WO2020221365A1 - 高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 - Google Patents
高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 Download PDFInfo
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Definitions
- the present invention relates to the field of biotechnology, in particular to an aminoacyl-tRNA synthetase that efficiently introduces lysine derivatives.
- the unnatural amino acid integrated protein obtained by replacing the amino acid residue at the desired position in the protein with the 20 types of amino acids (unnatural amino acids) other than those involved in normal protein synthesis can be used as the structure of the protein Effective means of functional analysis.
- aaRS aminoacyl-tRNA synthetase
- tyrRS tyrosyl-tRNA synthetase
- the key is its orthogonal relationship, that is, aaRS in the two groups of eubacteria, archaea, and eukaryotes acylate tRNA in their respective groups, but cannot acylate the others.
- Group of tRNA is aminoacylated.
- Pyrrolysine is a lysine derivative with a bulky methylpyrroline moiety on the side chain. Wild-type PylRS can bind N ⁇ -Boc-L-lysine to tRNAPyl in E. coli.
- LysRS has strict recognition of lysine, it has been difficult to introduce lysine derivatives with functional groups of various sizes and shapes into proteins site-specifically.
- the art needs to modify the wild-type lysyl-tRNA synthetase and develop an aminoacyl-tRNA synthetase that efficiently introduces lysine derivatives into the protein.
- the purpose of the present invention is to provide an aminoacyl-tRNA synthetase and method for efficiently introducing lysine derivatives into proteins.
- a mutein of aminoacyl-tRNA synthetase is at one or more positions selected from the following group corresponding to the amino acid sequence of wild-type aminoacyl-tRNA synthetase Points are missing: amino acid residues 102, 128-140, and 159-179.
- the plurality includes: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 1, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34.
- the 128th-140th position includes the 128th position, the 129th position, the 130th position, the 131th position, the 132th position, the 133th position, the 134th position, the 135th position, and the 136th position. , 137th, 138th, 139th and 140th.
- the positions 159-179 include positions 159, 160, 161, 162, 163, 164, 165, 166, and 167 , 168th, 169th, 170th, 171th, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th and 180 people.
- the mutant protein has at least truncated the following amino acid residues of the amino acid sequence of wild-type aminoacyl-tRNA synthetase:
- the X is a positive integer between 128-132, such as 128, 129, 130, 131 or 132.
- the X is 128.
- Y is a positive integer between 133-140, such as 133, 134, 135, 136, 137, 138, 139 or 140.
- the Y is 140.
- the A is a positive integer between 159-164, such as 159, 160, 161, 162, 163 or 164.
- the A is 159.
- the B is a positive integer between 165-179, such as 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 or 164 .
- the B is 179.
- the wild-type aminoacyl-tRNA synthetase is derived from Methanosarcina mazei, Methanosarcina barkeri, or Methanosarcina barkeri of the methanogenic archaea. Coccus (Methanosarcina acetivorans).
- amino acid sequence of the wild-type aminoacyl-tRNA synthetase is shown in SEQ ID NO.: 1 or 2.
- the number of the deleted amino acid residues is based on SEQ ID NO.: 1 or 2.
- the mutant protein is a truncated aminoacyl-tRNA synthetase.
- the length of the amino acid sequence of the mutant protein is at least 90%, preferably 92%-99%, of the length of the sequence shown in SEQ ID NO.: 1 or 2.
- the mutein (truncated form) has at least 90%, 95%, 96%, 97%, 98% of the peptide fragment of the corresponding length in SEQ ID NO.: 1 or 2. Or 99% sequence identity.
- the mutant protein has at least truncated amino acid residues 128-140 and 159-179 of the amino acid sequence of wild-type aminoacyl-tRNA synthetase.
- the mutant protein has at least truncated amino acid residues 102, 128-140, and 159-179 of the amino acid sequence of wild-type aminoacyl-tRNA synthetase.
- amino acid sequence of the mutant protein is shown in SEQ ID NO.:3.
- amino acid sequence of the mutant protein is shown in SEQ ID NO.:4.
- the mutant protein has an amino acid mutation selected from the group consisting of histidine at position 29 in the mutant aminoacyl-tRNA synthetase whose sequence is shown in SEQ ID NO.: 4 (H), aspartic acid at position 76 (D), serine at position 89 (S), asparagine at position 91 (N), arginine at position 96 (R), serine at position 121 (S) , Asparagine at position 129 (N), Serine at position 145 (S), Alanine at position 148 (A), Leucine at position 274 (L), Cysteine at position 313 (C), Phenylalanine (F) at position 349, or a combination thereof.
- amino acid number of the mutation site is based on SEQ ID NO.:4.
- the mutant protein is a mutant aminoacyl-tRNA synthetase whose sequence is shown in SEQ ID NO.: 4, and based on the sequence shown in SEQ ID NO.: 4, there is also a mutant protein selected from the following group Amino acid mutations: histidine at position 29 (H), aspartic acid at position 76 (D), serine at position 89 (S), asparagine at position 91 (N), arginine at position 96 (R ), the 121st serine (S), the 129th asparagine (N), the 145th serine (S), the 148th alanine (A), the 274th leucine (L), the 313th Cysteine at position (C), phenylalanine at position 349 (F), or a combination thereof.
- Amino acid mutations histidine at position 29 (H), aspartic acid at position 76 (D), serine at position 89 (S), asparagine at position 91 (N), arginine at position
- the histidine (H) at position 29 is mutated to tyrosine (Y); and/or
- the aspartic acid (D) at position 76 is mutated to glycine (G); and/or
- the 89th serine (S) is mutated to glycine (G); and/or
- the asparagine (N) at position 91 is mutated to threonine (T); and/or
- the arginine (R) at position 96 is mutated to lysine (K); and/or
- the 121st serine (S) is mutated to proline (P); and/or
- the asparagine (N) at position 129 is mutated to aspartic acid (D); and/or
- the 145th serine (S) is mutated to proline (P); and/or
- the 148th alanine (A) is mutated to threonine (T); and/or
- the leucine (L) at position 274 is mutated to alanine (A); and/or
- the 313th cysteine (C) is mutated to serine (S); and/or
- the 349th phenylalanine (F) is mutated to tyrosine (Y).
- the mutation of the mutant protein in the mutant aminoacyl-tRNA synthetase whose sequence is shown in SEQ ID NO.: 4 is selected from the following group: H29Y, D76G, S89G, N91T, R96K , S121P, N129D, S145P, A148T, L274A, C313S, F349Y, or a combination thereof.
- the mutant protein corresponding to the mutant aminoacyl-tRNA synthetase shown in SEQ ID NO.: 4 further includes a mutation selected from the following group: H29Y, D76G, S89G , N91T, R96K, S121P, N129D, S145P and A148T.
- mutant protein corresponding to the mutant aminoacyl-tRNA synthetase shown in SEQ ID NO.: 4 further includes a mutation selected from the following group: L274A, C313S and F349Y .
- amino acid sequence of the mutant protein is shown in SEQ ID NO.: 5 or 6.
- the remaining amino acids of the mutant protein are the same or substantially the same as the sequence shown in SEQ ID NO.: 1 or SEQ ID NO.: 2.
- the said substantially identical is that at most 50 (preferably 1-20, more preferably 1-10) amino acids are different, wherein the said differences include amino acid Substitution, deletion or addition, and the mutant protein still has the activity of aminoacyl-tRNA synthetase.
- the amino acid sequence of the mutant protein has at least 70% compared with SEQ ID NO. 1 or SEQ ID NO.: 2, preferably at least 75%, 80%, 85%, 90%, more preferably At least 95%, 96%, 97%, 98%, 99% or more sequence identity.
- the mutant protein is formed by mutation of the wild-type aminoacyl-tRNA synthetase shown in SEQ ID NO.: 1 or SEQ ID NO.: 2.
- the mutein is selected from the following group:
- amino acid sequence of the mutant protein is shown in any one of SEQ ID NO.: 3-6.
- the mutant protein is a non-natural protein.
- the mutant protein is used to introduce predetermined modified amino acids into the target protein.
- the mutant protein has one or more of the following characteristics:
- the second aspect of the present invention provides an isolated polynucleotide, which encodes the mutein of the first aspect of the present invention.
- polynucleotide encodes a polypeptide shown in any one of SEQ ID NO.: 3-6.
- polynucleotide encodes the polypeptide shown in SEQ ID NO.: 4, and the nucleotide sequence is shown in SEQ ID NO.: 9.
- the polynucleotide includes a DNA sequence, an RNA sequence, or a combination thereof.
- the polynucleotide additionally contains auxiliary elements selected from the group consisting of signal peptides, secretory peptides, tag sequences (such as 6His), flanking the ORF of the mutant aminoacyl-tRNA synthetase, Or a combination.
- auxiliary elements selected from the group consisting of signal peptides, secretory peptides, tag sequences (such as 6His), flanking the ORF of the mutant aminoacyl-tRNA synthetase, Or a combination.
- the third aspect of the present invention provides a vector containing the polynucleotide according to the second aspect of the present invention.
- the vectors include expression vectors, shuttle vectors, and integration vectors.
- the vector is selected from the group consisting of pET, pCW, pUC, pPIC9k, pMA5, or a combination thereof.
- the vector is pEvol vector and/or pBAD vector.
- the vector is used to express the mutein of the first aspect of the present invention.
- the fourth aspect of the present invention provides a host cell, which contains the vector of the third aspect of the present invention, or its genome integrates the polynucleotide of the second aspect of the present invention, or expresses There is the mutant protein described in the first aspect of the present invention.
- the host cell is selected from the group consisting of prokaryotic cells, eukaryotic cells, or a combination thereof.
- the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or an animal cell (such as a mammalian cell).
- the host cell is a prokaryotic cell, such as Escherichia coli.
- the host cell is Escherichia coli Top10 or BL21.
- the host cell comprises:
- the host cell further comprises:
- the first nucleic acid sequence contains a codon recognized by the artificial tRNA at a position for introducing a predetermined modified amino acid.
- the predetermined modified amino acid is a substrate of the mutein.
- the predetermined modified amino acid is a lysine with a modified group.
- the modified amino acid is selected from the following group: alkynyloxycarbonyl lysine derivative, tert-butoxycarbonyl (BOC)-lysine derivative, fatty acylated lysine derivative, or Its combination.
- the structure of the alkynyloxycarbonyl lysine is as shown in formula I:
- n 0-8.
- the artificial tRNA is suppressor tRNA, preferably amber suppressor tRNA.
- the coding nucleic acid sequence of the artificial tRNA is shown in SEQ ID NO.:7.
- the codon recognized by the artificial tRNA is UAG (amber), UAA (ochre), or UGA (opal), preferably an amber codon.
- the mutein specifically catalyzes the formation of an "artificial tRNA-Xa" complex from the artificial tRNA, wherein Xa is the predetermined modified amino acid in the form of an aminoacyl group.
- the host cell is a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli.
- the target protein is selected from the group consisting of insulin, human insulin precursor protein, insulin lispro precursor protein, insulin glargine precursor protein, parathyroid hormone, cortirelin, Calcitonin, bivalirudin, glucagon-like peptides and their derivatives Exenatide and Liraglutide, Somaglutide, Ziconotide, Sermorelin, Somarelin, Secretion Hormone, teduglutide, hirudin, growth hormone, growth factor, growth hormone releasing factor, corticotropin, releasing factor, deserelin, desmopressin, ecalcitonin, glucagon, Leuprolide, luteinizing hormone releasing hormone, somatostatin, thyroid stimulating hormone releasing hormone, triptorelin, vasoactive intestinal peptide, interferon, parathyroid hormone, BH3 peptide, amyloidosis peptide, or the above Fragments of peptides, or combinations thereof.
- an expression system in the fifth aspect of the present invention, includes:
- the first nucleic acid sequence contains a codon recognized by the artificial tRNA at a position for introducing a predetermined modified amino acid.
- the expression system further includes:
- the predetermined modified amino acid is a lysine with a modified group.
- the modified amino acid is selected from the following group: alkynyloxycarbonyl lysine derivative, tert-butoxycarbonyl (BOC)-lysine derivative, fatty acylated lysine derivative, or Its combination.
- the structure of the alkynyloxycarbonyl lysine is as shown in formula I:
- n 0-8.
- the artificial tRNA is suppressor tRNA, preferably amber suppressor tRNA.
- nucleic acid sequence of the artificial tRNA is shown in SEQ ID NO.:7.
- the codon recognized by the artificial tRNA is UAG (amber), UAA (ochre), or UGA (opal), preferably an amber codon.
- the expression system is a cell or a cell extract.
- the expression system is used to introduce a predetermined modified amino acid into a target protein or prepare a target protein containing unnatural amino acids.
- the sixth aspect of the present invention provides a plasmid system, the plasmid system comprising:
- a first expression cassette said first expression cassette containing a first coding sequence encoding a protein of interest, said first coding sequence containing a codon for introducing a predetermined modified amino acid, said codon being UAG( Amber), UAA (ochre), or UGA (opal); and
- a second expression cassette the second expression cassette containing a second coding sequence encoding an aminoacyl-tRNA synthetase, wherein the aminoacyl-tRNA synthetase is the mutant protein according to the first aspect of the present invention
- the system further contains a third expression cassette, the third expression cassette contains a third coding sequence encoding an artificial tRNA, wherein the artificial tRNA contains an anticodon corresponding to the codon;
- aminoacyl-tRNA synthetase specifically catalyzes the artificial tRNA to form an "artificial tRNA-Xa" complex, wherein Xa is the predetermined modified amino acid in the aminoacyl form.
- the plasmid system is a single plasmid system or a multiple plasmid system.
- the multiple plasmid system includes a double plasmid system, a three plasmid system and a four plasmid system.
- the codon is UAG (amber) or UGA (opal).
- the codon includes a three-base nucleotide sequence corresponding to an amino acid on mRNA or DNA.
- the predetermined modified amino acid is a lysine with a modified group.
- the modified amino acid is selected from the following group: alkynyloxycarbonyl lysine derivative, tert-butoxycarbonyl (BOC)-lysine derivative, fatty acylated lysine derivative, or Its combination.
- the first expression cassette, the second expression cassette and the third expression cassette are located in different plasmids respectively.
- any two or three of the first expression cassette, the second expression cassette and the third expression cassette are located in the same plasmid.
- the plasmid is an expression vector selected from the group consisting of pBAD/gIII ABC, pBAD/His ABC, pET28a, pETDuet-1, or pEvol-pBpF vector.
- the plasmid further contains a resistance gene, a tag sequence, a repressor gene (araC), a promoter gene (araBAD), or a combination thereof.
- the resistance gene is selected from the group consisting of ampicillin resistance gene (AmpR), chloramphenicol resistance gene (CmR), kanamycin resistance gene (KanaR), tetracycline resistance Gene (TetR), or a combination thereof.
- AmR ampicillin resistance gene
- CmR chloramphenicol resistance gene
- KanaR kanamycin resistance gene
- TetR tetracycline resistance Gene
- the plasmid system is a double plasmid system.
- the first expression cassette is located in a first plasmid
- the second expression cassette is located in a second plasmid
- the third expression cassette is located in the first plasmid or the second plasmid.
- the third expression cassette is located in a second plasmid.
- the first plasmid is an expression vector selected from the group consisting of pBAD-His ABC, pBAD/His ABC, pET28a, and pETDuet-1.
- the first plasmid further contains a resistance gene, a tag sequence, a repressor gene (araC), a promoter gene (araBAD), or a combination thereof.
- the second plasmid is a pEvol-pBpF vector.
- the second plasmid further contains a resistance gene, a tag sequence, a repressor gene (araC), a promoter gene (araBAD), or a combination thereof.
- the positions of the first expression cassette, the second expression cassette and the third expression cassette in the plasmid are not limited in any way.
- any two or three of the first expression cassette, the second expression cassette and the third expression cassette can be combined into one.
- any two of the first expression cassette, the second expression cassette and the third expression cassette may be connected by a connecting sequence (such as IRES, P2A, T2A, etc.).
- the first expression cassette, the second expression cassette and/or the third expression cassette may or may not contain a promoter.
- the first expression cassette, the second expression cassette and/or the third expression cassette further comprise one or more promoters, and the promoters are operably linked to the first coding sequence, the The second coding sequence, the third coding sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site are connected.
- the first expression cassette further includes a first promoter, and preferably the first promoter is an inducible promoter.
- the first promoter is selected from the group consisting of arabinose promoter (araBAD), lactose promoter (Plac), pLacUV5 promoter, pTac promoter, or a combination thereof.
- the first expression cassette includes a promoter (such as araBAD), a ribosome binding site RBS, the first coding sequence, a terminator or a tag sequence in order from 5'to 3'.
- a promoter such as araBAD
- RBS ribosome binding site
- the second expression cassette further comprises a second promoter, and preferably the second promoter is an inducible promoter.
- the second promoter is selected from the group consisting of arabinose promoter (araBAD), glnS promoter, proK promoter, or a combination thereof.
- the second expression cassette includes in order from 5'-3': a promoter (such as araBAD or glnS), a ribosome binding site RBS, the second coding sequence and a terminator (rrnB or glnS T).
- a promoter such as araBAD or glnS
- RBS ribosome binding site
- RBS ribosome binding site
- rrnB or glnS T glnS
- the third expression cassette further includes a third promoter, and preferably the third promoter is a constitutive promoter.
- the third promoter is a reverse transcription promoter proK.
- the third expression cassette includes a promoter (such as proK), a ribosome binding site RBS, a third coding sequence, and a terminator or tag sequence in order from 5'-3'.
- a promoter such as proK
- RBS ribosome binding site
- RBS ribosome binding site
- terminator or tag sequence in order from 5'-3'.
- the seventh aspect of the present invention provides a host cell or cell extract containing the expression system according to the fifth aspect of the present invention or the plasmid system according to the sixth aspect of the present invention.
- the host cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, yeast cells, insect cells, mammalian cells, or a combination thereof.
- the cell extract is derived from cells selected from the group consisting of Escherichia coli, Bacillus subtilis, yeast cells, insect cells, mammalian cells, or a combination thereof.
- the eighth aspect of the present invention provides a method for introducing unnatural amino acids into a target protein or preparing a target protein containing unnatural amino acids, including the steps:
- the target protein is selected from the group consisting of: human insulin precursor protein, insulin lispro precursor protein, insulin glargine precursor protein, parathyroid hormone, cortirelin, blood lowering Calcin, bivalirudin, glucagon-like peptides and their derivatives exenatide and liraglutide, ziconotide, sermorelin, somatorelin, secretin, teduglutide, Hirudin, growth hormone, growth factor, growth hormone releasing factor, corticotropin, releasing factor, desserrellin, desmopressin, ecalcitonin, glucagon, leuprolide, luteinizing Hormone releasing hormone, somatostatin, thyroid stimulating hormone releasing hormone, triptorelin, vasoactive intestinal peptide, interferon, parathyroid hormone, BH3 peptide, amyloid peptide, or fragments of the foregoing peptides, or combinations thereof .
- the unnatural amino acid is lysine with a modified group.
- the unnatural amino acid is selected from the group consisting of alkynyloxycarbonyl lysine derivatives, tert-butoxycarbonyl (BOC)-lysine derivatives, fatty acylated lysine derivatives, Or a combination.
- the method includes the steps:
- the cell or cell extract is cultured, so that the lysine derivative is introduced into the object through the mutein and artificial tRNA pair according to the first aspect of the present invention Protein.
- the ninth aspect of the present invention provides a kit comprising (a) a container, and (b) the mutein of the first aspect of the present invention, or the second aspect of the present invention in the container.
- the kit further includes a cell extract.
- the plasmid system is a multi-plasmid system, and the plasmids are located in the same or different containers.
- a translation system in a tenth aspect of the present invention, includes:
- the eleventh aspect of the present invention provides the mutein according to the first aspect of the present invention, or the host cell according to the fourth aspect of the present invention, or the plasmid system according to the sixth aspect of the present invention, or the first aspect of the present invention.
- the use of the kit described in the ninth aspect is to incorporate unnatural amino acids into a target protein or to prepare a target protein containing unnatural amino acids.
- the twelfth aspect of the present invention provides a method for producing the mutant protein of the first aspect of the present invention, including the steps of: (i) culturing the host cell of the fourth aspect of the present invention to express the mutation protein.
- the thirteenth aspect of the present invention provides an enzyme preparation containing the mutein described in the first aspect of the present invention.
- the dosage form of the pharmaceutical preparation includes: a lyophilized preparation, a liquid preparation, or a combination thereof.
- Figure 1 shows the plasmid map of pEvol-suppylRs-pylT.
- Figure 2 shows the plasmid map of pEvol-IPYEpylRs-pylT.
- Figure 3 shows the plasmid map of pEvol-optpylRs-pylT.
- Figure 4 shows the pEvol-IPYEpylRs (L274A, C313S, F349Y)-pylT plasmid map.
- Figure 5 shows the plasmid map of pBAD-araBAD[A1-u4-u5-TEV-R-MiniINS]-glnS[IPYEpylRs]-proK[pylT].
- the mutant lysyl-tRNA synthetase of the present invention has high activity, high expression and good solubility, and can significantly increase the amount of unnatural amino acid insertion and the target protein containing unnatural amino acid. The amount of expression.
- the mutant lysyl-tRNA synthetase of the present invention can also improve the stability of the target protein so that it is not easily broken. On this basis, the inventor completed the present invention.
- the term “about” may refer to a value or composition within an acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
- the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
- Sequence identity is passed along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) ) Compare two aligned sequences and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
- a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
- aminoacyl-tRNA synthetase and “lysyl-tRNA synthetase” are used interchangeably.
- wild-type lysyl-tRNA synthetase refers to a naturally-occurring aminoacyl-tRNA synthetase that has not been artificially modified. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerization For enzyme chain reaction (PCR), etc., the amino acid sequence can be deduced from the nucleotide sequence.
- the source of the wild-type lysyl-tRNA synthetase is not particularly limited. A preferred source is Methanosarcina mazei, Methanosarcina barkeri and Methanosarcina barkeri of the methanogenic archaea. Methanosarcina acetivorans, etc., but not limited to this.
- amino acid sequence of the wild-type lysyl-tRNA synthetase is shown in SEQ ID NO.:1.
- amino acid sequence of the wild-type lysyl-tRNA synthetase is shown in SEQ ID NO.: 2.
- mutant protein As used herein, the terms "mutant protein”, “mutant protein of the present invention”, “mutated aminoacyl-tRNA synthetase of the present invention”, “mutant lysyl-tRNA synthetase”, “mutant enzyme”, “ammonia "Mutant of acyl-tRNA synthetase” can be used interchangeably, and both refer to a non-naturally occurring mutant aminoacyl-tRNA synthetase, and the mutant aminoacyl-tRNA synthetase is a reference to SEQ ID NO.: 1 or SEQ ID NO.: A protein artificially modified by the polypeptide shown in 2. Specifically, the mutant aminoacyl-tRNA synthetase is as described in the first aspect of the present invention.
- amino acid numbering in the mutant lysyl-tRNA synthetase of the present invention is based on the wild-type lysyl-tRNA synthetase (preferably, SEQ ID NO.: 1 or SEQ ID NO.: 2).
- the amino acid number of the mutant protein may be relative to SEQ ID NO.:1
- the misalignment of the amino acid numbering of SEQ ID NO.: 2 such as misalignment of positions 1-5 to the N-terminus or C-terminus of the amino acid, and using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misalignment is Mutant proteins with the same or similar glycosyltransferase activity that are within a reasonable range and should not have a homology of 80% (such as 90%, 95%, 98%) due to misplacement of amino acid numbering are not in the present invention Within the range of mutant proteins.
- the mutant protein of the present invention is a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, plants) using recombinant technology.
- a prokaryotic or eukaryotic host for example, bacteria, yeast, plants
- the mutein of the present invention may be glycosylated or non-glycosylated.
- the mutein of the present invention may also include or exclude the starting methionine residue.
- the present invention also includes fragments, derivatives and analogs of the mutein.
- fragment refers to a protein that substantially retains the same biological function or activity as the mutein.
- the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
- the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
- the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs fall within the scope of those skilled in the art.
- conservatively substituted amino acids are preferably generated by amino acid substitutions according
- the recognition of the amino acid substrate of PylRS is related to the three-dimensional structure of the catalytically active functional domain.
- the size of lysine derivatives that can be activated by wild-type PylRS is limited, and lysine derivatives with large functional groups cannot be introduced into proteins. Therefore, By mutating the PylRS site, avoiding the steric hindrance of the binding substrate, or the interaction of the mutant amino acid with the substrate amino acid or the main chain part, to improve the effect.
- the mutant protein is shown in any one of SEQ ID NO.: 3-6.
- the mutant protein of the present invention generally has higher homology (identity).
- the mutant protein is The peptide fragments of corresponding length present in SEQ ID NO: 1 or 2 have at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.
- mutant protein of the present invention can also be modified.
- Modified (usually not changing the primary structure) forms include: chemically derived forms of mutein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes mutant proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.
- polynucleotide encoding the mutant protein of the present invention may include a polynucleotide encoding the mutant lysyl-tRNA synthetase of the present invention, or may also include additional coding and/or non-coding sequences.
- the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or muteins having the same amino acid sequence as the present invention.
- These nucleotide variants include substitution variants, deletion variants and insertion variants.
- an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the encoding of the mutant protein.
- the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
- the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
- stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
- the muteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, are purified to homogeneity.
- the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
- primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
- the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
- the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
- artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
- the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
- the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
- mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
- the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used.
- the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
- the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
- construct or "vector” generally refers to a nucleic acid capable of transporting the coding sequence of the target protein to which it is attached.
- vector One type of vector is a "plasmid”, which refers to a circular double-stranded DNA loop that can connect additional DNA segments.
- the vector can be transformed or transfected into a suitable host cell to provide protein expression.
- the process may include culturing the host cell transformed with the expression vector under conditions that provide for expression of the vector encoding the target nucleic acid sequence of the protein, and optionally recovering the expressed protein.
- the vector may be, for example, a plasmid or viral vector provided with an origin of replication, a promoter optionally used to express the target nucleic acid sequence, and an optional regulator of the promoter.
- the vector may contain one or more selectable marker genes, such as kanamycin resistance genes.
- the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
- the polynucleotide sequence of the present invention can be used to express or produce recombinant mutein. Generally speaking, there are the following steps:
- the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector.
- recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors well known in the art. As long as it can replicate and stabilize in the host, any plasmid and vector can be used.
- An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
- the methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the mutein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
- the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
- promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
- the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- a prokaryotic cell such as a bacterial cell
- a lower eukaryotic cell such as a yeast cell
- a higher eukaryotic cell such as a mammalian cell.
- Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as ginseng cells).
- Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples that can be cited include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
- Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
- the host is a prokaryotic organism such as Escherichia coli
- competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method.
- the steps used are well known in the art.
- Another method is to use MgCl 2 .
- transformation can also be performed by electroporation.
- the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
- the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
- the medium used in the culture can be selected from various conventional mediums.
- the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
- the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
- operably linked means that the target gene to be transcribed and expressed is linked to its control sequence in a conventional manner in the art to be expressed.
- the present invention provides a plasmid system, as described in the sixth aspect of the present invention.
- the plasmid system includes:
- a first expression cassette said first expression cassette containing a first coding sequence encoding a protein of interest, said first coding sequence containing a codon for introducing a predetermined modified amino acid, said codon being UAG( Amber), UAA (ochre), or UGA (opal); and
- a second expression cassette the second expression cassette containing a second coding sequence encoding an aminoacyl-tRNA synthetase, wherein the aminoacyl-tRNA synthetase is the mutant protein according to the first aspect of the present invention
- the system further contains a third expression cassette, the third expression cassette contains a third coding sequence encoding an artificial tRNA, wherein the artificial tRNA contains an anticodon corresponding to the codon;
- aminoacyl-tRNA synthetase specifically catalyzes the artificial tRNA to form an "artificial tRNA-Xa" complex, wherein Xa is the predetermined modified amino acid in the aminoacyl form.
- the plasmid system is a single plasmid system or a multiple plasmid system.
- the multiple plasmid system includes a double plasmid system, a three plasmid system and a four plasmid system.
- the first expression cassette, the second expression cassette and the third expression cassette are located in different plasmids respectively.
- any two or three of the first expression cassette, the second expression cassette and the third expression cassette are located in the same plasmid.
- the plasmid is an expression vector selected from the group consisting of pBAD/gIII ABC, pBAD/His ABC, pET28a, pETDuet-1, or pEvol-pBpF vector.
- the plasmid further contains a resistance gene, a tag sequence, a repressor gene (araC), a promoter gene (araBAD), or a combination thereof.
- the resistance gene is selected from the group consisting of ampicillin resistance gene (AmpR), chloramphenicol resistance gene (CmR), kanamycin resistance gene (KanaR), tetracycline resistance Gene (TetR), or a combination thereof.
- AmR ampicillin resistance gene
- CmR chloramphenicol resistance gene
- KanaR kanamycin resistance gene
- TetR tetracycline resistance Gene
- the DNA sequence of the promoter glnS is shown in SEQ ID NO.: 10.
- the DNA sequence of the terminator glnS T is shown in SEQ ID NO.: 11.
- the DNA sequence of the promoter proK is shown in SEQ ID NO.: 12.
- the DNA sequence of the terminator proK T is shown in SEQ ID NO.: 13.
- the present invention mainly has the following advantages:
- the present invention found for the first time that positions 102, 128-140, and 159-179 are missing in the wild-type aminoacyl-tRNA synthetase (such as SEQ ID NO.: 1 or 2) Amino acid residues, the resulting truncated mutant aminoacyl-tRNA synthetase (such as SEQ ID NO.: 3) not only can still introduce lysine derivatives with large functional groups into the protein, but the enzyme activity is significantly increased, and the expression level High, good solubility, can significantly increase the amount of unnatural amino acids inserted and the expression of target proteins containing unnatural amino acids.
- the wild-type aminoacyl-tRNA synthetase such as SEQ ID NO.: 1 or 2
- Amino acid residues such as SEQ ID NO.: 3
- the enzyme activity is significantly increased, and the expression level High, good solubility, can significantly increase the amount of unnatural amino acids inserted and the expression of target proteins containing unnatural amino acids.
- mutant lysyl-tRNA synthetase of the present invention can also improve the stability of the target protein so that it is not easily broken.
- the present invention also found that other sites of the truncated mutant aminoacyl-tRNA synthetase are mutated, and the resulting mutant protein (such as SEQ ID NO.: 4-6) can further increase the amount of unnatural amino acid insertion and contain unnatural amino acid.
- the amount of the target protein of amino acids and/or the stability of the target protein can further increase the amount of unnatural amino acid insertion and contain unnatural amino acid.
- the DNA sequence of pylRs (SEQ ID NO.:8) was synthesized and cloned for expression.
- the SpeI-SalI site downstream of the araBAD promoter of the vector plasmid pEvol-pBpF (purchased from NTCC, chloramphenicol resistant), where the SpeI restriction site is increased by PCR, and the SalI site is possessed by the vector itself.
- the original glutamine promoter glnS of the expression vector plasmid pEvol-pBpF is retained.
- the DNA sequence (SEQ ID NO.: 7) of the unnatural tRNA (pylT) was inserted by PCR.
- the sequence was cut from the cloning vector with restriction enzymes SpeI and SalI, and the plasmid pEvol-pylRs-pylT was cut with SpeI and SalI at the same time (the target DNA fragment was a large 4.3kb fragment), and separated by nucleic acid electrophoresis , Extracted with agarose gel DNA recovery kit, ligated with T4 DNA Ligase, chemically transformed (CaCl 2 method) into large E.coli Top10 competent cells, and the transformed cells were cultured in chloramphenicol-containing Culture on LB agar medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar) at 37°C overnight.
- LB agar medium 10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar
- a single live colony was picked and cultured in liquid LB medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl) containing chloramphenicol at 37°C at 220 rpm overnight.
- the plasmid was extracted with a small amount of plasmid extraction kit, and the plasmid was named pEvol-pylRs-pylT.
- the amino acid sequence of wild-type lysyl-tRNA synthetase pylRs (SEQ ID NO.: 1), the original three sequences (ie 102T, 128Q ⁇ 140V, 159I ⁇ 179M, 35 amino acids in total) were knocked out.
- the amino acid sequence (SEQ ID NO.: 3) of the synthetase suppylRs is obtained, and the plasmid is named pEvol-suppylRs-pylT.
- the plasmid map is shown in Figure 1.
- the 1st to 184th amino acids are replaced with another 149 amino acids, and the above plasmid pEvol-suppylRs- is also deleted.
- the 3 amino acid sequences of pylT knocking out wild-type lysyl-tRNA synthetase pylRs ie 102T, 128Q ⁇ 140V, 159I ⁇ 179M, 35 amino acids in total).
- the amino acid sequence (SEQ ID NO.: 4) of the synthetase IPYEpylRs was obtained, and the DNA sequence of IPYEpylRs (SEQ ID NO.: 9) was synthesized according to the codon preference of E. coli.
- the plasmid was named pEvol-IPYEpylRs-pylT, and the plasmid map is shown in Figure 2.
- IPYEpylRs SEQ ID NO.: 9
- the mutant lysyl-tRNA synthetase IPYEpylRs According to the amino acid sequence (SEQ ID NO.: 4) of the mutant lysyl-tRNA synthetase IPYEpylRs, the mutations H29Y, D76G, D89G, N91T, R96K, D121P, N129D, S145P, A148T were introduced, and the amino acid sequence was obtained as SEQ ID NO .:
- the plasmid was named pEvol-optpylRs-pylT, and the plasmid map is shown in Figure 3.
- the mutant lysyl-tRNA synthetase IPYEpylRs According to the amino acid sequence (SEQ ID NO.: 4) of the mutant lysyl-tRNA synthetase IPYEpylRs, the mutations L274A, C313S, F349Y are introduced, and the mutant lysyl-tRNA with the amino acid sequence shown in SEQ ID NO.: 6 is obtained Synthetase IPYEpylRs (L274A, C313S, F349Y)
- the plasmid is named pEvol-IPYEpylRs (L274A, C313S, F349Y)-pylT, the plasmid map is shown in Figure 4.
- amino acid sequence (SEQ ID NO.: 4) of the mutant lysyl-tRNA synthetase IPYEpylRs according to the DNA sequence of the unnatural tRNA (pylT) (SEQ ID NO.: 7), according to the sequence derived from the pEvol-pBpF vector Glutamine promoter glnS and promoter proK and their corresponding terminator DNA sequences (SEQ ID NO.: 10-13), according to the codon preference of E.
- coli synthesize glnS[pylRs]-proK[pylT] DNA sequence (SEQ ID NO.: 14), cloned into the plasmid pBAD-A1-u4-u5-TEV-R-MiniINS (plasmid constructed by our company, kana resistant), the AvrII-XbaI site downstream of the terminator rrnB , Where AvrII and XbaI restriction sites are increased by PCR.
- SEQ ID NO.: 14 The sequence shown in SEQ ID NO.: 14 was cut from the cloning vector pUC57-glnS[pylRs]-proK[pylT] with the restriction enzymes AvrII and XbaI, and the plasmid pBAD-A1-u4 was simultaneously cut with AvrII and XbaI -u5-TEV-R-MiniINS is cut, separated by nucleic acid electrophoresis, extracted with agarose gel DNA recovery kit, connected with T4 DNA Ligase, and transformed into E.coli Top10 competent by chemical method (CaCl 2 method) Among the cells, the transformed cells were cultured on LB agar medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar) containing kanamycin at 37°C overnight.
- LB agar medium 10g/L yeast peptone, 5g/L yeast extract, 10g/
- a single live colony was picked and cultured in liquid LB medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl) containing kanamycin at 37°C at 220 rpm overnight. Add 20% glycerol to the final concentration to preserve the strain.
- the plasmid was extracted with a small amount of plasmid extraction kit, and the obtained plasmid containing wild-type lysyl-tRNA synthetase was named pBAD-araBAD[A1-u4-u5-TEV-R-MiniINS]-glnS[IPYEpylRs]-proK[ pylT].
- the plasmid map is shown in Figure 5.
- the fusion protein is expressed in the form of insoluble "inclusion bodies".
- inclusion bodies In order to release the inclusion bodies, the E. coli cells were disrupted with a high-pressure homogenizer. Nucleic acid, cell debris and soluble protein are removed by 10000g centrifugation. The inclusion bodies containing the fusion protein were washed with pure water, and the obtained inclusion body precipitate was used as the raw material for folding.
- the inclusion bodies were dissolved in a pH 10.5 7.5 M urea solution containing 2-10 mM mercaptoethanol, so that the total protein concentration after dissolution was 10-25 mg/mL. Dilute the sample 5-10 times, and perform conventional folding for 16-30 hours at 4-8°C and pH 10.5-11.7. At 18-25°C, the pH value is maintained at 8.0-9.5, the fusion proteolysis is hydrolyzed with trypsin and carboxypeptidase B for 10-20 hours, and then 0.45M ammonium sulfate is added to terminate the enzymatic hydrolysis reaction. The results of reverse phase HPLC analysis showed that the yield of this enzymatic hydrolysis step was higher than 90%.
- the insulin analog obtained after digestion with trypsin and carboxypeptidase B was named BOC-lysine insulin.
- Boc-lysine insulin cannot be enzymatically digested under the above conditions.
- the sample was clarified by membrane filtration, and 0.45 mM ammonium sulfate was used as a buffer, and initially purified by hydrophobic chromatography, the purity of SDS-polyacrylamide gel electrophoresis reached 90%.
- the obtained Boc-human insulin was analyzed by MALDI-TOF mass spectrometry, and its molecular weight was found to be consistent with the theoretical molecular weight of 5907.7 Da.
- the samples were collected by hydrophobic chromatography, and hydrochloric acid was added to carry out the Boc-human insulin deprotection reaction.
- Sodium hydroxide solution was added to control the pH to 2.8-3.2 to terminate the reaction.
- the recombinant human insulin was obtained. The rate is higher than 85%.
- Wild type pylRs 360 suppylRs 715 IPYEpylRs 740 optpylRs 760 IPYEpylRs(L274A, C313S, F349Y) 370 Single plasmid IPYEpylRs 630
- Plasmid pEvol-pylRs-pylT, plasmid pEvol-IPYEpylRs-pylT, plasmid pEvol-optpylRs-pylT and plasmid pEvol-IPYEpylRs (L274A, C313S, F349Y)-pylT and insulin fusion protein expression vector pBAD-INS Plasmid pEvol-pylRs-pylT, plasmid pEvol-IPYEpylRs-pylT, plasmid pEvol-optpylRs-pylT and plasmid pEvol-IPYEpylRs (L274A, C313S, F349Y)-pylT and insulin fusion protein expression vector pBAD-INS (plasmid constructed by our company (CaCl 2 method) was used to co-transform large E.
- the fusion protein is expressed in the form of insoluble "inclusion bodies".
- the E. coli cells were disrupted with a high-pressure homogenizer. Nucleic acid, cell debris and soluble protein are removed by 10000g centrifugation.
- the inclusion bodies containing the fusion protein were washed with pure water, and the obtained inclusion body precipitate was used as the raw material for folding.
- the inclusion bodies were dissolved in a pH 10.5 7.5 M urea solution containing 2-10 mM mercaptoethanol so that the total protein concentration after dissolution was 10-25 mg/mL. Dilute the sample 5 to 10 times, and perform conventional folding for 16 to 30 hours at 4 to 8° C. and pH 10.5 to 11.7.
- the pH value is maintained at 8.0-9.5, the fusion proteolysis is hydrolyzed with trypsin and carboxypeptidase B for 10-20 hours, and then 0.45M ammonium sulfate is added to terminate the enzymatic hydrolysis reaction.
- the results of reverse phase HPLC analysis showed that the yield of the enzymatic hydrolysis step was higher than 90%.
- the insulin analog obtained after digestion with trypsin and carboxypeptidase B is named butynoxycarbonyl-lysine insulin. Butynyloxycarbonyl-lysine insulin cannot be enzymatically hydrolyzed under the above conditions.
- the sample was clarified by membrane filtration, and 0.45 mM ammonium sulfate was used as a buffer, and initially purified by hydrophobic chromatography, the purity of SDS-polyacrylamide gel electrophoresis reached 90%. And the obtained butynyloxycarbonyl-human insulin was analyzed by MALDI-TOF mass spectrometry, and it was found that its molecular weight was consistent with the theoretical molecular weight of 5907.7 Da.
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Abstract
Description
最初的残基 | 代表性的取代 | 优选的取代 |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Asn | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
酶 | 人胰岛素的产量(mg/L发酵液) |
野生型pylRs | 360 |
suppylRs | 715 |
IPYEpylRs | 740 |
optpylRs | 760 |
IPYEpylRs(L274A,C313S,F349Y) | 370 |
单质粒IPYEpylRs | 630 |
酶 | 丁炔氧羰基人胰岛素的产量(mg/L发酵液) |
野生型pylRs | 530 |
suppylRs | 720 |
IPYEpylRs | 700 |
optpylRs | 730 |
IPYEpylRs(L274A,C313S,F349Y) | 1050 |
单质粒IPYEpylRs | 590 |
Claims (10)
- 一种氨酰基-tRNA合成酶的突变蛋白,其特征在于,所述突变蛋白在对应于野生型氨酰基-tRNA合成酶的氨基酸序列的选自下组的一个或多个位点发生缺失:第102位、第128-140位和第159-179位氨基酸残基。
- 如权利要求1所述的突变蛋白,其特征在于,所述突变蛋白至少截掉了野生型氨酰基-tRNA合成酶的氨基酸序列的以下氨基酸残基:(a)第102位氨基酸残基;(b)第X位至第Y位氨基酸残基,其中X为128-132之间的正整数,Y为133-140之间的正整数;和/或(c)第A位至第B位氨基酸残基,其中A为159-164之间的正整数,B为165-179之间的正整数。
- 如权利要求1所述的突变蛋白,其特征在于,所述突变蛋白在对应于序列如SEQ ID NO.:4所示的突变型氨酰基-tRNA合成酶中还存在选自下组的氨基酸突变:第29位组氨酸(H)、第76位天冬氨酸(D)、第89位丝氨酸(S)、第91位天冬酰胺(N)、第96位精氨酸(R)、第121位丝氨酸(S)、第129位天冬酰胺(N)、第145位丝氨酸(S)、第148位丙氨酸(A)、第274位亮氨酸(L)、第313位半胱氨酸(C)、第349位苯丙氨酸(F)、或其组合。
- 如权利要求1所述的突变蛋白,其特征在于,所述突变蛋白具有如SEQ ID NO.:3-6任一所示的氨基酸序列。
- 一种载体,其特征在于,所述载体含有如权利要求4所述的多核苷酸。
- 一种宿主细胞,其特征在于,所述的宿主细胞含有权利要求5所述的载体,或其基因组中整合有权利要求4所述的多核苷酸,或表达有权利要求1所述的突变蛋白。
- 如权利要求6所述的宿主细胞,其特征在于,所述宿主细胞包含:(a)权利要求1所述的突变蛋白;和(b)在所述突变蛋白的存在下能够与预定的修饰氨基酸结合的人工tRNA;以及任选地(c)编码目的蛋白的第一核酸序列,所述第一核酸序列含有由所述人工tRNA识别的密码子。
- 一种质粒系统,其特征在于,所述质粒系统包含:(1)第一表达盒,所述第一表达盒含有编码目的蛋白的第一编码序列,所述第一编码序列中含有用于引入预定的修饰氨基酸的密码子,所述密码子为UAG(琥珀)、UAA(赭石)、或UGA(蛋白石);和(2)第二表达盒,所述第二表达盒含有编码氨酰基-tRNA合成酶的第二编码序列,其中所述氨酰基-tRNA合成酶为权利要求1所述的突变蛋白;并且,所述系统还含有第三表达盒,所述第三表达盒含有编码人工tRNA的第三编码序列,其中所述人工tRNA含有对应于所述密码子的反密码子;并且所述的氨酰基-tRNA合成酶特异性催化所述人工tRNA形成“人工tRNA-Xa”复合物,其中Xa为氨酰基形式的所述预定的修饰氨基酸。
- 一种试剂盒,其特征在于,所述试剂盒含有(a)容器,以及(b)位于所述容器内的 权利要求1所述的突变蛋白、或权利要求4所述的多核苷酸、或权利要求5所述的载体、或权利要求8所述的质粒系统。
- 如权利要求1所述的突变蛋白、或权利要求6所述的宿主细胞、或权利要求8所述的质粒系统、或权利要求9所述的试剂盒的用途,其特征在于,用于将非天然氨基酸掺入目的蛋白中或制备含非天然氨基酸的目的蛋白。
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EP20798501.1A EP3964570A4 (en) | 2019-04-30 | 2020-04-30 | Aminoacyl-trna synthetase efficiently introducing lysine derivatives |
US17/594,841 US20220290202A1 (en) | 2019-04-30 | 2020-04-30 | Aminoacyl-trna synthetase efficiently introducing lysine derivatives |
BR112021021859A BR112021021859A2 (pt) | 2019-04-30 | 2020-04-30 | Aminoacil-trna sintetase introduzindo derivados de lisina de forma eficaz |
AU2020265979A AU2020265979A1 (en) | 2019-04-30 | 2020-04-30 | Aminoacyl-tRNA synthetase efficiently introducing lysine derivatives |
JP2021565005A JP2022530917A (ja) | 2019-04-30 | 2020-04-30 | リシン誘導体を効率的に導入するアミノアシル-tRNAシンテターゼ |
CN202080032606.7A CN113767168A (zh) | 2019-04-30 | 2020-04-30 | 高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 |
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