Patent Application
20230313168
The contents of the electronic sequence listing (BGU-P-098-US.xml; Size: 139,200 bytes; and Date of Creation: Jun. 20, 2023) is herein incorporated by reference in its entirety.
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.
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.
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.
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.
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 (λtrans→cis), the azobenzene molecule undergoes a dramatic switch from the trans to the cis configuration (shortening by at least ˜3.5 Å), 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 (λcis→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. 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.
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.
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.
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.
According to some embodiments, the mutant is selected from the group consisting of:
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.
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.
According to some embodiments, the mutant aaRS of the invention comprises:
According to some embodiments, the mutant aaRS of the invention further comprises alanine 167 mutated to phenylalanine.
According to some embodiments, the mutant aaRS of the invention further comprises tyrosine 32 mutated to leucine or tyrosine 32 mutated to glycine.
According to some embodiments, the mutant aaRS of the invention further comprises leucine 65 mutated to valine or leucine 65 mutated to glycine.
According to some embodiments, the mutant is selected from the group consisting of:
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.
According to some embodiments, the amino acid sequence of an aaRS is SEQ ID NO: 1.
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.
According to another aspect, there is provided a nucleic acid molecule comprising a coding region encoding a mutant aaRS of the invention.
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.
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.
According to another aspect, there is provided an orthogonal translation system, comprising,
According to some embodiments, orthogonal translation system of the invention further comprises a non-standard amino acid (nsAA) recognized by the mutant aaRS.
According to some embodiments, the nsAA is an unnatural amino acid (uAA).
According to some embodiments, the uAA comprises a biorthogonal chemical moiety.
According to some embodiments, the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azide or an alkyne group.
According to some embodiments, the mutant aaRS is the mutant aaRS of the invention and the uAA comprises an azobenzene group.
According to some embodiments, the nsAA is a modified phenylalanine.
According to some embodiments, the modified phenylalanine is 4-propargyloxy-L-phenylalanine (pPR).
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.
According to some embodiments, the stop codon is a TAG stop codon.
According to another aspect, there is provided a cell comprising an orthogonal translation system of the invention.
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.
According to some embodiments, the ORF comprises a plurality of stop codons.
According to some embodiments, the ORF comprises at least 10 stop codons.
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.
According to some embodiments, the cell is devoid of native TAG stop codons and does not express release factor 1 (RF1).
According to some embodiments, the cell comprises RF1 and at least one native TAG stop codon.
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.
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.
According to some embodiments, the converting comprises addition of a detectable moiety by Click chemistry.
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.
According to another aspect, there is provided a protein comprising a nsAA produced by a method of the invention.
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.
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.
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 well 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
By a first aspect, the present invention provides a mutant aminoacyl-tRNA synthetase (aaRS).
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.
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.
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.
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.
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.
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.
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.
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.
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-1-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.
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.
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.
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”.
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.
In some embodiments, the aaRS into which the mutation is introduced comprises or consists of the amino acid sequence
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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:
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.
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.
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.
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.
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.
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.
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.
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.
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: 19 or a fragment, analog or derivative thereof.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, the vector is introduced into a cell by standard methods including electroporation (e.g., as described in From et al., 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 al., 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.
Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., 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, 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.
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.
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 herpesviral 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.
In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, 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.
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-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-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.
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.
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 hsp17.5-E or hsp17.3-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.
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.
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).
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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 TD NO: 11.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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 TD NO: 20.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, a sequence of the nucleic acid molecule comprises or consists of the sequence:
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.
In some embodiments, each of SEQ ID NO:7-11 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.
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.
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.
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).
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.
In another aspect, there is provided an orthogonal translation system, comprising:
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.
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.
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.
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.
In another aspect, there is provided a cell comprising a mutant aaRS of the invention.
In another aspect, there is provided a cell comprising a nucleic acid molecule of the invention.
In another aspect, there is provided a cell comprising an orthogonal translation system of the invention.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
By another aspect, there is provided a protein produced by a method of the invention.
By another aspect, there is provided a protein comprising a nsAA.
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.
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.
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.
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.
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.
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.
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.
Other terms as used herein are meant to be defined by their well-known meanings in the art.
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.
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.
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 al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “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 al., “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, C T (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: The uAA para-propargyloxy-1-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 (CuSO4) and Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) were purchased from Sigma-Aldrich, BugBuster protein extraction reagent was purchased from Merck.
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.ΔA (Isaacs lab) and pEvol-pAzFRS.1.t1 were a gift from Farren Isaacs (Addgene plasmids #73581 and #73547). Isomerization experiments were performed with a 365 nm 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).
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.t1, pAcFRS.2.t1 and pAzFRS.2.t1. Prior to MAGE cycling, cultures were established by inoculating the liquid medium with a single bacterial colony or by adding 30 μl 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 15,000 g 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 μM 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.
Both colE1 (negative) or SDS (positive) selections were optimized by testing LB broth containing various concentrations of colE1 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 E1 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/μl 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.
Plasmid construction: Plasmids bearing GFP-based reporter genes were known in the art. Plasmids bearing the OTS variants for pPR incorporation were constructed by insertion of aaRS genes to a previously described plasmid harboring a p15A 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 BglII and SalI, 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 XhoI and HindIII restriction enzymes, and ligated to a similarly cut reporter plasmid. The ELP60 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-uAA incorporation were constructed by inserting aaRS genes into a previously described plasmid (pEvol) harboring a p15A 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 BglII and SalI 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 μg ml−1) and analyzed by Sanger sequencing.
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 μg/ml kanamycin. For cells harboring plasmid-based orthogonal translation system and GFP reporter proteins, the media was also supplemented with 25 μg/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/μl anhydrotetracycline, 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.
Alternatively, Cultures were then inoculated at a 1:50 dilution in 2xYT medium supplemented with kanamycin (30 μg ml−1). For cells harboring the plasmid-based orthogonal translation system and GFP reporter proteins, the media were also supplemented with chloramphenicol (25 μg ml−1). Cells were allowed to grow at 34° C. to an OD600 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 OD600 reading.
ELP expression and purification: Before batch expression, starter cultures (1:25 v/v of final expression volume) of 2xYT media supplemented with 30 μg/ml kanamycin and 25 μg/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 Mut1-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 mM uAA. Cells were incubated at 34° C. for 4-5 h and then reporter protein expression was induced with 60 μg/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.
Protein concentration was calculated by measuring the OD280 of purified protein according to the following extinction coefficients: Tyr (WT protein): 33,935, ELP(1pPR)-GFP: 33,645, ELP(5pPR)-GFP: 32,485, ELP(10pPR)-GFP: 31,035, based on extinction coefficient of pPR (1200 M·cm−1).
Alternatively, before batch expression, starter cultures (1:40 v/v of final expression volume) of 2xYT media, supplemented with kanamycin (30 μg ml−1) and chloramphenicol (25 μg 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-uAA (0.25 mM). For the expression of ELP60(10TAG), ELP60(6TAG), and ELP60(2TAG) by AzoRS-4, the C321.ΔRF1 strain[40], 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 β-d-1-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.
Protein concentrations were calculated by measuring the OD280 of the purified protein according to the following extinction coefficients: ELP60(tyrosine×10): 16,390, ELP60(1×10): 26,900, ELP60(1×6): 16,736, and ELP60(2×10): 6,572, based on the extinction coefficient of 1 (2,541 M cm−1); ELP60(2×10): 41590, ELP60(2×6): 25550, and ELP60(2×10): 9510, based on the extinction coefficient of 2 (4010 M cm−1); and ELP60(3×10): 79122, ELP60(3×6): 74546, and ELP60(3×10): 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.
In-vivo click reaction: Before the reaction, ELP(1TAG) and ELP(10TAG), (both without GFP) were expressed in the genomically recoded organism or in the BL21 strain, by the parent-pPR-RS or evolved Mut1-RS. Starter cultures of 2xYT media supplemented with 30 μg/ml kanamycin and 25 μg/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 μg/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 CuSO4 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.
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 μM TAMRA, 3 mM THPTA premixed for 20 min with 0.4 mM CuSO4, 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. Laemmli sample buffer was added to supernatant, and denaturation was performed by heating at 100° C. for 5 min.
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 FLA-9500, using 532 nm laser. Equal lysis of all samples was validated by Coomassie blue staining of the gel.
Phase transition analysis: To characterize the inverse transition temperature of ELP variants, the OD600 of the ELP 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).
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.
Circular Dichroism (CD) analysis: The secondary structure of ELPs was studied using an Jasco J-715 spectropolarimeter (Tokyo) equipped with a PTC-348WI temperature controller, 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 μM in water. Data were considered for analysis whenever the Dynode voltage was below 800 V.
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 2.5 μl 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 200 kV maintained at −180° C.; and images are recorded on a FEI Ceta 16M camera (4k×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).
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.
In order to improve multi-site pPR incorporation, a modified MAGE-based protein evolution strategy was employed to identify improved mutants of the M. jannaschii 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 E1 (ColE1)-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 (
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) (
The evolved variants were now evaluated for multisite (3-30) pPR incorporation. C321.ΔRF1 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 (
The best-performing variant, Mut1-RS (
Given the high incorporation efficiency of the evolved pPR-RS variants in C321.ΔRF1, 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 Mut1-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 Mut1-RS in the BL21 strain showed production rates of ˜140{circumflex over ( )}% and ˜44{circumflex over ( )}%, respectively, compared to WT proteins (
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.ΔA); incorporation of six uAAs yielded ˜6-9{circumflex over ( )}% of WT proteins in the presence of release factor inhibitors; and proteins bearing ten uAAs were produced with yields of ˜3{circumflex over ( )}% 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.
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 ELPs with one or ten instances of pPR expressed in C321.ΔRF1 by using Mut1-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 (
In addition to the expected fluorescent band of labeled ELP(10TAG), 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.ΔRF1 with the Mut1-RS plasmid alone generated the same labeled double bands only when the expression of Mut1-RS was induced, and extraction of these bands from the gel, followed by tryptic digestion and LC-MS analysis, validated the presence of Mut1-RS, confirming the above premise (
It is well known that the application of CuAAC bioconjugation in living systems is hindered due to the CuI-induced generation of reactive oxygen species (ROS), with consequent cellular toxicity. Therefore, the cell viability and the labeling efficiency of ELP(10TAG) produced by Mut1-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(10pPR) with a reduced copper concentration of 50 μM, without a significant decrease in labeling efficiency—evaluated as 56 and 35{circumflex over ( )}% in the presence of 25 μM and 10 μM copper, respectively (
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.
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.
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 kDa 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.
The performance of evolved nsAA-RSs was characterized for the ability to incorporate azobenzene-containing nsAA into proteins. Mutants are shown in
Next, the ability of the Azo-RS variant, to incorporate azobenzene uAAs 1, 2, or 3 (
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.ΔRF1. 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 tolC-mediated negative-positive-negative selection cycles (ColE1-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 (
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 ELP variants was designed to analyze the effect of azobenzene incorporation on the ELP Tt. The design was based on previously described hydrophilic ELPs which have a high Tt (>90° C. for a 25 μM 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 ELP60WT, ELP60(2TAG), ELP60(6TAG), and ELP60(10TAG), 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, ELP60(1×10) is the protein product of the ELP60(10TAG) gene, wherein 1 was incorporated in 10 encoded TAG codons.
The ELP60 protein series was first produced in the C321.ΔRF1 strain by using AzoRS-4 and azobenzene-uAA 1. To determine protein yields, small batches of ELP60(1×2), ELP60(1×6), and ELP60(1×10) 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 ELP60(WT). The accuracy of incorporating 1 was evaluated by intact mass-spectrometry (MS) (
Tables 4-7. Sequence and signal intensities of peptides identified LC-MS of tryptic fragments. X denoted the azobenzene-uAA.
QGVPGGGK (45)
To determine the ability of a light-mediated isomerization of 1 to engender a difference in the Tt of the ELP (indicated as ΔTtcis/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 ΔTtcis/trans induced by the isomerization process increased with the number of incorporated instances of 1, from zero [for the control protein ELP60(tyrosine×10);
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 ΔTtcis/trans of 10° C. In “Spiropyran derivative of an elastin-like bielastic polymer: photoresponsive molecule machine to convert sunlight into mechanical work”, Alonso et al., 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 ΔTtcis/trans 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 ELP60(1×2) to 16.7% for ELP60(1×10), can generate a Tt difference of ˜5-12° C. (when examined at 25 μM). These findings suggest that the natural amino acids incorporated in the remaining X-guest residue positions may also affect the ΔTtcis/trans 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.
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 ΔTtcis/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.ΔRF1 strain. The protein yields of ELP60(2×10) and ELP60(3×10) 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 ELP60(10TAG)MS, expressed as ELP60(2×10)MS and ELP60(3×10)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) (
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 (
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 (
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.
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 ELP60(WT)-ELP60(10TAG), consisting of the gene for ELP60(WT) (the hydrophilic block) fused at the genetic level to the gene for ELP60(10TAG) (the hydrophobic block) was generating, thus setting a 1:1 hydrophilic:hydrophobic block ratio. ELP60(WT)-ELP60(1×10) and ELP60(WT)-ELP60(2×10) 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 ELP60(WT)-ELP60(1×10) and ELP60(WT)-ELP60(2×10) (
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 ΔTtcis/trans was ˜−5° C. and ˜−1° C. for ELP60(WT)-ELP60(1×10) and ELP60(WT)-ELP60(2×10), 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-ELP60(WT)-ELP60(1×10) were slightly (˜5-10 nm) but consistently larger than those assembled from the cis-ELP60(WT)-ELP60(1×10) (
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.
This application is a bypass continuation of PCT Patent Application No. PCT/IL2021/050194 having International filing date of Feb. 18, 2021, which claims the benefit of priority to U.S. Provisional Application No. 62/978,895 titled “MUTANT AMINOACYL-TRNA SYNTHETASES” filed Feb. 20, 2020, the contents of which are all incorporate herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL21/50194 | Feb 2021 | US |
| Child | 17892163 | US |
