-4 ’-azobenzene (AzoPhe). In some embodiments, the azobenzene-containing amino acid is AzoPhe or tri-fluorinated azobenzene (Azo3F). In some embodiments, the azobenzene-containing amino acid is AzoPhe, Azo3F or tetra-ortho- fluorinated azobenzene (Azo4F). In some embodiments, the azobenzene-containing amino acid is AzoPhe. In some embodiments, the azobenzene-containing amino acid is Azo3F. In some embodiments, the azobenzene-containing amino acid is Azo4F. In some embodiments, the aaRS transfers 4-propargyloxy-L-phenylalanine (pPR) to the tRNA molecule. In some embodiments, the aaRS transfers phenylalanine-4’ -azobenzene (AzoPhe), tri-fluorinated azobenzene (Azo3F) or tetra-ortho-fluorinated azobenzene (Azo4F) to the tRNA molecule. In some embodiments, the aaRS transfers phenylalanine-4 ’-azobenzene (AzoPhe) to the tRNA molecule. In some embodiments, the aaRS transfers tri-fluorinated azobenzene (Azo3F) to the tRNA molecule. In some embodiments, the aaRS transfers tetra-ortho-fluorinated azobenzene (Azo4F) to the tRNA molecule.
[085] In some embodiments, the tRNA molecule is an orthogonal tRNA (o-tRNA). In some embodiments, the tRNA molecule comprises a stop anticodon. In some embodiments the tRNA molecule comprises an amber anticodon. In some embodiments, the aaRS does not recognize a canonical tRNA in a cell. In some embodiments, the canonical tRNA comprises an anticodon with complementarity to a tyrosine codon. In some embodiments, the cell is a target cell. In some embodiments, the cell is a cell comprising the mutant aaRs. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is an Escherichia coli cell. In some embodiments, the cell is selected from a bacterium, an Escherichia coli cell, a eukaryotic cell, a yeast cell a fungal cell, a plant cell, an animal cell.
[086] The term "orthogonal" as used herein refers to molecules (e.g.,
"orthogonal tRNA synthetase" and "orthogonal tRNA" pairs) that can process information in parallel with wild-type molecules (e.g., tRNA synthetases and tRNAs), but that do not engage in crosstalk with the wild-type molecules of a cell. As a non-limiting example, the orthogonal tRNA synthetase preferentially aminoacylates a complementary orthogonal tRNA (O-tRNA), but no other cellular tRNAs, with a non-canonical amino acid (e.g., Propargyl-l-Lysine), and the orthogonal tRNA is a substrate for the orthogonal synthetase but is not substantially aminoacylated by any endogenous tRNA synthetases. In some embodiments, orthogonal is with respect to a target cell. In some embodiments, the target cell is a cell of the invention.
[087] In the context of tRNAs and aminoacyl-tRNA synthetases, the term "orthogonal" refers to an inability or reduced efficiency, e.g., less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency, of an O-tRNA to function with an endogenous tRNA synthetase (RS) compared to an endogenous tRNA to function with the endogenous tRNA synthetase, or of O-tRNA synthetase (O-RS) to function with an endogenous tRNA compared to an endogenous tRNA synthetase to function with the endogenous tRNA. For a non-limiting example, an O-tRNA in a cell is aminoacylated by any endogenous RS of the cell with reduced or even zero efficiency, when compared to aminoacylation of an endogenous tRNA by the endogenous RS. In another non-limiting example, an O-tRNA synthetase aminoacylates any endogenous tRNA a cell of interest with reduced or even zero efficiency, as compared to aminoacylation of the endogenous tRNA by an endogenous RS.
[088] In some embodiments, the O-tRNA anticodon loop recognizes a codon, which is not recognized by endogenous tRNAs, on the mRNA and incorporates the UAA at this site in the polypeptide, details of which are further described, for example, in U.S. Pat. No.
2006/0160175, which is hereby incorporated by reference in its entirety. For a non-limiting example, the unique codon may include nonsense codons, such as, stop codons, four or more base codons, rare codons, codons derived from natural or unnatural base pairs and/or the like.
In some embodiments, the unique codon is the TAG stop codon.
[089] As used herein, aaRS recognition of a tRNA molecule refers to the association of an aaRS with a specific tRNA molecule including but not limited to contact at the anticodon or the acceptor stem of the tRNA molecule. As used herein, transfer to a tRNA molecule, refers to the process by which an amino acid or an amino acid derived molecule is associated with an aaRS or a mutant aaRS and moved onto the 3 ’-hydroxyl group on the CCA tail of the tRNA molecule. The process is also referred to in the art as “charging the tRNA molecule”.
[090] As used herein, the term “canonical” describes an endogenous molecule that is present in a cell without any transgenic manipulation to the cell or to the progenitors of the cell.
[091] In some embodiments, the aaRS into which the mutation is introduced comprises or consists of the amino acid sequence
MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMEQRKI HMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 1) or a sequence with at least 95% identity thereto. In some embodiments, an amino acid sequence of Mj aaRS consists of SEQ ID NO: 1. In some embodiments, an amino acid sequence of wild-type aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto. In some embodiments, an amino acid sequence of aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto. In some embodiments, the an amino acid sequence of a non-mutant aaRS comprises or consists of SEQ ID NO: 1 or a sequence with 95% identity thereto. In some embodiments, the amino acid numbering provided herein is with respect to the sequence of SEQ ID NO: 1. In some embodiments, SEQ ID NO: 1 comprises a wildtype sequence for an aaRS and the isolated peptide is a mutant aaRS.
[092] In some embodiments, the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; leucine 65 mutated to valine; glutamic acid 107 mutated to alanine; phenylalanine 108 mutated to tyrosine; glutamine 109 mutated to methionine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid; leucine 162 mutated to lysine; leucine 162 mutated to valine; leucine 162 mutated to arginine; leucine 162 mutated to serine; leucine 162 mutated to cysteine; alanine 167 mutated to histidine, alanine 167 mutated to aspartic acid and alanine 167 mutated to tyrosine.
[093] In some embodiments the mutation is tyrosine 32 mutated to leucine, or threonine. In some embodiments the mutation is tyrosine 32 mutated to leucine. In some embodiments the mutation is tyrosine 32 mutated to threonine. In some embodiments the mutation is leucine 65 mutated to valine. In some embodiments, the mutation is glutamic acid 107 mutated to alanine. In some embodiments, the mutation is phenylalanine 108 mutated to tyrosine. In some embodiments, the mutation is glutamine 109 mutated to methionine. In some embodiments, the mutation is aspartic acid 158 mutated to serine, or glycine. In some embodiments, the mutation is aspartic acid 158 mutated to serine. In some embodiments, the mutation is aspartic acid 158 mutated to glycine. In some embodiments, the mutation is isoleucine 159 mutated to alanine, methionine, cysteine, or tyrosine. In some embodiments, the mutation is isoleucine 159 mutated to alanine. In some embodiments, the mutation is isoleucine 159 mutated to methionine. In some embodiments, the mutation is isoleucine 159 mutated to cysteine. In some embodiments, the mutation is isoleucine 159 mutated to tyrosine. In some embodiments, the mutation is leucine 162 mutated to glutamic acid, lysine, valine, arginine, serine or cysteine. In some embodiments, the mutation is leucine 162 mutated to glutamic acid. In some embodiments, the mutation is leucine 162 mutated to lysine. In some embodiments, the mutation is leucine 162 mutated to valine. In some embodiments, the mutation is leucine 162 mutated to arginine. In some embodiments, the mutation is leucine 162 mutated to serine. In some embodiments, the mutation is leucine 162 mutated to cysteine. In some embodiments, the mutation is alanine 167 mutated to histidine, aspartic acid or tyrosine. In some embodiments, the mutation is alanine 167 mutated to histidine. In some embodiments, the mutation is alanine 167 mutated to aspartic acid. In some embodiments, the mutation is alanine 167 mutated to tyrosine. It will be understood by a skilled artisan that any combination of the above recited mutations is envisioned and may be present in the mutant aaRS of the invention.
[094] In some embodiments, the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; leucine 162 mutated to arginine; and alanine 167 mutated to phenylalanine.
[095] In some embodiments, the mutation is selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to alanine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to tyrosine; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid; leucine 162 mutated to lysine; leucine 162 mutated to valine; leucine 162 mutated to arginine; leucine 162 mutated to serine; leucine 162 mutated to cysteine; alanine 167 mutated to histidine, alanine 167 mutated to aspartic acid, alanine 167 mutated to phenylalanine and alanine 167 mutated to tyrosine.
[096] In some embodiments, the mutation is tyrosine 32 mutated to leucine or glycine. In some embodiments, the mutation is tyrosine 32 mutated to leucine. In some embodiments, the mutation is tyrosine 32 mutated to glycine. In some embodiments, the mutation is leucine 65 mutated to valine or glycine. In some embodiments, the mutation is leucine 65 mutated to valine. In some embodiments, the mutation is leucine 65 mutated to glycine. In some embodiments, the mutation is glutamic acid 107 mutated to serine, asparagine or aspartic acid. In some embodiments, the mutation is glutamic acid 107 mutated to serine. In some embodiments, the mutation is glutamic acid 107 mutated to asparagine. In some embodiments, the mutation is glutamic acid 107 mutated to aspartic acid. In some embodiments, the mutation is phenylalanine 108 mutated to arginine. In some embodiments, the mutation is glutamine 109 mutated to methionine, serine, leucine or cysteine. In some embodiments, the mutation is glutamine 109 mutated to methionine. In some embodiments, the mutation is glutamine 109 mutated to serine. In some embodiments, the mutation is glutamine 109 mutated to leucine. In some embodiments, the mutation is glutamine 109 mutated to cysteine. In some embodiments, the mutation is aspartic acid 158 mutated to glycine. In some embodiments, the mutation is isoleucine 159 mutated to tyrosine. In some embodiments, the mutation is leucine 162 mutated to serine or arginine. In some embodiments, the mutation is leucine 162 mutated to serine. In some embodiments, the mutation is leucine 162 mutated to arginine. In some embodiments, the mutation is alanine 167 mutated to phenylalanine.
[097] In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, and isoleucine 159 mutated to tyrosine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to serine or arginine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to serine.
In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine and leucine 162 mutated to arginine.
[098] In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to leucine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to leucine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and tyrosine 32 mutated to glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to valine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to valine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, and leucine 65 mutated to glycine.
[099] In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to leucine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to leucine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and tyrosine 32 mutated to glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to valine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to valine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine mutated to tyrosine, leucine 162 mutated to serine or arginine, alanine 167 mutated to phenylalanine, and leucine 65 mutated to glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to valine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to valine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine or glycine and leucine 65 mutated to glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to valine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to valine or glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to valine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to leucine and leucine 65 mutated to glycine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to valine. In some embodiments, the mutant aaRS comprises aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to serine or arginine, tyrosine 32 mutated to glycine and leucine 65 mutated to glycine.
[0100] In some embodiments, the aaRS further comprises mutation of arginine 257 to glycine, mutation of aspartic acid 286 to arginine, or both. In some embodiments, the aaRS further comprises mutation of arginine 257 to glycine. In some embodiments, the aaRS further comprises mutation of aspartic acid 286 to arginine. In some embodiments, the aaRS further comprises mutation of both arginine 257 to glycine and aspartic acid 286 to arginine. In some embodiments, SEQ ID NO: 1 further comprises these two known mutations. In some embodiments, the sequence into which the mutations of the invention are introduced comprises or consists of
MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLGHYLQIKKMIDLQNAGF
DIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV
YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMEQRKI
HMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG
VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMR
LKNAVAEELIKILEPIRKRL (SEQ ID NO: 28) or a sequence with 95% identity thereto. In some embodiments, the sequence into which the mutations of the invention are introduced consists of SEQ ID NO: 28.
[0101] In some embodiments, the mutant aaRS comprises tyrosine 32 mutated to leucine, aspartic acid 158 mutated to serine, isoleucine 159 mutated to methionine, leucine 162 mutated to lysine, alanine 167 mutated to histidine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNSMHYKGVDVHVGGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 2), or a fragment, a derivative or analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 2.
[0102] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to alanine, leucine 162 mutated to glutamic acid, alanine 167 mutated to histidine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGAHYEGVDVHVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 3), or a fragment, a derivative or analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 3.
[0103] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to threonine, leucine 65 mutated to valine, glutamic acid 107 mutated to alanine, phenylalanine 108 mutated to tyrosine, glutamine 109 mutated to methionine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to cysteine, leucine 162 mutated to arginine, alanine 167 mutated to aspartic acid, arginine 257 mutated to glycine and aspartic acid 286 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSAYMLDKDYTLN VYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 4), or a fragment, a derivative or analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 4.
[0104] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to methionine; leucine 162 mutated to serine, alanine 167 mutated to histidine, arginine 257 mutated to glycine and aspartic acid 286 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGMHYSGVDVHV GGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 5), or a fragment, a derivative or analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 5.
[0105] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, leucine 162 mutated to cysteine, alanine 167 mutated to tyrosine, arginine 257 mutated to glycine, and aspartic acid 286 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF
DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYCGVDVYVGGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 6), or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 6.
[0106] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 12) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 12.
[0107] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV YRL ALKTTLKR ARRSMELI AREDENPKV AE VI YPIMQ VN G YH Y S G VD VF V GGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 13) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 13. [0108] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEKLREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSVSLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 14) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 14.
[0109] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to asparagine, phenylalanine 108 mutated to valine, glutamine 109 mutated to leucine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHA YLN QKGELDEIRKIGD YNKKVFEAMGLKAKYV Y GSNVLLDKD YTLN V YRL ALKTTLKR ARRSMELIAREDENPKV AE VI YPIMQ VN G YH Y S G VD VF V GGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 15) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 15.
[0110] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to aspartic acid, aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSDFQLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 16) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 16.
[0111] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence: MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSVCLDKDYTLNV YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYSGVDVFVGGMEQRK IHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAG VVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAE ELIKILEPIRKRL (SEQ ID NO: 17) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 17.
[0112] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; and leucine 162 mutated to arginine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGF
DIIIVLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNV
YRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYRGVDVAVGGMEQR
KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA
GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 18) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 18.
[0113] In some embodiments, the mutant aaRS comprises: tyrosine 32 mutated to leucine, lysine 65 mutated to glycine; glutamic acid 107 mutated to aspartic acid, phenylalanine 108 mutated to arginine, glutamine 109 mutated to methionine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence:
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGF DIIIGLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSDRMLDKDYTLN V YRL ALKTTLKR ARRSMELI AREDENPKV AE VIYPIMQ VN G YH Y S G VD VF V GGMEQR KIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPA GVVEGNPIMEIAKYFLEYPLTIKGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVA EELIKILEPIRKRL (SEQ ID NO: 19) or a fragment, a derivative or an analog thereof. In some embodiments, the mutant aaRS comprises or consists of the amino acid sequence of SEQ ID NO: 19.
[0114] In some embodiments, the fragment, derivative or analog comprises at least one of the recited mutations. In some embodiments, the fragment, derivative or analog is an active fragment, derivative or analog. In some embodiments, active refers to possessing an aaRS activity. In some embodiments, the aaRS activity is the ability to catalyzes the attachment of an amino acid to its cognate tRNA. In some embodiments, the aaRS activity is the ability to recognize an amino acid. In some embodiments, the aaRS activity is the ability to recognize a tRNA. In some embodiments, the aaRS activity is the ability to transfer an amino acid to a tRNA.
[0115] The term “derivative” as used herein, refers to any polypeptide that is based off the polypeptide of the invention and still comprises the recited mutations. A derivative is not merely a fragment of the polypeptide, nor does it need to have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the polypeptide of the invention. In some embodiments, a derivative of a sequence comprises at least 70, 75, 80, 85, 90, 92, 93, 95, 97, 99 or 100% identity to that sequence. Each possibility represents a separate embodiment of the invention. In some embodiments, a derivative of a sequence comprises at least 90% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 95% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 97% identity to that sequence. In some embodiments, a derivative of a sequence comprises at least 99% identity to that sequence.
[0116] In some embodiments, a fragment comprises at least 50, 100, 150, 200, or 250 amino acids of the aaRS. Each possibility represents a separate embodiment of the invention. In some embodiments, a fragment is a functional fragment. In some embodiments, a fragment comprises at least 50 amino acids of the aaRS. In some embodiments, a fragment comprises at least 100 amino acids of the aaRS.
[0117] In some embodiments, the fragment is a portion of the polypeptide comprises any one of a leucine at position 32, a threonine at position 32, a valine at position 65, an alanine at position 107, a tyrosine at position 108, a methionine at position 109, a serine at position 158, a glycine at position 158, an alanine at position 159, a methionine at position 159, a cysteine at position 159, a tyrosine at position 159, a glutamic acid at position 162, a lysine at position 162, a valine at position 162, an arginine at position 162, a serine at position 162, a cysteine at position 162, a histidine at position 167, an aspartic acid at position 167, and a tyrosine at position 167. Such a fragment will still be recognizable as being from the polypeptide of the invention, and as such will be at least 10 amino acids in length. As such, any fragment of the isolated polypeptide of the invention will still comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 80, or at least 100 amino acids surrounding position 32, position 65, position 107, position 108, position 109, position 158, position 159, position 162, or position 167 of the polypeptide. Each possibility represents a separate embodiment of the present invention.
[0118] In some embodiments, the fragment is a portion of the polypeptide comprises any one of a leucine at position 32, a glycine at position 32, a valine at position 65, a glycine at position
65, a serine at position 107, an asparagine at position 107, a aspartic acid at position 107, a valine at position 108, a arginine at position 108, a methionine at position 109, a serine at position 109, a leucine at position 109, a cysteine at position 109, a glycine at position 158, a tyrosine at position 159, a an alanine at position 162, a serine at position 162, and a phenylalanine at position 167. Such a fragment will still be recognizable as being from the polypeptide of the invention, and as such will be at least 10 amino acids in length. As such, any fragment of the isolated polypeptide of the invention will still comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 80, or at least 100 amino acids surrounding position 32, position 65, position 107, position 108, position 109, position 158, position 159, position 162, or position 167 of the polypeptide. Each possibility represents a separate embodiment of the present invention.
[0119] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.
[0120] In some embodiments, the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a fragment, analog or derivative thereof. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a fragment, analog or derivative thereof.
[0121] In some embodiments, the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. In some embodiments, the mutant aaRS comprises or consists of an amino acid sequence selected from: SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 or a fragment, analog or derivative thereof. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. In some embodiments, the mutant aaRS consists of an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19or a fragment, analog or derivative thereof.
[0122] By another aspect, the present invention provides an isolated polypeptide, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0123] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In some embodiment, the peptides, polypeptides and proteins described herein have modifications rendering them more stable while in the body, more capable of penetrating into cells or capable of eliciting a more potent effect than previously described. In some embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
[0124] As used herein, the term “isolated polypeptide” refers to a peptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature. Typically, a preparation of isolated peptide contains the peptide in a highly-purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. Each possibility represents a separate embodiment of the invention.
Nucleic acid molecules
[0125] In another aspect, there is provided a nucleic acid molecule encoding a mutant aaRS of the invention, or a fragment, a derivative or an analog thereof. [0126] In another aspect, there is provided a nucleic acid molecule comprising a coding region encoding a mutant aaRS of the invention, or a fragment, a derivative or an analog thereof.
[0127] In some embodiments, the nucleic acid molecule encodes a mutant aaRS of the invention. In some embodiments, the nucleic acid molecule comprises a coding region encoding a mutant aaRS of the invention.
[0128] In some embodiments, the nucleic acid molecule is selected from DNA, RNA, cDNA, genomic DNA (gDNA), vector DNA, vector RNA, LNA, PNA and a combination thereof. In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the nucleic acid molecule is cDNA. In some embodiments, the nucleic acid molecule is gDNA. In some embodiments, the nucleic acid molecule is LNA. In some embodiments, the nucleic acid molecule is PNA. In some embodiments, the nucleic acid molecule is a hybrid molecule comprising more than one type of nucleic acid.
[0129] As used herein, the phrases "coding sequence" and "coding region" are interchangeable and refer to the region that when translated results in the production of an expression product, such as a polypeptide, protein, or enzyme, and specifically the mutant aaRS. In some embodiments, the coding region is operably linked to at least one regulatory element. In some embodiments, the regulatory element is configured to express the coding region in a target cell. In some embodiments, the regulatory element is configured to express a protein encoded by the coding region in a target cell. In some embodiments, the regulatory element is a promoter. In some embodiments, the regulatory element is an enhancer. In some embodiments, the regulatory element is a silencer. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, expression of the coding region refers to a state in which mRNA is transcribed from the coding region acting as a template. In some embodiments, expression of the coding region refers to a state in which polypeptide is translated from the mRNA transcribed from the coding region.
[0130] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
[0131] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is an RNA vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is configured for expression in a bacterial cell. In some embodiments, the expression vector is configured for expression in a mammalian cell. In some embodiments, the expression vector is configured for expression in a target cell.
[0132] Expressing of a gene or protein within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector.
[0133] In some embodiments, the vector is introduced into a cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82,
5824 (1985)), Heat shock, 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 ah, Nature 327. 70-73 (1987)), and/or the like. A vector of the invention may be introduced into a target cell by any method known in the art, including but not limited to those provided herein. In some embodiments, the introducing produces a cell of the invention.
[0134] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et ah, Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et ah, Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
[0135] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
[0136] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpes viral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.
[0137] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF/myc/cyto, pCM V /myc/cy to , pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
[0138] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDS VE, and any other vector allowing expression of proteins under the direction of the S V -40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[0139] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
[0140] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
[0141] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
[0142] A person with skill in the art will appreciate that a gene or protein can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex vivo gene therapy). [0143] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATTCTATGCATTATAAGGGCGTTGATGTTCAT
GTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAA
AAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAG
ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAG
GGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATA
ATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAA
ATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAA
ATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAA
G ATTTT AG AGCC A ATTAG A A AG AG ATT AT A (SEQ ID NO: 7). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 7. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 7. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 7.
[0144] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA GTTATCTATCCAATAATGCAGGTTAATGGGGCTCATTATGAGGGCGTTGATGTTCA TGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCA AAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAA GATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTA GGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAAT AATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAA A ATTTGGTGG AG ATTTG AC AGTT A AT AGCT ATG AGG AGTT AG AG AGTTT ATTT AAA AATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAA AG ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 8). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 8. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 8. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 8.
[0145] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACTATAGGGTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGCGTATATGCTTGATA
AGG ATT AT AC ACTG A ATGTCTAT AG ATTGGCTTT A A A A ACT ACCTT A A A A AG AGC A
AGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAG
TTATCTATCCCATAATGCAGGTTAATGGTTGTCATTATAGGGGCGTTGATGTTGATG
TTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAA
AAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGA
TGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGG
GCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAA
TGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAAA
TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAA
TAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAAG ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 9). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 9. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 9. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 9.
[0146] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
A AGTGGT A A A AT AC ATTT AGGGC ATT ATCTCC A A AT A A A A A AG ATG ATTGATTT AC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATGGTATGCATTATTCGGGCGTTGATGTTCAT
GTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAA
AAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAG
ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAG
GGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATA
ATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAA
ATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAA
ATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAA
G ATTTT AG AGCC A ATTAG A A AG AG ATT AT A (SEQ ID NO: 10). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 10. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 10. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 10.
[0147] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATGGGTATCATTATTGTGGCGTTGATGTTTAT
GTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAA
AAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAG
ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAG
GGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATA
ATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAA
ATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAA
ATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAA
G ATTTT AG AGCC A ATTAG A A AG AG ATT AT A (SEQ ID NO: 11). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 11. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 11. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 11.
[0148] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTT
GTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAA
AAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAG
ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAG GGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATA ATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAA ATTTGGTGG AG ATTTG AC AGTT A AT AGCT ATG AGG AGTT AG AG AGTTT ATTT A A A A ATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAA G ATTTT AG AGCC A ATTAG A A AG AG ATT AT A (SEQ ID NO: 20). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 20. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 20. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 20.
[0149] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTT
GTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAA
AAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAG
ATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAG
GGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATA
ATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAA
ATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAA
ATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAA
G ATTTT AG AGCC A ATTAG A A AG AG ATT AT A (SEQ ID NO: 21). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 21. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 21. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 21. [0150] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTAGTGTTTCTCTTGATA
AGG ATT AT AC ACTG A ATGTCTAT AG ATTGGCTTT A A A A ACT ACCTT A A A A AG AGC A
AGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAG
TTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTTG
TTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAA
AAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGA
TGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGG
GCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAA
TGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAAA
TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAA
TAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAAG
ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 22). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 22. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 22. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 22.
[0151] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
A AGTGGT A A A AT AC ATTT AGGGC ATT ATCTCC A A AT A A A A A AG ATG ATTG ATTT AC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTAATGTTTTGCTTGATA
AGG ATT AT AC ACTG A ATGTCTAT AG ATTGGCTTT A A A A ACT ACCTT A A A A AG AGC A AGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAG
TTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTTG
TTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAA
AAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGA
TGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGG
GCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAA
TGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAAA
TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAA
TAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAAG
ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 23). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 23. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 23. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 23.
[0152] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGATTTCCAGCTTGATA
AGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCA
AGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAG
TTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTTG
TTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAA
AAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGA
TGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGG
GCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAA
TGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAAA
TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAA
TAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAAG ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 24). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 24. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 24. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 24.
[0153] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTTCTGTGTGTCTTGATA
AGG ATT AT AC ACTG A ATGTCTAT AG ATTGGCTTT A A A A ACT ACCTT A A A A AG AGC A
AGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAG
TTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTTTG
TTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAA
AAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGA
TGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGG
GCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAA
TGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAAAA
TTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAA
TAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAAAG
ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 25). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 25. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 25. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 25.
[0154] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGGATAGGGTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC AAAATGCTGGATTTGATATAATTATAGTTTTGGCTGATTTACACGCCTATTTAAACC
AGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTT
TGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGAT
AAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGC
AAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAA
GTTATCTATCCAATAATGCAGGTTAATGGGTATCATTATAGGGGCGTTGATGTTGC
GGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCA
AAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAA
GATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTA
GGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAAT
AATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAA
A ATTTGGTGG AG ATTTG AC AGTT A AT AGCT ATG AGG AGTT AG AG AGTTT ATTT AAA
AATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAA
AG ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 26). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 26. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 26. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 26.
[0155] In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAG
AGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACC
AAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTAC
AAAATGCTGGATTTGATATAATTATAGGTTTGGCTGATTTACACGCCTATTTAAAC
CAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTT
TTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGATAGGATGCTTGA
TAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAG
CAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGA
AGTTATCTATCCAATAATGCAGGTTAATGGTTATCATTATTCGGGCGTTGATGTTTT
TGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCA
AAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAA
GATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTA GGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAAT AATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAGGGCCAGAAA A ATTTGGTGG AG ATTTG AC AGTT A AT AGCT ATG AGG AGTT AG AG AGTTT ATTT AAA AATAAGGAATTGCATCCAATGCGCTTAAAAAATGCTGTAGCTGAAGAACTTATAA AG ATTTT AG AGCC A ATT AG A A AG AG ATT AT A (SEQ ID NO: 27). In some embodiments, the sequence of the nucleic acid molecule comprises SEQ ID NO: 27. In some embodiments, the coding region of the nucleic acid molecule comprises or consists of SEQ ID NO: 27. In some embodiments, the coding region of the nucleic acid molecule consists of SEQ ID NO: 27.
[0156] In some embodiments, each of SEQ ID NO:7-l 1 comprises a coding sequence for a mutant aaRS. In some embodiments, each of 20-27 comprises a coding sequence for a mutant aaRS. In some embodiments, each of the nucleic acid molecules comprises a coding sequence coding for a mutant aaRS. It will be understood by a skilled artisan, that as the protein is the active molecule any substitution to the nucleic acid sequence that does not alter the protein encoded is also envisioned. As the codons for amino acids are degenerate, one codon may be switched for a synonymous codon.
[0157] In some embodiments, the coding region encodes a recombinant protein. In some embodiments, the recombinant protein is a mutant aaRS. As used herein, the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. In some embodiments, the polypeptide is a recombinant protein. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector used to express the recombinant protein in a cell.
[0158] In general, and throughout this specification, the term “vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector, wherein virally- derived DNA or RNA sequences are present in the virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfecting into host cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
[0159] Recombinant expression vectors can comprise a nucleic acid coding for the protein of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0160] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
Orthogonal translation systems
[0161] In another aspect, there is provided an orthogonal translation system, comprising: a. a mutant aaRS of the invention or a nucleic acid molecule of the invention; and b. a tRNA compatible with the aaRS.
[0162] In some embodiments, the orthogonal translation system is configured for translation in a target cell. In some embodiments, the orthogonal translation system is configured for in vitro translation. In some embodiments, the orthogonal translation system is configured for administration to a subject. In some embodiments, the orthogonal translation system is configured for administration to a cell. In some embodiments, the orthogonal translation system is configured for transfection to a cell. In some embodiments, the orthogonal translation system comprises a mutant aaRS of the invention. In some embodiments, the orthogonal translation system comprises a nucleic acid molecule of the invention.
[0163] In some embodiments, the tRNA is an orthogonal tRNA. In some embodiments, the tRNA is a non-naturally occurring tRNA. In some embodiments, the tRNA is a Mj tRNA. In some embodiments, the Mj tRNA is the tRNA corresponding to a stop codon. In some embodiments, the tRNA corresponds to a stop codon. In some embodiments, the stop codon is a stop codon that is absent in a target cell. In some embodiments, the stop codon is a stop codon that is depleted in a target cell. In some embodiments, the tRNA is recognized by the aaRS. In some embodiments, the tRNA is compatible with the mutant aaRS. In some embodiments, the tRNA is recognized by the mutant aaRS. In some embodiments, the mutation does not affect the aaRS’s recognition of the tRNA. In some embodiments, the mutation enhances the aaRS’s recognition of the tRNA. In some embodiments, the tRNA comprises an anticodon. In some embodiments, the anticodon corresponds to a stop codon. In some embodiments, the anticodon recognizes a stop codon. In some embodiments, the anticodon anneals to a stop codon. In some embodiments, the stop codon is a TAG stop codon. In some embodiments, the stop codon is a TGA stop codon. In some embodiments, the stop codon is a TAA stop codon. In some embodiments, the stop codon is not a TGA stop codon. In some embodiments, the stop codon is not a TAA stop codon.
[0164] In some embodiments, the orthogonal translation system further comprises an nsAA. In some embodiments, the nsAA is a uAA. In some embodiments, the uAA comprises a chemical moiety. In some embodiments, the chemical moiety is a biorthogonal chemical moiety. In some embodiments, the uAA is not naturally found in a target cell. In some embodiments, the biorthogonal chemical moiety is not naturally found in a target cell. In some embodiments, the chemical moiety is an azide or an alkyne group. In some embodiments, the chemical moiety comprises an azide or an alkyne group. Unnatural amino acids comprising azide and/or alkyne groups are well known in the art and non-limiting example include 3-Azido-D alanine, 3-azido- L-alanine, 4-azido-D-homoalanine, 4-azido-L-homoalanaine, 5-azido-D-ornithine, 5-azido-L- ornithine, 6-azido-D lysine, 6-azido-L-lysine, Boc-(R)-4-(2-propynyl)-L-proline, Boc- propargyl-Glycine-OH, Fmoc-(S)-propargyl-alanine-OH, Fmoc-(R)-propargyl-alanine-OH, and pPR. In some embodiments, the chemical moiety is an azide group. In some embodiments, the chemical moiety is an alkyne group. In some embodiments, the chemical moiety is an azobenzene group. Unnatural amino acids comprising azobenzene groups are well known in the art and non limiting example include 4,4’-AMPB, 3,3’-AMPB, 3,4’-AMPB, 3,3’-APB, AzoPhe, Azo3F and Azo4F. In some embodiments, the uAA is a modified phenylalanine. In some embodiments, the modified phenylalanine is selected from 4-propargyloxy-L-phenylalanine (pPR), and phenylalanine-4’ -azobenzene (AzoPhe). In some embodiments, the modified phenylalanine is pPR. In some embodiments, the modified phenylalanine is AzoPhe. In some embodiments, a uAA comprising an azobenezene group is selected from AzoPhe, Azo3F and Azo4F. In some embodiments, Azo3F is 2,4,6-tri-fluorinated azobenzene. In some embodiments, a uAA comprising an azobenezene group is AzoPhe. In some embodiments, a uAA comprising an azobenezene group is Azo3F. In some embodiments, a uAA comprising an azobenezene group is Azo4F.
[0165] In some embodiments, the mutant aaRS comprises a mutation found in SEQ ID NO: 2-6 and the uAA comprises an azide or an alkyne group. In some embodiments, the mutant aaRS comprises a sequence of SEQ ID NO: 2-6 and the uAA comprises an azide or an alkyne group. In some embodiments, the mutant aaRS comprises a mutation found in SEQ ID NO: 12-19 and the uAA comprises an azobenzene group. In some embodiments, the mutant aaRS comprises a sequence of SEQ ID NO: 12-19 and the uAA comprises an azobenzene group.
Cells
[0166] In another aspect, there is provided a cell comprising a mutant aaRS of the invention.
[0167] In another aspect, there is provided a cell comprising a nucleic acid molecule of the invention.
[0168] In another aspect, there is provided a cell comprising an orthogonal translation system of the invention. [0169] In some embodiments, the cell is a target cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the bacterium is E. coli. In some embodiments, the cell is not an archaeal cell. In some embodiments, the cell is an unmodified cell. In some embodiments, the cell is unmodified with the exception of the presence of a protein, nucleic acid or system of the invention. In some embodiments, the cell is a genetically modified cell.
[0170] In some embodiments, the genome of the cell is unmodified. In some embodiments, the genome of the cell is modified. In some embodiments, the cell is devoid of TAG stop codons. In some embodiments, the TAG stop codons are endogenous TAG stop codons. In some embodiments, the TAG stop codons are native TAG stop codons. In some embodiments, the cell is depleted of TAG stop codons. In some embodiments, depleted comprises at least 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% of the stop codons of the cell having been removed. Each possibility represents a separate embodiment of the invention. In some embodiments, the TAG stop codons are mutated to TGA or TAA stop codons. In some embodiments, the TAG stop codons are mutated to TGA stop codons. In some embodiments, the TAG stop codons are mutated to TAA stop codons. In some embodiments, the stop codon that is depleted or absent from the cell is the stop codon that corresponds to the anticodon loop of the tRNA.
[0171] In some embodiments, the cell is devoid of release factor 1 (RF1). In some embodiments, the cell does not express RF1. In some embodiments, the cell has decreased expression of RF1. In some embodiments, decreased is with respect to a wild-type cell. In some embodiments, decreased is with respect to a non-modified cell. In some embodiments, decreased is at least a 50, 60, 70, 75, 80, 90, 95, 97, 99 or 100% reduction in expression. Each possibility represents a separate embodiment of the invention. In some embodiments, the RF1 gene has been genomically ablated from the cell. In some embodiments, the cell is an RF1 knockout cell.
[0172] In some embodiments, the cell is a wild-type cell. In some embodiments, the cell expresses RF1. In some embodiments, the cell expresses RF1 at normal levels. In some embodiments, the cell comprises at least one TAG stop codon. In some embodiments, the cell comprises its natural content of TAG stop codons. In some embodiments, the cell does not comprise a TAG stop codon mutated to a TGA or TAA stop codon. [0173] In some embodiments, the cell further comprises a vector comprising an open reading frame (ORF). In some embodiments, the ORF is a coding region. In some embodiments, the ORF comprises at least one stop codon within the open reading frame. In some embodiments, the stop codon is a stop codon that corresponds to the anticodon of the tRNA of the orthogonal translation system. In some embodiments, the at least one stop codon is not the last codon of the ORF. In some embodiments, at least one codon coding for an amino acid is present after the stop codon in the ORF. In some embodiments, the amino acid encoded after the stop codon is a natural amino acid. In some embodiments, the last codon of the ORF is a stop codon that does not correspond to the anticodon of the tRNA of the orthogonal translation system.
[0174] In some embodiments, the vector is an expression vector. In some embodiments, the vector is configured to express a protein encoded by the ORF in the cell. In some embodiments, the ORF is operatively linked to at least one regulatory element. In some embodiments, the regulatory element is configured to induce expression of the protein encoded by the ORF in the cell. In some embodiments, the regulatory element is capable of induce expression of the protein encoded by the ORF in the cell.
[0175] In some embodiments, the ORF comprises at least one stop codon. In some embodiments, the ORF comprises at least two stop codons. In some embodiments, the OFR comprises a plurality of stop codons. In some embodiments, the ORF comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 stop codons. Each possibility represents a separate embodiment of the invention. In some embodiments, the ORF comprises at least 10 stop codons. In some embodiments, the ORF comprises at least 30 stop codons. It will be understood by a skilled artisan that the number of stop codons recited herein does not refer to the stop codon at the end of the ORF that is responsible for stopping translation. The stop codon at the end of the ORF that stops translation will not correspond to the anticodon of the tRNA of the orthogonal translation system.
[0176] In some embodiments, the ORF encodes a protein of interest. In some embodiments, the ORF encodes a protein to comprise an nsAA. In some embodiments, the protein of interest is a protein to be tagged. In some embodiments, the protein or interest is a protein to be made light responsive. Methods of use
[0177] In another aspect, there is provided a method of producing a protein comprising an nsAA, the method comprising introducing into a cell an expression vector comprising an ORF encoding the protein, wherein the ORF comprises at least one stop codon, and wherein the cell comprises an orthogonal translation system of the invention, thereby producing a protein comprising an nsAA.
[0178] In some embodiments, the protein is a target protein. In some embodiments, the expression vector comprising an ORF encoding the protein is an expression vector as described herein above. In some embodiments, the orthogonal translation system is an orthogonal translation system comprising a nsAA. In some embodiments, the cell comprises the nsAA. In some embodiments, the orthogonal translation system is compatible with the nsAA. In some embodiments, the tRNA of the orthogonal translation system is compatible with the nsAA. In some embodiments, the mutant aaRS of the orthogonal translation system is compatible with the nsAA.
[0179] In some embodiments, the method further comprises introducing the orthogonal translation system into the cell. In some embodiments, the method further comprises introducing the nsAA into the cell. In some embodiments, introducing comprises transfection. In some embodiments, introducing comprises nucleofection. In some embodiments, introducing comprises genomic alteration. In some embodiments, introducing comprises genome editing.
[0180] In some embodiments, the method is for labeling a protein. In some embodiments, the method is for labeling and the nsAA is an azide or alkyne group containing nsAA. In some embodiments, the method is for labeling and the mutant aaRS comprises a mutation found in SEQ ID NO: 2-6. In some embodiments, the method is for labeling and the mutant aaRS comprises a sequence of SEQ ID NO: 2-6.
[0181] In some embodiments, the method is for labeling and further comprises converting the nsAA into a detectably labeled amino acid. In some embodiments, converting comprises addition of a detectable moiety by Click chemistry. In some embodiments, the Click chemistry is copper- catalyzed Click chemistry. In some embodiments, the Click chemistry is not copper-catalyzed Click chemistry. In some embodiments, the Click chemistry comprises azide and/or alkene cycloaddition.
[0182] As used herein, a “detectable moiety” is any molecule or portion of a molecule that can be specifically detected by a method known in the art. Examples of detectable moieties include, but are not limited to fluorescent moieties, radioactive moieties, bulky groups, dyes, and a tag. The term "moiety", as used herein, relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. The term "moiety" further means part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule. In some embodiments, the detectable moiety is a fluorescent moiety.
[0183] In some embodiments, method is for producing a light-responsive protein. In some embodiments, a light-responsive protein is a light-sensitive protein. In some embodiments, the method is for producing a light-responsive protein and the nsAA comprises an azobenzene group. In some embodiments, the method is for producing a light-responsive protein and the mutant aaRS of the orthogonal translation system comprises a mutation found in SEQ ID NO: 12-19. In some embodiments, the method is for producing a light-responsive protein and the mutant aaRS of the orthogonal translation system comprises a sequence of SEQ ID NO: 12-19. In some embodiments, the method further comprises irradiating the produced protein with light.
[0184] By another aspect, there is provided a protein produced by a method of the invention.
[0185] By another aspect, there is provided a protein comprising a nsAA.
[0186] In some embodiments, the protein is a protein comprising a nsAA. In some embodiments, the protein is a light-responsive protein. In some embodiments, the protein is a light-sensitive protein. In some embodiments, the protein is an ELP. In some embodiments, the protein is a self assembling protein. In some embodiments, the protein is a diblock. In some embodiments, the protein is a ELP diblock copolymer.
[0187] In some embodiments, the protein comprises at least one nsAA. In some embodiments, the protein comprises a plurality of nsAA. In some embodiments, the protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 nsAA. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises at least 5 nsAA. In some embodiments, the protein comprises at least 10 nsAA. In some embodiments, the protein comprises at least 15 nsAA. In some embodiments, the protein comprises at least 20 nsAA. In some embodiments, the protein comprises at least 30 nsAA. In some embodiments, the protein comprises at least 50 nsAA. In some embodiments, the protein comprises at least 100 nsAA.
[0188] In some embodiments, all the nsAA in the protein are the same nsAA. In some embodiments, the nsAA comprise at least two different nsAA. In some embodiments, the nsAA are present at predetermined positions in the protein. In some embodiments, at least one nsAA is inserted in a hydrophobic segment of an ELP diblock co-polymer. In some embodiments, all the nsAA are inserted in a hydrophobic segment of an ELP diblock co-polymer.
[0189] Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.
[0190] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.
[0191] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[0192] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
[0193] Other terms as used herein are meant to be defined by their well-known meanings in the art.
[0194] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
[0195] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
EXAMPLES
[0196] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et ah, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et ah, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et ah, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Materials and Methods
[0197] Materials: The uAA para-propargyloxy-l-phenylalanine (pPR) was purchased from Chem-Impex and from Iris-biotech. For in-vivo click reaction DPBS (without calcium and magnesium) was purchased from Thermo-Fisher scientific, TAMRA-azide (azide fluor 545), sodium ascorbate, copper(II) sulfate pentahydrate (CuS04) and Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA) were purchased from Sigma- Aldrich, BugBuster protein extraction reagent was purchased from Merck.
[0198] The azobenzene-uAAs 1 and 2 were purchased from Giotto Biotech and the azobenzene- uAA 3 was purchased from Chiroblock. Restriction endonucleases and ligation enzymes were purchased from New England Biolabs. DNA amplification was performed using The KAPA2G Fast HotStart ReadyMix or the KAPA HiFi PCR kit (Roche). Plasmid purification was conducted with Plasmid HiYield mini-prep (RBC Bioscience) and the PCR/restriction product was purified using a HiYield gel/PCR extraction kit (RBC Bioscience). Ligation was performed using the Quick Ligation™ Kit or with the T4 DNA Ligase, both purchased from New England Biolabs. Ligation products were transformed into 5 -alpha Competent E. coli (High Efficiency) or Stbl2 Competent E. coli (High Efficiency), purchased from New England Biolabs. SDS solution was purchased from Bio-Rad. Anhydrotetracycline hydrochloride was purchased from Sigma- Aldrich. C321.AA (Isaacs lab) and pEvol-pAzFRS.l.tl were a gift from Farren Isaacs (Addgene plasmids # 73581 and # 73547). Isomerization experiments were performed with a 365nm UV lamp (VL-6.LC, 12W, VILBER LOURMAT) and green, and blue mounted LEDs: (1) 405 nm, 1000 mW, 800 mA (M405L4, Thor-Labs) and (2) 530 nm, 350 mM (M530L3, Thor-Labs).
[0199] Diversification and selection of pPR-RS and AzoRS variants AARS libraries were generated by MAGE-based diversification of previously isolated genomically integrated mutants, pAcF-RS.tl, pAcFRS.2.tl and pAzFRS.2.tl. Prior to MAGE cycling, cultures were established by inoculating the liquid medium with a single bacterial colony or by adding 30 mΐ of a confluent liquid culture (1: 100 dilution) at 34 °C to mid-logarithmic growth (OD at 600 nm of 0.6 - 0.7) in a shaking incubator. To induce expression of the lambda-red recombination proteins cell cultures were shifted to 42 °C for 15 min and then immediately chilled on ice. In a 4 °C environment, 1 ml of cells was centrifuged at 15,000g for 30 s. Supernatant medium was removed and cells were resuspended in milli-Q water. The cells were spun down, the supernatant was removed, and washing procedure was repeated. After a final 30 s spin, the supernatant was removed and MAGE oligos (5-6 mM in DNase-free water) were added to the cell pellet. MAGE oligos are known in the art, and are provided for example in Amiram et al., 2015, “Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids”, Nature Biotechnology, 22, 1272-1279, herein incorporated by reference in its entirety. The oligo-cell mixture was transferred to a pre-chilled 1 mm gap electroporation cuvette (Bio- Rad) and electroporated under the following parameters: 1.8 kV, 200 V and 25 mF. LB media (3 ml) was immediately added to the electroporated cells. The cells were recovered from electroporation and grown at 34 °C for 3-3.5 h. Once the cells reached mid-log stage, they were used in additional MAGE cycles, subjected to negative and positive selection cycles, or frozen for further use.
[0200] Both colEl (negative) or SDS (positive) selections were optimized by testing LB broth containing various concentrations of colEl or SDS before addition of cells. Control strains with or without the TolC gene were used to verify selection conditions. Growth curves of libraries and controls in both selection experiments were monitored in real- time using kinetic measurements of the OD (600 nm) on a shaking and incubating plate reader. Screening of improved pPR variants after rounds of diversification with MAGE and negative (using varying concentrations of colicin El in the absence of pPR) and positive (using varying concentrations of SDS in the presence of pPR) selection was performed by plating cells on LB plates supplement with proper antibiotics, 0.2% L- Arabinose, 60 ng/mΐ anhydrotetracycline, and pPR at a concentration of 1 mM. Colonies expressing high levels of GFP were selected and subjected to further analysis of GFP expression by intact cell fluorescence measurements in the presence or absence of pPR. The aaRS genes of the best-performing colonies were analyzed by Sanger sequencing. [0201] Plasmid construction: Plasmids bearing GFP -based reporter genes were known in the art. Plasmids bearing the OTS variants for pPR incorporation were constmcted by insertion of aaRS genes to a previously described plasmid harboring a pl5A origin of replication and a chloramphenicol resistance marker. The gene encoding for the parent-pPR-RS OTS was chemically synthetized (IDT), and aaRS genes were PCR-amplified from chromosomal templates. All variants were inserted sequentially using the flanking restriction sites restriction sites Bglll and Sail, to produce inducible expression under the control of araBAD promoter and the rrnB terminator. The second constitutive copy of the aaRS, typically found in the pEvol system was removed.
[0202] The GFP(2TAG) reporter gene was chemically synthesized (IDT), restricted with Xhol and Hindlll restriction enzymes, and ligated to a similarly cut reporter plasmid. The ELP6o genes were chemically synthesized as half-proteins, ELP30 genes (GeneArt, Thermo Fisher), restricted with BseRI, and ligated sequentially using PreRDL, under the control of the pTac promoter in a pet24 modified vector (GeneScript). Plasmids bearing the OTS variants for azobenzene -u A A incorporation were constmcted by inserting aaRS genes into a previously described plasmid (pEvol) harboring a pi 5 A origin of replication and a chloramphenicol resistance marker. The gene encoding for the AzoRS OTS was synthetized (IDT), and the evolved genomic aaRS genes were PCR-amplified from chromosomal templates. All variants were inserted sequentially by using the flanking restriction sites Bglll and Sail to obtain inducible expression under the control of the araBAD promoter and the rrnB terminator. The second constitutive copy of the aaRS typically found in the pEvol system was removed. Ligation was conducted with the Quick Ligation™ Kit (NEB®) and the ligation products were transformed into NEB® 5- alpha Competent E. coli (High Efficiency), later plated on LB-agar plates supplemented with chloramphenicol (25 pg ml 1) and analyzed by Sanger sequencing.
[0203] Analysis of GFP expression by intact cell fluorescence measurements: For 96-well plate- based assays, strains harboring chromosomally integrated orthogonal translation systems and GFP reporter plasmids were inoculated from frozen stocks and grown to confluence overnight. Cultures were then inoculated at 1:15 dilution in 2xYT media supplemented with 30 pg/ml kanamycin. For cells harboring plasmid-based orthogonal translation system and GFP reporter proteins, the media was also supplemented with 25 pg/ml chloramphenicol. Cells and allowed to grow at 34 °C to an OD600 of 0.5-0.8 in a shaking plate incubator at 567 c.p.m. (~3 h). aaRS expression was then induced by the addition of 0.2% arabinose, GFP expression was induced by the addition of 60 ng/mΐ anhydrotetracy cline, and the uAA was added at a concentration of 1 mM. For GFP activity assays in BL21 strain, inducers for aaRS and GFP expression were added immediately after inoculation in the plate. Cultures and inducers were added individually to each well. Cells were incubated at 34 °C overnight. Following expression, cells were centrifuged at 4,000 g for 5 min. Supernatant medium was removed and cells were resuspended in PBS. GFP fluorescence was measured on a Biotek spectrophotometric plate reader using excitation and emission wavelengths of 485 and 528 nm, respectively. Fluorescence signals were normalized by dividing the fluorescence counts by the OD600 reading.
[0204] Alternatively, Cultures were then inoculated at a 1:50 dilution in 2xYT medium supplemented with kanamycin (30 pg ml 1). For cells harboring the plasmid-based orthogonal translation system and GFP reporter proteins, the media were also supplemented with chloramphenicol (25 pg ml 1). Cells were allowed to grow at 34 °C to an ODeoo of 0.5-0.8 in a shaking plate incubator at 567 rpm (~3 h). The expression of aaRS was then induced by adding arabinose (0.2%); GFP expression was induced by adding anhydrotetracycline (60 ng ml 1); and the uAA was added at a concentration of 0.25 mM. Following expression, the cells were centrifuged at 4,000 g for 5 min, the supernatant medium was removed, and the cells were resuspended in PBS. GFP fluorescence was measured on a Biotek spectrophotometric plate reader by using excitation and emission wavelengths of 485 nm and 528 nm, respectively. Fluorescence signals were normalized by dividing the fluorescence counts by the ODeoo reading.
[0205] ELP expression and purification: Before batch expression, starter cultures (1:25 v/v of final expression volume) of 2xYT media supplemented with 30 pg/ml kanamycin and 25 pg/ml chloramphenicol were inoculated with transformed cells from a fresh agar plate or from stocks stored at -80 °C, and incubated overnight at 34 °C while shaking at 220 r.p.m. Cells were centrifuged at 4,000 g for 10 min, supernatant medium was removed and cells were resuspended in remaining media, and transferred to expression flasks (containing 2xYT media, antibiotics, 0.2% arabinose and the uAA). For the expression of ELP(10TAG)-GFP by Mutl-RS in the genomically recoded organism, cells were supplemented with 0.25 mM of the uAA. For expression of ELP(30TAG)- GFP or for expression in BL21, cells were supplemented with 1 irM uAA. Cells were incubated at 34 °C for 4-5 h and then reporter protein expression was induced with 60 pg/ml anhydrotetracycline. Cells were harvested 24 h after inoculation by centrifugation at 4,000 g for 30 min at 4 °C. The cell pellet was resuspended by vortex in ~2 ml PBS buffer and stored at -80 °C or immediately purified. For purification, resuspended pellets were lysed by ultrasonic disruption (18 cycles of 10 s sonication separated by 40 s intervals). Poly(ethyleneimine) (0.2 ml of 10% solution) was added to each lysed suspension before centrifugation at 4,000 g for 15 min at 4 °C to separate cell debris from the soluble cell lysate. All ELP constructs were purified by a modified inverse transition cycling (ITC) protocol consisting of multiple “hot” and “cold” spins using sodium citrate to trigger the phase transition. Before purification, the soluble cell lysate was incubated for 1-2 min at 75 °C to denature native E. coli proteins. The cell lysate was then cooled on ice, centrifuged for 2 min at -14,000 r.p.m and the pellet was discarded. For “hot” spins, the ELP phase transition was triggered by adding sodium citrate to the cell lysate or the product of a previous cycle of ITC at a final concentration of -0.5 M. The solutions were then centrifuged at -14,000 r.p.m for 2 min and the pellets were resuspended in PBS, followed by a 2 min “cold” spin performed without addition of sodium citrate to remove denatured contaminant. Additional rounds of ITC were carried out as needed, using a saturated solution of sodium citrate until sufficient purification was achieved.
[0206] Protein concentration was calculated by measuring the OD280 of purified protein according to the following extinction coefficients: Tyr (WT protein): 33,935, ELP(lpPR)-GFP: 33,645, ELP(5pPR)-GFP: 32,485, ELP(10pPR)-GFP: 31,035, based on extinction coefficient of pPR (1200 M-cm-1).
[0207] Alternatively, before batch expression, starter cultures (1:40 v/v of final expression volume) of 2xYT media, supplemented with kanamycin (30 pg ml 1) and chloramphenicol (25 pg ml 1), were inoculated with transformed cells from either a fresh agar plate or from stocks stored at -80 °C, incubated overnight at 34 °C while shaking at 220 rpm, and transferred to expression flasks containing 2xYT media, antibiotics, arabinose (0.2%), and azobenzene -u A A (0.25 mM). For the expression of ELP6o(10TAG), ELP6o(6TAG), and ELP6o(2TAG) by AzoRS- 4, the C321.ARF1 strain1401, supplemented with azobenzene-uAA (0.25 mM) and arabinose (0.2%), was incubated at 34 °C for 4-5 h and then protein expression was induced with isopropyl b-d-l-thiogalactopyranoside (IPTG, 1 mM). The cells were harvested 24 h after inoculation by centrifugation at 4,000 g for 30 min at 4 °C. The cell pellet was then resuspended by vortex in milli-Q water (~4 ml) and either stored at -80 °C or purified immediately. For purification, resuspended pellets were lysed by ultrasonic disruption (18 cycles of 10 s sonication, separated by 40 s intervals of rest). Poly(ethyleneimine) was added (0.2 ml of a 10% solution) to each lysed suspension before centrifugation at 4,000 g for 15 min at 4 °C to separate cell debris from the soluble cell lysate. All ELP constructs were purified by a modified inverse transition cycling (ITC) protocol[20b] consisting of multiple “hot” and “cold” spins by using sodium chloride to trigger the phase transition. Before purification, the soluble cell lysate was incubated for 1-2 min at 42-55 °C to denature the native E.coli proteins. The cell lysate was then cooled on ice, centrifuged for 2 min at -14,000 rpm, and the pellet was discarded. For “hot” spins, the ELP phase transition was triggered by adding sodium chloride to the cell lysate or to the product of a previous cycle of ITC at a final concentration of -5 M. The solutions were then centrifuged at -14,000 rpm for 10 min and the pellets were resuspended in milli-Q water, after which a 2 min “cold” spin was performed without sodium chloride to remove denatured contaminant. Additional rounds of ITC were conducted as needed using a saturated solution of sodium chloride until sufficient purification was achieved.
[0208] Protein concentrations were calculated by measuring the OD280 of the purified protein according to the following extinction coefficients: ELP6o(tyrosinexlO): 16,390, ELP6o(lxlO): 26,900, ELP6O(1X6): 16,736, and ELP6o(2xlO): 6,572, based on the extinction coefficient of 1 (2,541 M cm 1); ELP6o(2xlO): 41590, ELP60(2x6): 25550, and ELP60(2xlO): 9510, based on the extinction coefficient of 2 (4010 M cm 1); and ELP6o(3xlO): 79122 , ELP6o(3x6): 74546, and ELP6O(3X 10): 25482, based on the extinction coefficient of 3 (123250 M cm 1).
[0209] Intact mass measurements: Intact mass measurements of the proteins were performed using the MALDI-TOF instrument (MALDI-TOF/TOF autoflex speed), at the Ilse Katz Institute for Nanoscale Science and Technology (Ben-Gurion University of the Negev). Spectrum analysis was performed by the Flexanalysis software.
[0210] In-vivo click reaction: Before the reaction, ELP(ITAG) and ELP(IOTAG), (both without GFP) were expressed in the genomically recoded organism or in the BL21 strain, by the parent - pPR-RS or evolved Mutl-RS. Starter cultures of 2xYT media supplemented with 30 pg/ml kanamycin and 25 pg/ml chloramphenicol were inoculated with transformed cells from a fresh agar plate or from stocks stored at -80°C, and incubated overnight at 34 °C while shaking at 220 r.p.m. Cells were then inoculated in 2 ml 2xYT media, supplemented with antibiotics, 0.2% arabinose and 1 mM of the uAA. Cells were incubated at 34 °C for 4-5 h and then reporter protein expression was induced with 60 pg/ml anhydrotetracycline. When comparing the click reaction in the GRO and BL21, induction of all proteins was performed immediately after seeding. Cells were grown overnight, harvested by centrifugation at -14,000 r.p.m for 2 min, resuspended in DPBS buffer and immediately used. Proteins were conjugated in-vivo to azide-fluor 545 (TAMRA azide). E. coli cells were added to a final OD600=1 in the reaction. Concentrations of other reagents in the reaction were as following: 2% v\v DMSO, 0.1 mM TAMRA, 0.5 mM THPTA premixed with 0.1 mM CuS04 for 20 min, 2.5 mM sodium ascorbate. DPBS solution was added up to desired volume. Reaction was performed for 1 hour at 25 °C, in a shaking incubator at 400 r.p.m in the dark. Cells were washed by cycles of 3 min centrifugation at -14,000 r.p.m followed by pellet resuspension in PBS, until the supernatant was colorless. Cells were then lysed using BugBuster protein extraction reagent according to manufacturer's instructions, and lysed cells were centrifuged for 10 min at 4 °C to separate cell debris from soluble proteins. Laemmli sample buffer was added to the reaction, and denaturation was performed by heating at 100 °C for 5 min.
[0211] For in vitro click reaction performed on cell lysates, cells were lysed by BugBuster protein extraction reagent after protein expression and cell harvesting. Cell lysate, equivalent to final OD600=5.5 was added to the reaction. Final concentration for other reagents were as following: 50 mM TAMRA, 3 mM THPTA premixed for 20 min with 0.4 mM CuS04, 5 mM sodium ascorbate. DPBS was added up to desired volume. Reaction was performed for 1 hour at 25 °C, in a shaking incubator at 400 r.p.m in the dark. Faemmli sample buffer was added to supernatant, and denaturation was performed by heating at 100 °C for 5 min.
[0212] All clicked products were analyzed on SDS-PAGE until free TAMRA was eluted from the gel. In-gel fluorescence analysis was performed on the Typhoon FFA-9500, using 532 nm laser. Equal lysis of all samples was validated by Coomassie blue staining of the gel.
[0213] Phase transition analysis: To characterize the inverse transition temperature of EFP variants, the ODeoo of the EFP solution (in milli-Q water, unless otherwise noted) was monitored as a function of temperature, with heating and cooling performed at a rate of 1 °C min 1 on a UV- vis spectrophotometer equipped with a multicell thermoelectric temperature controller (Thermo Scientific).
[0214] Dynamic light scattering (DLS) analysis: ELP self-assembly was analyzed using a Zetasizer Nano ZS (Malvern Pananalytical). For each sample, 12-17 acquisitions (determined automatically by the instrument) were obtained at 10 °C for single-block ELPs or at 1-2 °C increments from 18 to 50 °C for diblock ELPs. Populations comprising less than 1% of the total mass (by volume) were excluded from the analysis.
[0215] Circular Dichroism (CD) analysis: The secondary structure of ELPs was studied using an Jasco J-715 spectropolarimeter (Tokyo) equipped with a PTC-348WI temperature controler, using a 1 -mm quartz cuvette instrument by scanning from 280 nm to 180 nm at either 10 °C or 30 °C. Purified constructs were diluted to 7.5 mM in water. Data were considered for analysis whenever the Dynode voltage was below 800 V.
[0216] Transmission electron microscopy: TEM at cryogenic temperature (Cryo-TEM) was used for direct imaging of solutions and dispersions. Vitrified specimens were prepared on a copper grid coated with a perforated lacey carbon 300 mesh (Ted Pella Inc.). A typically 2.5m1 drop from the solution was applied to the grid and blotted with a filter paper to form a thin liquid film of solution. The blotted sample were immediately plunged into liquid ethane at its freezing point (-183°C). The procedure was performed automatically in the Plunger (Lieca EM GP). The vitrified specimens were then transferred into liquid nitrogen for storage. The samples were studied using a FEI Talos F200C TEM, at 200kV maintained at - 180 °C; and images are recorded on a FEI Ceta 16M camera (4k x 4k CMOS sensor) at low dose conditions, to minimize electron beam radiation damage. The measurements were done at the Ilse Katz Institute for Nanoscale Science and Technology (Ben-Gurion University of the Negev).
[0217] Statistical analysis: Statistical analysis was performed using GraphPad Prism software. Significance is reported where P was found to be <0.05. For multiple comparisons, GFP production by each aaRS variant at the presence of the uAA was compared with GFP production by the parent-pPR-RS system, using ANOVA followed by Dunnett test, under the assumptions of Gaussian distribution and unequal variability of differences. For single comparison analysis, a one-tailed heteroscedastic t-test was performed. GFP production by each aaRS variant in the presence of azobenzene-uAAs was compared with GFP production by the AzoRS system using ANOVA followed by Dunnett’s post-hoc test, assuming a Gaussian distribution and unequal variability of differences.
EXAMPLE 1: Evolution and performance of chromosomally integrated nsAA-RS variants
[0218] In order to improve multi-site pPR incorporation, a modified MAGE- based protein evolution strategy was employed to identify improved mutants of the M. janaschii tyrosyl-tRNA synthetase (MjTyrRS) that efficiently charge an amber suppressor tRNA with pPR in Escherichia coli. In brief, mutants of the MjTyrRS were subjected to 5 or 10 rounds of MAGE -based diversification followed by tolC- mediated (1) negative, (2) positive, and (3) negative selections (colicin El (ColEl) -mediated negative selection, or SDS-mediated positive selections cycles). Individual colonies harboring genomically integrated mutant aaRSs from the remaining orthogonal library were screened for increased GFP(3TAG) production in the presence of pPR to identify improved variants. The best performing variants in isolated colonies were analyzed by Sanger sequencing, to identify the resulting mutations in the aaRS sequence (Fig. 1A). A similar MAGE- based protein evolution strategy was also employed to identify mutants of the MjTyrRS that efficiently charge an amber suppressor tRNA with azide -containing nsAAs in Escherichia coli. The best performing variants in isolated colonies were analyzed by Sanger sequencing, to identify the resulting mutations in the aaRS sequence (Fig. IB).
[0219] The ability of a previously-described pPR-RS (hereafter named “parent-pPR-RS”) to incorporate 3-30 instances of the uAA per protein in the GRO was now characterized. Three reporter proteins for multi-site uAA incorporation were used: GFP(3TAG), ELP(10TAG)-GFP and ELP(30TAG)-GFP, and their WT protein controls (GFP WT, ELP(10Tyrosine)-GFP, ELP(30Tyrosine)-GFP) (Fig. 2A). A GFP fluorescence assay indicated that multi-site pPR incorporation by parent-pPR-RS, expressed from a multi-copy plasmid, in the GRO produced ~5%, ~2% and -24.5% of pPR-containing GFP(3TAG), ELP(IOTAG)- GFP and ELP(30TAG)- GFP, respectively, as compared to WT proteins (Fig. 2B). In addition, the inventors also compared the efficacy of the parent-pPR-RS, which was integrated into a permissive region in the GRO genome so that the aaRS is expressed from only a single chromosomal copy. As expected, the reduction in copy number caused by genomic integration of the OTS resulted in further decrease in protein yields, with -0.5%, -0.75% and -9.4% production of GFP(3TAG), ELP(10TAG)-GFP and ELP(30TAG)-GFP, respectively, as compared with WT proteins (Fig. 2B).
EXAMPLE 2: Efficient multi-site uAA incorporation by evolved pPR-RSs
[0220] The evolved variants were now evaluated for multisite (3—30) pPR incorporation. C321.ARF1 was co-transformed with plasmids carrying the above-mentioned reporter proteins and episomal versions of each evolved pPR-RS variant. As expected, the expression of the evolved pPR-RSs from multi-copy plasmids led to higher protein production in the presence of pPR (Fig. 2C-E). Of note, only truncated ELP(30TAG)-GFP was produced by the parent pPR- RS in these conditions (Fig. 2B). Notably, when expressed from multi-copy plasmids in the absence of uAAs, most evolved variants showed increased protein production compared with the parent enzyme (Fig. 2C-E), in keeping with similar findings for other chromosomally evolved aaRSs (Fig. 2F). However, kinetic analysis demonstrated a reduced rate of AA incorporation in the absence of the uAA, which was also somewhat reduced when protein expression was induced in minimal medium (Fig. 2G-Z). Accurate incorporation of pPR was confirmed by intact mass analysis, as well as by LC-MS of tryptic fragments of ELP(10TAG)-GFPMS (~%L93 of the total ions by Mutl-RS, ~94L% of the total ions by Mut2-RS, and ~87L% of total ions produced by the parent-pPR-RS) and ELP(30TAG)MS (~64— 92.5L% of the total ions, by Mutl-RS, depending on pPR concentration and ~36L% of the total ions, by Mut2-RS using ImM pPR), with the latter methodology providing improved sensitivity of misincorporation events (Fig. 23, 24A, 24C, 24G-I, Fig. 3A-D). Therefore, it appears that the parent pPR-RS, Mutl-RS (which shows the highest level of background incorporation of all of the evolved variants), and Mut2- RS (which shows the lowest level of background incorporation of all of the evolved variants), all have similar fidelities when challenged to incorporate ten instances of pPR per protein. Likewise, when incorporating 30 pPR residues per protein, Mutl-RS shows higher fidelity than Mut2-RS, despite having higher background incorporation in the absence of pPR.
[0221] The best-performing variant, Mutl-RS (Fig. 1C), was further evaluated in the production of proteins with three to 30 instances of the uAA in the presence of twofold or fourfold reduced concentrations of pPR (lMmM is typically added to the growth medium; Fig. 4A). This analysis revealed that Mutl-RS is able to efficiently produce GFP(3TAG) and ELP(10TAG)-GFP in the presence of twofold or fourfold reduced pPR concentrations with only a minor loss of protein yield (0 to ~8L%) and ~8L% loss in pPR incorporation fidelity in the presence of fourfold reduced pPR concentrations (Fig. 24C-D). In contrast, production of ELP(30TAG)-GFP resulted in protein losses of ~40L% and ~70L% in the presence of two- or fourfold reduced pPR concentrations, respectively. Importantly, our evolved aaRS outperformed the parent synthetase by 20- to 200-fold improved protein yields at all pPR concentrations [except for ELP(30TAG)- GFP, which could not be produced by the parent in these conditions]. Small-batch expression of ELP( 10TAG)-GFP and ELP(30TAG)-GFP produced using Mutl-RS in the C321.ARF1 strain was performed. Detected protein yields were 24.52±1.9 and 54.42±5.7AAmg/L for ELP(IOTAG)- GFP and ELP(30TAG)-GFP, respectively, when expressed with 1 AAmM pPR in the growth medium (compared with 8.98±0.88AAmg/L and 14.97±0.85AAmg/L, respectively, of the equivalent WT proteins).
[0222] Given the high incorporation efficiency of the evolved pPR-RS variants in C321.ARF1, it was posited that they would also be capable of enhancing multisite pPR incorporation efficacy in non-recoded E. coli. To test this premise, the ability of Mutl-RS to incorporate three or ten pPR instances in a single protein was evaluated using the commonly utilized, non-recoded BL21 protein-expression strain. Fluorescence analysis of GFP(3TAG) and ELP(10TAG)-GFP expression by Mutl-RS in the BL21 strain showed production rates of ~140A% and ~44A%, respectively, compared to WT proteins (Fig. 4B). To determine protein yields and purity, a similar small-batch expression of ELP(10TAG)-GFP by using Mutl-RS in BL21 was performed. Protein yields were 6.33±0.2AAmg/L by Mutl-RS (or ~50A% of the equivalent WT protein, which expressed at 12.26±0.35AAmg/L). Correct incorporation of pPR was confirmed by intact mass analysis (Fig. 23 and Fig. 3A-D) as well as by LC-MS of tryptic fragments (~88A% and ~72A% of the total ions by Mutl-RS when expressed using 1 and 0.25AAmM pPR in the growth media, compared with ~88A% of total ions produced by the parent -pPR-RS, using 1 AAmM pPR in the growth medium, Fig. 24B, 24E, 24F).
[0223] The above results should be viewed in the wider context of a number of previously reported findings, particularly the following: three uAAs were incorporated per protein at WT levels by using a partially recoded and RF-1 deficient BL21 strain (B-95.AA); incorporation of six uAAs yielded ~6— 9A% of WT proteins in the presence of release factor inhibitors; and proteins bearing ten uAAs were produced with yields of ~3L% compared to WT protein yields in an RF-1 deficient, non-recoded E. coli strain. It has also been previously reported that multisite incorporation can be achieved by AA replacement, but this method replaces all instances of the relevant natural amino acid with the uAA and, as such, is not site specific. It can therefore result in labeling in unintended sites in the target proteins and/or loss of function as a result of unintended AA to uAA substitutions. The methodology disclosed herein, based on high- efficiency evolved aaRS variants that facilitate the production of proteins with ten pPRs in high yields in the non-recoded BL21 strain, can therefore be used to complement AA substitution, recoding, or RF1 attenuation efforts. Although ELPs and GFP are well-expressed, soluble proteins, we expect that our evolved aaRSs will also enhance the expression of additional proteins bearing multiple pPR residues, albeit in varying degrees.
[0224] The next step in this study was to determine whether efficient multisite pPR incorporation could be exploited for the bioorthogonal fluorescent labeling of proteins in vitro and in bacteria. It was posited that the multiple conjugated fluorophores and increased target protein yields of the pPR incorporation method disclosed herein could improve the fluorescent signal that is generated following a click reaction of pPR-containing proteins with an azide-fluorescent molecule, thereby enabling short reaction times, lower Cu concentrations, and improved biocompatibility. First, the fluorescent signal generated upon conjugation of purified EFPs with one or ten instances of pPR expressed in C321.ARF1 by using Mutl-RS was compared. As expected, improved signals and an improved limit of detection were obtained for purified proteins harboring ten pPR instances compared to those with only a single pPR residue (Fig. 5A). To test whether this increase in signal strength also translated into an improved signal following a click reaction in bacteria, we conjugated TAMRA-azide fluor to the pPR-containing proteins expressed by either the parent-pPR-RS or the evolved Mutl-RS (following induction of protein expression) in the presence of 100LLmM copper for (only) 1 hour at room temperature. At the end of the reaction, the cells were washed and then imaged by fluorescent microscopy to evaluate the labeling efficiency and to verify that the conjugation reaction had indeed occurred inside the cells. Analysis of the microscopy images indicated that ~90L% of the bacteria were labeled by expression of EFP(IOTAG) with Mutl-RS, as compared with ~25L% labeled cells obtained by expression of EFP(ITAG) or EFP(IOTAG) with parent pPR-RS, and that the fluorescence per cell increased by approximately five- to sevenfold using the former condition as compared to the latter (Fig. 5B-E, Fig. 25). In addition, the labeled cells were lysed, and the proteins were then resolved by SDS-PAGE and analyzed by in-gel fluorescence to image the TAMRA-labeled proteins. In keeping with the microscopy results, labeling in the bacteria resulted in a noticeable fluorescent band only for cells expressing the ELP(lOpPR) proteins produced by the Mutl-RS, while all other conjugation reactions produced signals that were below the limit of detection under these conditions (Fig. 6A). Next, the identical labeling experiment was performed in the BL21 strain, which, as expected, returned similar results (Fig. 6B-C). Notably, a comparison of the size and number of labeled bands following labeling in the C321 ARFl and in BL21 cells indicated that only the full-length protein was produced by Mutl- RS in both strains. However, a closer examination of the labeled products obtained after the more efficient in-vitro TAMRA labeling of the cell lysates indicated that various truncated proteins were also produced by the BL21 strain, presumably due to competition with RF-1, which remained present in this non-recoded strain. In-vitro TAMRA labeling of ELP(lOpPR) also revealed that while Mutl-RS produced mainly the full-length protein, only short truncated proteins were visible by the parent-pPR-RS (Fig. 6D).
[0225] In addition to the expected fluorescent band of labeled ELP(IOTAG), an additional, fluorescent double band was detected in all cells harboring the evolved pPR-RS (regardless of the reporter protein) in both E. coli strains. It was posited that the double bands represented the aminoacylated aaRS and aminoacylated aaRS-tRNA complex (~35 kDa), in which the complexed pPR was also fluorescently labeled by the TAMRA azide, as has previously been described in other cell types. As anticipated, transformation of C321.ARF1 with the Mutl-RS plasmid alone generated the same labeled double bands only when the expression of Mutl-RS was induced, and extraction of these bands from the gel, followed by tryptic digestion and LC- MS analysis, validated the presence of Mutl-RS, confirming the above premise (Fig. 6E-F, Fig. 24 J).
EXAMPLE 3: Evolved pPR-RSs enable rapid and non-toxic protein labeling in vivo
[0226] It is well known that the application of CuAAC bioconjugation in living systems is hindered due to the Cul-induced generation of reactive oxygen species (ROS), with consequent cellular toxicity. Therefore, the cell viability and the labeling efficiency of ELP(IOTAG) produced by Mutl-RS was tested in the recoded E. coli in the presence of different copper concentrations. The efficiency of the evolved aaRS enabled labeling of ELP(lOpPR) with a reduced copper concentration of 50 m M, without a significant decrease in labeling efficiency — evaluated as 56 and 35L% in the presence of 25 mM and 10 mM copper, respectively (Fig. 7A). These results exemplify the efficacy of multisite protein labeling for signal amplification when compared with those of previous studies, which reported extensive modification with 100LLmM or 1 mM copper (in an overnight reaction at 4A°C) but a complete loss of labeling when the copper concentration was reduced to 10 mM. We then determined the effect of copper toxicity on the viability of C321.ARF l in the presence of 10-100 mM copper. Growth curves indicated that although the lag phase for recovery was lengthened as the copper concentration increased, the cells were able to recover and reached an OD600 value similar to that of untreated cells in the stationary phase (Fig. 7B), in agreement with previous studies in mammalian and E. coli cells.
[0227] Several methodologies have described the use of Cu ligands to reduce Cu-mediated toxicity to cells by using an azide probe that contains an internal copper-chelating moiety or by using strained alkynes, which obviate the need for copper catalysts. However, unlike simple azides and alkynes, the azide probes that have an internal copper chelating moiety and the strained alkynes are large structures, and they might, therefore, interfere with the function of the labeled protein. Additional drawbacks of strain-promoted azide-alkyne cycloaddition (SPAAC) are that it is much slower than CuAAC and it is neither strictly bio-orthogonal nor regioselective. Here it is shows herein that the incorporation of multiple alkyne instances in ELP can be used to generate an enhanced fluorescent signal at a reduced copper concentration.
[0228] Commonly used fluorescent labeling methods include fusion to GFP variants or to self labeling enzymes (e.g., SNAP- and CLIP-tag and self-labeling tags (e.g., tetracysteine tag). However, these methods are limited, as the large size of the fused proteins (-20-27 kDa) may perturb the cellular localization, structure, or function of the fused protein, while the utilization of small, genetically encoded labeling tags often results in nonspecific staining of the membrane and hydrophobic pockets and thiols in off-target proteins. In contrast, it is shown herein that site- specific pPR incorporation in ELP-fusion proteins enables labeling at multiple, precise positions with minimal changes to the target protein sequence. Although other studies have demonstrated the detection of proteins that were each conjugated in vivo to a single fluorescent molecule, herein, the conjugation of a single TAMRA molecule failed to generate a detectable signal in a SDS-PAGE protein gel, under the examined conditions. This could be the result of comparatively lower expression levels of the target proteins (due to the proteins themselves, the strength of the promotor, or to other factors that affect expression) or of the moderate extinction coefficient and quantum yield of TAMRA compared to other fluorophores. The data nonetheless suggest that the ability to conjugate multiple fluorophores to the target protein can compensate for low protein expression or fluorophore properties.
[0229] This is the first study to use ELP fusion proteins as scaffolds for fluorophore conjugation sites. ELPs have already been successfully fused to a variety of proteins and typically do not reduce (and can even enhance) protein yields. Herein is shown that they can also enable the conjugation of multiple fluorophore labels while preventing or minimizing perturbation of proper protein folding or function, which can be caused by internal labeling. Herein, every third pentapeptide contained an X-guest residue that encoded for pPR, which resulted in a ~ 12 kPa ELP protein. Thus, the ability to precisely control the length, composition and number of sites for conjugation of the fluorescent molecules suggests that other (e.g., smaller) ELP fusions can also be used, and that the ELP-family can be exploited as valuable fusion partners, permitting site-specific conjugation of multiple fluorescent molecules for increased labeling efficiency and improved detection of the target proteins. Moreover, ELPs containing natural amino acids or uAAs have previously been designed and utilized for various applications, such as protein purification, hydrogel formation, drug delivery, tumor targeting and tissue engineering.
EXAMPLE 4: Efficient multi-site AzoPhe incorporation by evolved nsAA-RS
[0230] The performance of evolved nsAA-RSs was characterized for the ability to incorporate azobenzene-containing nsAA into proteins. Mutants are shown in Figure IB. To evaluate the mutants for multi-site (3-30) AzoPhe incorporation, a GRO was transfected with plasmids carrying the reporter proteins of elastin-like protein (ELP)-GFP fusion, whereby ELP coding sequence harbors 1, 5, 10 or 30 TAGs or WT equivalents (harboring 10 or 30 tyrosine substitutions), and episomal versions of one selected evolved AzoPhe-RS variant (Mut 7). Plate- based fluorescence analysis indicated that expression of the evolved nsAA-RS (AzoPhe-RS) from multi-copy plasmids lead to higher multi-site-specific incorporation in the presence of AzoPhe compared with the literary-RS and to the endogenous system of tyrosine with 10 TAGs
(Fig. 8A).
[0231] Next, the ability of the Azo-RS variant, to incorporate azobenzene uAAs 1, 2, or 3 (Fig. 9B; 1-10 instances per protein) in the Escherichia coli strain C321.ARF1, which lacks all the native TAG codons and their associated release factor (RF-1). To this end, the GFP- and EFP- based reporter proteins were utilized to indicate the multi-site incorporation of uAAs in the reassigned TAG codons GFP(2TAG), ELP(1TAG)-GFP, ELP(5TAG)-GFP, and ELP(IOTAG)- GFP, which are identical to or slightly modified from previously described GFP and EFP -based reporter proteins for uAA incorporation (Fig. 9D, Table 1). A GFP fluorescence assay indicated that the multi-site incorporation of 1, 2, or 3 by AzoRS, when expressed from a multi-copy plasmid in C321.ARF1, produced up to -96%, 14%, and -4% of EFP(1 TAG)-GFP, EFP(5TAG)- GFP, and EFP(10TAG)-GFP, respectively, as compared with control GFP and EFP proteins, which contained tyrosines incorporated by the wild-type MjTyrRS system (Fig. 9E-G).
[0232] To enable the multi-site incorporation of azobenzene -uAAs, a modified protein-evolution strategy was used that was previously developed to identify improved MjTyrRS mutants, which can efficiently charge an amber suppressor tRNA with azobenzenes 1, 2, or 3 in C321.ARF1. Briefly, genomically integrated aaRS variants were subjected to 5-10 rounds of multiplex automated genome engineering (MAGE)-based diversification, using degenerate ssDNA oligonucleotides (Table 2), followed by successive to/C-mediated negative-positive-negative selection cycles (ColEl -mediated negative selection or SDS-mediated positive selection). The first (negative) selection cycle was used to eliminate non-orthogonal variants generated in the diversification process, which, even if rare, would otherwise be enriched in the subsequent positive selection cycle; the second (positive) selection cycle was used to enrich the efficient aaRS variants; and the third (negative) selection cycle was used to eliminate “cheater” non- orthogonal clones generated in response to the stress applied in the positive selection step. The production of GFP(2TAG) in the presence of 1 was used to evaluate activity in genomically integrated individual clones. Several improved variants were identified that, when expressed from a single chromosomal copy, were capable of 14-56-fold higher GFP(2TAG) production compared with the parent enzyme (Fig. 10A). After Sanger-sequencing all the evolved variants (Table 3), we evaluated them for the multi-site (1-10) incorporation of 1, 2, or 3 by co- transforming C321 . VRF l with plasmids carrying the above-mentioned reporter proteins and episomal versions of each evolved variant. As expected, the expression of the evolved aaRSs from multi-copy plasmids increased their ability to incorporate multiple (1, 5, or 10) azobenzene- uAAs. Notably, one of the evolved variants, designated AzoRS-4, increased the expression of ELP(10TAG)-GFP containing 1, 2, or 3, by -40-, -13-, and ~7-fold, respectively, as compared with the abovementioned AzoRS (Fig. 10B-D). Although the substituents of 1, 2, and 3 differ only in hydrogen-to-fluorine substitutions, which are not expected to significantly alter the size of the azobenzene molecule, the efficiencies of incorporating these uAAs were different between the aaRS variants. Similar findings have been reported for other Methanosarcina- derived azobenzene aaRSs.
[0233] Table 1: GFP and ELP sequences
Figure imgf000076_0001
[0234] Table 2: Degenerate ssDNA MAGE oligonucleotides
Figure imgf000077_0001
Single-stranded DNA oligonucleotides with two phosphorothioate bonds at the 5' end (denoted by *) were purchased from Integrated DNA Technologies. The degenerate base n represents all four bases, and k represents G/T.
[0235] Table 3: Annotations of specific mutations in evolved aaRS variants, as compared with the WT Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS) sequence. In addition to the indicated mutations, all mutants also harbor the R257G and D286R mutations, which have been shown to improve tRNA binding.
Figure imgf000077_0002
EXAMPLE 5: ELPs with a UV-light-responsive phase-separation behavior
[0236] While the above-mentioned ELP-GFP fusion proteins are valuable reporters for assessing the efficiency and ability of the aaRSs to incorporate multiple uAA instances, their transition temperature (Tt) is either above (in the form of GFP-fused proteins) or below (as unfused proteins) the typical temperature range (10-90 °C) used for Tt analysis and characterization. Therefore, a new set of EFP variants was designed to analyze the effect of azobenzene incorporation on the EFP Tt. The design was based on previously described hydrophilic ELPs which have a high Tt (>90 °C for a 25 mM solution) and are composed of glycine and alanine amino acids alternating in the X-guest residue position. These ELPs were selected as hosts for azobenzene incorporation since the hydrophobic azobenzene molecule was expected to dramatically reduce the Tt when incorporated in multiple sites in the ELPs. To analyze the effect of varying the number of azobenzene groups per ELP, four ELP genes were designed, each containing 60 pentapeptides and either 0, 2, 6, or 10 TAG codons (for azobenzene incorporation) distributed equally along the guest residue of the ELP; these ELP genes were termed ELPeoWT, ELP6O(2TAG), ELP6O(6TAG), and ELPeo(lOTAG), respectively (Table 1). Proteins expressed from the above-mentioned ELP genes are named according to the number and identity of the amino acid incorporated in the TAG codons. For example, ELP6o(lxlO) is the protein product of the ELP6O( 10TAG) gene, wherein 1 was incorporated in 10 encoded TAG codons.
[0237] The ELP60 protein series was first produced in the C321.ARF1 strain by using AzoRS-4 and azobenzene-uAA 1. To determine protein yields, small batches of ELP6o(lx2), ELP6o(lx6), and ELP6O(1X10) were purified, and the protein yields were 35.69 ± 3.69, 22.9 ± 1.27, and 24.34 ± 1.69 mg L 1, respectively, as compared with 39.72 ± 0.68 mg L 1 of ELP6o(WT). The accuracy of incorporating 1 was evaluated by intact mass-spectrometry (MS) (Fig. 11), and we also analyzed tryptic fragments of the MS-optimized reporter protein ELP6O(10TAG)MS (Table 1) expressed with 1 (i.e., ELP6O(1X10)MS) by liquid chromatography-MS (LC-MS), which identifies and assesses the extent of natural amino acid misincorporation more accurately than intact MS. The incorporation of 1 by AzoRS-4 was detected in -97% of the total ions, as compared with ~22% detected by AzoRS (Tables 4-6).
[0238] Tables 4-7. Sequence and signal intensities of peptides identified LC-MS of tryptic fragments. X denoted the azobenzene-uAA. [0239] Table 4: ELP6O(10X1)MS, expressed by AzoRS in the C321.ARF1 strain.
Figure imgf000079_0001
[0240] Table 5: ELP6O(10X1)MS, expressed by AzoRS-4 in the C321.ARF1 strain.
Figure imgf000079_0002
[0241] Table 6: ELP6O(10X2)MS, expressed by AzoRS-4 in the C321.ARF1 strain.
Figure imgf000079_0003
[0242] Table 7: ELP6O(10X3)MS, expressed by AzoRS-4 in the C321.ARF1 strain.
Figure imgf000079_0004
[0243] To determine the ability of a light-mediated isomerization of 1 to engender a difference in the Tt of the ELP (indicated as ATtds/trans), the ELPs were irradiated at 365 nm or 405 nm to induce isomerization to the cis (more hydrophilic) or trans (more hydrophobic) configuration, respectively. As expected, ELPs bearing mostly the cis isomers exhibited a higher Tt than ELPs bearing mostly the trans isomers. The ATtds/trans induced by the isomerization process increased with the number of incorporated instances of 1, from zero [for the control protein ELP6o(tyrosinexlO); Fig. 12] to ~12 °C for ELPeo(lxlO) (Fig. 13A-C). It is also evident that the Tt of the ELPs decreases with increasing azobenzene content. Next, the secondary structure of light-irradiated ELPs was examined by using circular dichroism (CD) spectroscopy at various temperatures. All ELPs — including the control ELPs — showed the characteristic disordered negative peak at around -190 nm, which decreased in magnitude as the temperature increased (Fig. 13D-F). Notably, the magnitude of the negative peak was greater in the control ELP6o(tyrosinexlO) than in ELPs containing 1, and it was similar in the control ELP6o(benzophenonexlO), which contains an uAA with two aromatic rings, and in ELPeo(lxlO) (Fig. 13J-K). The effect of isomerization was also evident in the CD spectra and increased with increasing numbers of 1 incorporated per ELP chain. The irradiation time required to induce the change in the Tt for ELPeo(lxlO), which showed the largest ATtcis/u-ans, was further investigated. Under these experimental conditions, 30 s of irradiation with blue or UV light were sufficient to generate -90% and -60%, respectively, of the maximum ATtds/trans (defined as the ATtcis/u-ans observed after 30 min of irradiation) in this ELP (Fig. 13G-H). In addition, increasing the concentration of ELP6o(lx10) (from 12.5 mM to 50 mM) also increased the observed ATtds/trans (Fig. 13L-N). Interestingly, ELPs bearing cis isomers of 1, but not those bearing the trans isomer, exhibited a moderate thermal hysteresis, i.e., the Tt observed when heating the ELP solution was lower from that observed while cooling it (Fig. 130). The OD changes in the ELP60(lxl0) solution were nearly identical throughout 10 successive 30 second- or 3 -minute irradiation cycles (Fig. 131), indicating that the effect of isomerization on the Tt was reversible throughout multiple (and short) irradiation cycles. Finally, we confirmed that light irradiation indeed induced the isomerization of 1 within the ELP by examining the UV-vis spectrum of ELP(lxlO) after light irradiation. Indeed, the characteristic peaks associated with the cis and trans isomers of 1 were clearly visible (Fig. 13P-R) and reversible throughout 10 successive irradiation cycles (Fig. 131). [0244] There are two previous studies in which ELPs containing 1 (or any azobenzene) have been produced, in both cases by the polymerization of chemically synthesized peptides containing various quantities of 1 in the X-guest residue. In “Photomodulation of the inverse temperature transition of a modified elastin poly(pentapeptide)”, Strzegowski, et al, Journal of the American Chemical Society 1994, 116, 813-814, an ELP was synthesized with the sequence (VPGXG)n, in which the mole fraction of X was 68% valine (V) and 32% azobenzene and n > 120. This ELP exhibited a ATtds/u-ans of 10 °C. In “Spiropyran derivative of an elastin-like bielastic polymer: photoresponsive molecule machine to convert sunlight into mechanical work”, Alonso et ah, Macromolecules 2001, 34, 8072-8077, ELP polymers were chemically synthesized and polymerized with the sequence [fv(VPGVG), fx(VPGXG)]n, where X represents an azobenzene -bearing amino acid and fv and fx represent the mole-fraction of each pentapeptide. This study also demonstrated that the ELP Tt can be manipulated by triggering the cis-trans isomerization of the azobenzene groups, with a ATtcis/u-ans 0.5 or 5 °C for ELPs with a 5% or 15% mole fraction of azobenzene incorporated in the X position, respectively. However, the molecular weight distribution of these ELPs was not reported and only the VPGVG motif was explored. In contrast, the incorporation of 1 by the evolved aaRSs disclosed herein enabled the precise production of ELPs bearing various numbers of 1, allowing us to evaluate the effect of increasing azobenzene incorporation on the Tt of the ELP by comparing it across ELPs comprising exactly 60 pentapeptides for each construct. The findings presented herein demonstrate that the incorporation of 1 into more hydrophilic ELPs, at mole fractions ranging between 3.3% for ELP6O(1X2) to 16.7% for ELP6o(lxlO), can generate a Tt difference of -5-12 °C (when examined at 25 mM). These findings suggest that the natural amino acids incorporated in the remaining X- guest residue positions may also affect the ATtcis/u-ans generated upon azobenzene isomerization. Thus, the evolved aaRSs described in this study, which permit the precise positioning of azobenzene units alongside other, natural amino acids in various ELP and ELP-inspired sequences, will facilitate the examination of additional sequence determinants for encoding light responsiveness in ELPs.
Example 6: Producing ELPs with a visible-light-responsive phase-separation behavior
[0245] Since UV light is less preferable than visible light for biological applications, the effects of azobenzene substituents, previously shown to permit isomerization with visible light, on the ELP ATtcis/trans were examined next. To this end, ELPs (as described above) were produced containing the visible light-responsive azobenzene-uAAs 2 and 3, using AzoRS-4 in the C321.ARF1 strain. The protein yields of ELP6o(2xlO) and ELP6o(3xlO) were 10.7 ± 0.44 and 2.63 ± 0.77 mg L 1, respectively, as determined by small-batch expression. The fidelity of incorporation was assessed by the LC-MS of tryptic fragments of ELP6O(10TAG)MS, expressed as ELP6O(2X10)MS and ELP6O(3X 10)MS (~97% of the total ions for 2 and ~82% of the total ions for 3; Tables 6-7). The visible-light irradiation of 2, as compared with 1, produces lower quantities of each isomer in the photo-stationary state (PSS) (Fig. 9B). Nevertheless, since the ELP phase transition involves both intra and inter-chain interactions, and since multiple instances of 2 are incorporated per ELP chain, it was hypothesized that the isomerization of even a small majority of 2 would suffice to change the overall ELP hydrophobicity and, therefore, its Tt upon isomerization. Indeed, the light-irradiation of ELP6o(2x2), ELP6o(2x6), and ELP6o(2xlO) produced a more modest - but reproducible - ATtcis/u-ans (namely, a difference of between ~ 1 °C and ~3 °C for a 25 mM solution) upon light irradiation (Fig. 14A-C), which was also slightly affected by ELP concentration (Fig. 14G-I), which can be attributed to the lower PSS composition of 2 compared with 1. Nevertheless, a 30 s irradiation was sufficient to induce -75% of the maximum ATtcis/u-ans for ELP6o(2xlO) (Figure 4D-E). The effect of isomerization on the Tt was reversible throughout 10 successive irradiation cycles (Fig. 14F) and a light-induced isomerization was clearly visible by the UV-vis spectrum of the visible-light irradiated ELP6O(2X10) (Fig. 13P-R). The CD signature of ELP6o(2xlO) confirmed its disordered conformation, albeit with smaller variations in the magnitude of the negative peak (around 190 nm) between ELPs bearing mostly cis or mostly trans isomers, as compared with ELP60(1X10) (Fig. 14J-L).
[0246] Next, the light-responsive properties of ELPs bearing multiple instances of 3 were examined. These ELPs have excellent photoisomerization efficiencies, reaching PSS compositions of 91% and 84% in the cis and trans configurations, respectively (Fig. 9B). Surprisingly, the visible-light irradiation of ELP6o(3xlO) produced the smallest effect on the ELP Tt (~0 °C for a 12.5 pM solution) of all of the examined azobenzene-uAAs (Fig. 15), and the incorporation of 3 appeared to decrease the ELP Tt to a greater extent than the incorporation of 1 or 2 (which prevented analysis of ELP6o(3xlO) and ELP6o(3><6) at 25 pM). This finding was apparently unrelated to the PSS ratios, as an efficient isomerization was clearly visible by the UV-vis spectrum of the light-irradiated ELP6o(3xlO) (Fig. 13P-R). The CD signature of ELP6O(3X10) also revealed that the magnitude of the negative peak observed around 190 nm was different between ELPs bearing mostly cis or mostly trans isomers, and these differences were of a similar extent to those observed in ELP6o(lxlO) at 10 °C (Fig. 14J-L).
[0247] Azobenzene molecules are known to self-assemble and stimulate the self-assembly of various azobenzene conjugates. Therefore, it was hypothesized that azobenzene molecules also engender ELP self-assembly, which, in turn, may affect the local ELP concentration and, therefore, its Tt. Indeed, even when present as an amino acid side-chain, molecule 1 clearly self- assembled, in both the cis and trans configurations, and in different geometries depending on the isomerization state (Fig. 16A-B). To examine the effects of azobenzene properties and content on ELP self-assembly, first a dynamic light-scattering (DLS) analysis of all azobenzene- containing ELPs and of control ELPs containing 10 instances of tyrosine or benzophenone- bearing uAAs was performed. While ELPs bearing either 6 or 10 azobenzene -uAAs assembled into structures as large as a few hundred nanometers, ELPs bearing only two instances of azobenzene -uAAs 1 or 2 did not appear to self-assemble (control ELPs containing 10 instances of tyrosine or benzophenone-bearing uAAs also did not appear to assemble). In contrast, ELPs bearing only two instances of 3 did appear to self-assemble (Fig. 16A-V). To examine the geometries of the self-assembled nanostructures, we imaged ELP6o(lxlO), ELP6o(2xlO), and ELP6O(3X10) by cryo-transmission electron microscopy (cryo-TEM). All azobenzene-ELPs self- assembled into thin sheets, but ELP6o(2xlO) and ELP6o(3xlO) also formed clusters of aggregates. Notably, the number of aggregates of ELP6o(3xlO) was higher than the number of self-assembled sheets and much higher than the number of ELP6o(2xlO) aggregates (Fig. 17A-F). The control ELP6o(tyrosinexlO) did not show a self-assembly behavior (not shown). These findings raise the possibility that the somewhat different geometries and the greater self-assembly/aggregation tendency of ELP6o(3xlO) structures increase the local concentrations of these ELPs and, thereby, decrease their Tt and eliminate Tt differences in the cis- and trans- azobenzene isomerization state. Additionally, the ability of 3 to engender light-responsiveness and promote self-assembly may be attributed to the non-planar geometry of the trans configuration of 3, as it has been reported that changes in hydrogel elasticity were more modest when using the azobenzene side- chain of 3 than when using the azobenzene side-chain of 1, presumably because the trans configuration of 3 deviates significantly from planarity, which weakens its p-stacking ability. [0248] Genetically encoding various azobenzene-uAAs has allowed the comparison of ELPs of identical sequences and molecular weights while varying only the type of the incorporated azobenzene. Thereby, additional factors were identified related to the properties of azobenzene (derived from their substituent pattern), which may affect the self-assembly and phase-transition behavior of azobenzene-containing ELPs.
Example 7: Producing ELP diblock copolymers with a light-responsive self-assembly behavior
[0249] Numerous studies demonstrated that the genetic fusion of two or more ELP blocks of varying hydrophilicities can generate self-assembled nanostructures with various geometries. Building on this knowledge, self-assembling ELPs composed of a hydrophilic block and a hydrophobic block were designed, namely without and with azobenzene-uAAs, respectively. It was hypothesized that the change in the hydrophobicity of the azobenzene-uAAs upon isomerization would also modulate nanostructure assembly. The design was based on previously characterized ELP diblock copolymers that were shown to self-assemble to form spherical micelles at ratios between 1 :2 and 2: 1 of the hydrophilic: hydrophobic ELP blocks. An ELP fusion protein, termed ELP6o(WT)-ELP6o(10TAG), consisting of the gene for ELP6o(WT) (the hydrophilic block) fused at the genetic level to the gene for ELP6o(10TAG) (the hydrophobic block) was generating, thus setting a 1:1 hydrophilic :hydrophobic block ratio. ELP6o(WT)- ELP6O(1X10) and ELP6o(WT)-ELP6o(2xlO) were then expressed and their light-responsive self- assembly behavior characterized using UV-vis spectrometry and DLS. It was previously hypothesized that the diblock ELP assembly into nanostructures is due to the transition of the hydrophobic block, which is visible as a slight increase in the optical density of the solution at temperatures below the Tt, as was also apparent for ELP6o(WT)-ELP6o(lxlO) and ELP6o(WT)- ELP6O(2X10) (Fig. 18A, solid lines, Fig. 18G). The DLS confirmed the formation of self- assembled nanostructures for all proteins and the light-dependent assembly of these structures, with a ATSELF-ASSEMBLY of ~5 for ELP6o(WT)-ELP6o(lxlO) and of ~1 °C for ELP6o(WT)- ELP6O(2X10) (Fig. 18A, colored dots, Fig. 18G). Thus, it appears that identical azobenzene bearing ELPs (i.e., ELP6o(lxlO) and ELP6o(2xlO)) generate a larger ATt of mono-block phase transition than a ATSELF-ASSEMBLY as hydrophobic segments in diblock ELPs. Notably, while nanostructures of -25^15 nm were the predominant species observed in all proteins, small amounts (-2% by volume at the onset of micelle formation) of larger nanostructures (a several hundred nm) appeared to form as well, and their proportion increased with increasing temperatures (up to -15% or 25% for ELP6o(WT)-ELP6o(lxlO) and of ~1 °C for ELP6o(WT)- ELP6O(2X10), respectively, at 40 °C). Nevertheless, we were again able to confirm the reversibility of the isothermal light-mediated self-assembly of ELP6o(WT)-ELP6o(lxlO) during 10 successive irradiation cycles of only 30 s each, which generated populations of >95% monomer or self-assembled nanostructures by azobenzene isomerization (Fig. 18B, 18H).
[0250] Surprisingly, while diblocks bearing the more hydrophilic cis isomers were expected to demonstrate a higher Tt than that of diblocks bearing the more hydrophobic trans isomers, the opposite was observed: the ATtds/trans was — 5 °C and — 1 °C for ELP6o(WT)-ELP6o(lxlO) and ELP6O(WT)-ELP6O(2X 10), respectively. This finding suggests that the architecture and, accordingly, the properties of the self-assembled nanostructures depend on the azobenzene isomerization state. The DLS analysis also suggests that nanostructures assembled from the trans-ELP6o(WT)-ELP6o(lxlO) were slightly (-5-10 nm) but consistently larger than those assembled from the cis-ELP6o(WT)-ELP6o(lxlO) (Fig. 17A). To examine the self-assembled structures and how they differ upon irradiation, both the cis and trans configurations of the self- assembled ELP6O(WT)-ELP6O(1X 10) and ELP6o(WT)-ELP6o(2x 10) were examined by cryo-TEM at 38 °C, a temperature in all constructs self-assembled. It was found that both spherical nanostructures of -100-200 nm (similar in appearance to the spherical micelles generated by other ELP diblocks) and larger (several hundred nm), sheet-like structures were present in all samples, however spherical structures were rare in the cis-ELP6o(WT)-ELP6o(lxlO) and were less symmetrical (tear-drop shaped). In addition, aggregate clusters of -100-200 nm diameter were apparent in the cis-ELP6o(WT)-ELP6o(lxlO) but were rare in the trans-ELPeo(WT)- ELP6O(1X 10) sample (Fig. 18C-D, 18I-N). Thus, it is plausible that the isomerization of 1 affected not only the critical self-assembly temperature (thus generating a ATSELF- ASSEMBLY) but also the morphology of the self-assembled nanostructures across the entire self-assembly temperature range. In contrast, the light irradiation of 2 generated a more modest response, reflected both in the ATSELF-ASSEMBLY and in the indistinguishable morphologies of the nanostructures generated by mostly-cis or mostly-trans ELP diblock. Numerous studies demonstrated the design of ELP block-copolymers that self-assembled into a wide variety of geometries, both in vitro and inside cells. However, this is the first demonstration that previously established guidelines for thermal ELP assembly can be employed to generate light-responsive formation of nanostructures. These results indicate that, by genetically encoding azobenzene- uAAs in multi-block ELP polymers, engineering light-responsive self-assembly or change in structural properties of additional geometries can be informed by previously described guiding principles for generating such assemblies.
[0251] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

Claims:
1. A mutant aminoacyl-tRNA synthetase (aaRS) comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to threonine; leucine 65 mutated to valine; glutamic acid 107 mutated to alanine; phenylalanine 108 mutated to tyrosine; glutamine 109 mutated to methionine; aspartic acid 158 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to alanine; isoleucine 159 mutated to methionine; isoleucine 159 mutated to cysteine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to glutamic acid; leucine 162 mutated to lysine; leucine 162 mutated to valine; leucine 162 mutated to arginine; leucine 162 mutated to serine; leucine 162 mutated to cysteine; alanine 167 mutated to histidine, alanine 167 mutated to aspartic acid and alanine 167 mutated to tyrosine.
2. The mutant aaRS of claim 1, wherein said mutant is selected from the group consisting of: a. a mutant comprising tyrosine 32 mutated to leucine, aspartic acid 158 mutated to serine, isoleucine 159 mutated to methionine, leucine 162 mutated to lysine, and alanine 167 mutated to histidine; b. a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to alanine, leucine 162 mutated to glutamic acid, and alanine 167 mutated to histidine; c. a mutant comprising alanine 32 mutated to threonine, leucine 65 mutated to valine, glutamic acid 107 mutated to alanine, phenylalanine 108 mutated to tyrosine, glutamine 109 mutated to methionine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to cysteine, leucine 162 mutated to arginine, and alanine 167 mutated to aspartic acid; d. a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to methionine, leucine 162 mutated to serine, and alanine 167 mutated to histidine; and e. a mutant comprising tyrosine 32 mutated to leucine, leucine 65 mutated to valine, aspartic acid 158 mutated to glycine, isoleucine 159 mutated to tyrosine, alanine 162 mutated to cysteine, and alanine 167 mutated to tyrosine.
3. The mutant aaRS of claim 1 or 2, wherein said mutant aaRS comprises an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
4. A mutant aminoacyl-tRNA synthetase (aaRS) comprising an amino acid sequence of an aaRS comprising at least one amino acid mutation selected from the group consisting of: tyrosine 32 mutated to leucine, tyrosine 32 mutated to glycine; leucine 65 mutated to valine; leucine 65 mutated to glycine; glutamic acid 107 mutated to serine; glutamic acid 107 mutated to asparagine; glutamic acid 107 mutated to aspartic acid; phenylalanine 108 mutated to valine; phenylalanine 108 mutated to arginine; glutamine 109 mutated to methionine; glutamine 109 mutated to serine; glutamine 109 mutated to leucine; and glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; leucine 162 mutated to arginine; and alanine 167 mutated to phenylalanine.
5. The mutant aaRS of claim 4, comprising: a. aspartic acid 158 mutated to glycine; b. isoleucine 159 mutated to tyrosine; and c. leucine 162 mutated to serine or leucine 162 mutated to arginine.
6. The mutant aaRS of claim 5, further comprising alanine 167 mutated to phenylalanine.
7. The mutant aaRS of claim 5 or 6, further comprising tyrosine 32 mutated to leucine or tyrosine 32 mutated to glycine.
8. The mutant aaRS of any one of claims 5 to 7, further comprising leucine 65 mutated to valine or leucine 65 mutated to glycine.
9. The mutant aaRS of any one of claims 4 to 8, wherein said mutant is selected from the group consisting of: a. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; b. a mutant comprising tyrosine 32 mutated to glycine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; c. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to serine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; d. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to asparagine, phenylalanine 108 mutated to valine, glutamine 109 mutated to leucine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; e. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to aspartic acid, aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; f. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; glutamic acid 107 mutated to serine, phenylalanine 108 mutated to valine, glutamine 109 mutated to cysteine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine; g. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to valine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; and leucine 162 mutated to arginine; and h. a mutant comprising tyrosine 32 mutated to leucine, lysine 65 mutated to glycine; glutamic acid 107 mutated to aspartic acid, phenylalanine 108 mutated to arginine, glutamine 109 mutated to methionine; aspartic acid 158 mutated to glycine; isoleucine 159 mutated to tyrosine; leucine 162 mutated to serine; and alanine 167 mutated to phenylalanine.
10. The mutant aaRS of any one of claims 4 to 9, wherein said mutant comprises an amino acid sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
11. The mutant aaRS of any one of claims 1 to 10, wherein said amino acid sequence of an aaRS is SEQ ID NO: 1.
12. The mutant aaRS of any one of claims 1 to 11, further comprising a mutation of arginine 257 to glycine, a mutation of aspartic acid 286 to arginine or both.
13. A nucleic acid molecule comprising a coding region encoding the mutant aaRS of any one of claims 1 to 12.
14. The nucleic acid molecule of claim 13, wherein said coding region comprises a nucleic acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
15. The nucleic acid molecule of claim 13 or 14, wherein said coding region is operably linked to at least one regulatory element configured to express said coding region in a target cell.
16. An orthogonal translation system, comprising, a. a mutant aaRS of any one of claims 1 to 12, or a nucleic acid molecule of any one of claims 13 to 15, and b. an orthogonal tRNA compatible with said mutant aaRS and comprising an anticodon that corresponds to a stop codon.
17. The orthogonal translation system of claim 16, further comprising a non-standard amino acid (nsAA) recognized by said mutant aaRS.
18. The orthogonal translation system of claim 17, wherein said nsAA is an unnatural amino acid (uAA).
19. The orthogonal translation system of claim 18, wherein said uAA comprises a biorthogonal chemical moiety.
20. The orthogonal translation system of claim 19, wherein said mutant aaRS is the mutant aaRS of any one of claims 1 to 3 and said uAA comprises an azide or an alkyne group.
21. The orthogonal translation system of claim 19, wherein said mutant aaRS is the mutant aaRS of any one of claims 4 to 10 and said uAA comprises an azobenzene group.
22. The orthogonal translation system of any one of claims 17 to 21, wherein said nsAA is a modified phenylalanine.
23. The orthogonal translation system of claim 22, wherein said modified phenylalanine is 4-propargyloxy-L-phenylalanine (pPR).
24. The orthogonal translation system of claim 21, wherein said uAA comprising an azobenzene group is selected from phenylalanine-4’ -azobenzene (AzoPhe). tri- fluorinated azobenzene (Azo3F), and tetra-ortho-fluorinated azobenzene (Azo4F) amino acids.
25. The orthogonal translation system of any one of claims 16 to 24, wherein said stop codon is a TAG stop codon.
26. A cell comprising an orthogonal translation system of any one of claims 16 to 25.
27. The cell of claim 26, further comprising an expression vector comprising an open reading frame (ORF) comprising at least one of said stop codons within said open reading frame.
28. The cell of claim 27, wherein said ORF comprises a plurality of stop codons.
29. The cell of claim 27 or 28 wherein said ORF comprises at least 10 stop codons.
30. The cell of any one of claims 26 to 29, wherein said ORF is operatively linked to at least one regulatory element capable of inducing expression of said ORF within said cell.
31. The cell of any one of claims 26 to 30, wherein said cell is devoid of native TAG stop codons and does not express release factor 1 (RF1).
32. The cell of any one of claims 26 to 30, wherein said cell comprises RF1 and at least one native TAG stop codon.
33. A method of producing a protein comprising a nsAA, the method comprising introducing into a cell an expression vector comprising an open reading frame encoding said protein wherein said open reading frame comprises a stop codon, wherein said cell comprises an orthogonal translation system of any one of claims 16 to 32.
34. The method of claim 33 for labeling said protein, the method further comprising converting said nsAA into a detectably labeled amino acid and wherein said mutant aaRS is the mutant aaRS of any one of claims 1 to 3.
35. The method of claim 34, wherein said converting comprises addition of a detectable moiety by Click chemistry.
36. The method of claim 33 for producing a light-responsive protein, wherein said mutant aaRS is the mutant aaRS of any one of claims 4 to 10.
37. A protein comprising a nsAA produced by a method of any one of claims 33 to 36.
PCT/IL2021/050194 2020-02-20 2021-02-18 Mutant aminoacyl-trna synthetases WO2021165968A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP21757370.8A EP4107258A4 (en) 2020-02-20 2021-02-18 Mutant aminoacyl-trna synthetases
US17/892,163 US20230313168A1 (en) 2020-02-20 2022-08-22 Mutant aminoacyl-trna synthetases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202062978895P 2020-02-20 2020-02-20
US62/978,895 2020-02-20

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/892,163 Continuation US20230313168A1 (en) 2020-02-20 2022-08-22 Mutant aminoacyl-trna synthetases

Publications (1)

Publication Number Publication Date
WO2021165968A1 true WO2021165968A1 (en) 2021-08-26

Family

ID=77391696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2021/050194 WO2021165968A1 (en) 2020-02-20 2021-02-18 Mutant aminoacyl-trna synthetases

Country Status (3)

Country Link
US (1) US20230313168A1 (en)
EP (1) EP4107258A4 (en)
WO (1) WO2021165968A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114908066A (en) * 2022-05-17 2022-08-16 浙江大学 Orthogonal translation system and application thereof in restoring functional protein expression in PTC (Positive temperature coefficient) diseases by using redistributed codons

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244245A1 (en) * 2004-10-27 2013-09-19 The Scripps Research Institute Orthogonal Translation Components for the in Vivo Incorporation of Unnatural Amino Acids
WO2015120287A2 (en) * 2014-02-06 2015-08-13 Yale University Compositions and methods of use thereof for making polypeptides with many instances of nonstandard amino acids

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2444098C (en) * 2001-04-19 2016-06-21 The Scripps Research Institute Methods and composition for the production of orthogonal trna-aminoacyltrna synthetase pairs
CN102888387B (en) * 2011-07-21 2015-03-18 中国科学院生物物理研究所 3-chlorinated tyrosine translation system and application thereof
HUE042421T2 (en) * 2013-10-11 2019-07-29 Sutro Biopharma Inc NON-NATURAL AMINO ACID tRNA SYNTHETASES FOR PARA-METHYLAZIDO-L-PHENYLALANINE

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244245A1 (en) * 2004-10-27 2013-09-19 The Scripps Research Institute Orthogonal Translation Components for the in Vivo Incorporation of Unnatural Amino Acids
WO2015120287A2 (en) * 2014-02-06 2015-08-13 Yale University Compositions and methods of use thereof for making polypeptides with many instances of nonstandard amino acids

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4107258A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114908066A (en) * 2022-05-17 2022-08-16 浙江大学 Orthogonal translation system and application thereof in restoring functional protein expression in PTC (Positive temperature coefficient) diseases by using redistributed codons
CN114908066B (en) * 2022-05-17 2024-01-23 杭州嵌化合生医药科技有限公司 Orthogonal translation system and application thereof in redistribution codon recovery of functional protein expression in PTC disease

Also Published As

Publication number Publication date
US20230313168A1 (en) 2023-10-05
EP4107258A4 (en) 2024-04-17
EP4107258A1 (en) 2022-12-28

Similar Documents

Publication Publication Date Title
JP7474532B2 (en) Method for Rescuing Stop Codons via Genetic Reassignment Using ACE-tRNA
JP4229469B2 (en) Humanized green fluorescent protein gene and method
CN114672473B (en) Optimized Cas protein and application thereof
WO2013044792A1 (en) Nicotinamide adenine dinucleotide gene encoding fluorescent probe, preparation method therefor and application thereof
US20230313168A1 (en) Mutant aminoacyl-trna synthetases
CN114410609B (en) Cas protein with improved activity and application thereof
CN114507654B (en) Cas enzymes and systems and applications
CN109134644B (en) Far-red light fluorescent protein and fusion protein thereof
Israeli et al. Genetically encoding light‐responsive protein‐polymers using translation machinery for the multi‐site incorporation of photo‐switchable unnatural amino acids
US8679749B2 (en) Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation
WO2015184283A1 (en) Tethered ribosomes and methods of making and using thereof
US10597735B2 (en) Antibacterial and plasmid elimination agents
WO2011096501A1 (en) Photosensitizing fluorescent protein
KR101523834B1 (en) Red Fluorescence Protein Variants
EP3031821A1 (en) polyomavirus VLPs with a fusion protein
Mannervik Optimizing the heterologous expression of glutathione transferase
WO2020201115A1 (en) Optogenetic switches in bacteria
US20220267387A1 (en) Flavin mononucleotide-binding protein variants having improved fluorescence intensity derived from arabidopsis thaliana
US20230287065A1 (en) Split intein mediated protein polymerization for microbial production of materials
US20110288008A1 (en) Antibacterial and plasmid elimination agents
KR101973275B1 (en) Generation of fluorescent bacteria with lumazine protein and riboflavin biosynthetic genes
JP2022117439A (en) Method for linking multi-fragment dna and introducing the same into cell by using in vitro packaging
JP2022552137A (en) Chimeric Thermostable Aminoacyl-tRNA Synthetases for Enhanced Unnatural Amino Acid Incorporation
Abd Elhameed Artificial metallonucleases-molecular tools for gene therapy of cancer
WO2023049826A1 (en) Genetically encoded voltage indicators and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21757370

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021757370

Country of ref document: EP

Effective date: 20220920