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WO2020187269A1 - 在蛋白中高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 - Google Patents

在蛋白中高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 Download PDF

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Publication number
WO2020187269A1
WO2020187269A1 PCT/CN2020/080034 CN2020080034W WO2020187269A1 WO 2020187269 A1 WO2020187269 A1 WO 2020187269A1 CN 2020080034 W CN2020080034 W CN 2020080034W WO 2020187269 A1 WO2020187269 A1 WO 2020187269A1
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Prior art keywords
trna synthetase
lysyl
mutant
acid sequence
trna
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PCT/CN2020/080034
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English (en)
French (fr)
Inventor
查若鹏
吴松
张振山
刘慧玲
陈卫
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宁波鲲鹏生物科技有限公司
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Priority to CA3134163A priority Critical patent/CA3134163A1/en
Priority to US17/441,101 priority patent/US20220170030A1/en
Priority to CN202080023301.XA priority patent/CN113614226A/zh
Priority to BR112021018681A priority patent/BR112021018681A2/pt
Priority to JP2021559478A priority patent/JP7383306B2/ja
Priority to AU2020242724A priority patent/AU2020242724B2/en
Priority to EP20774776.7A priority patent/EP3943599A4/en
Publication of WO2020187269A1 publication Critical patent/WO2020187269A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01006—Lysine-tRNA ligase (6.1.1.6)

Definitions

  • the present invention relates to the field of biotechnology, in particular to an aminoacyl-tRNA synthetase that efficiently introduces lysine derivatives into proteins.
  • 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
  • tRNA pairs derived from various biological species more than 30 types of whole proteins have been synthesized.
  • the system that has the longest history and has been applied to the introduction of many useful unnatural amino acids is the pair of tyrosyl-tRNA synthetase (TyrRS) mutants and tRNA Tyr suppressed by amber.
  • 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.
  • the first aspect of the present invention provides a mutant lysyl-tRNA synthetase, which is the 19th amino acid sequence corresponding to the wild-type lysyl-tRNA synthetase. Arginine (R) and/or histidine (H) at position 29 are mutated.
  • the wild-type lysyl-tRNA synthetase is derived from Methanosarcina mazei, Methanosarcina barkeri, or Methanosarcina barkeri of the methanogenic archaea. Methanosarcina acetivorans.
  • 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.
  • the arginine (R) at position 19 is mutated to histidine (H) or lysine (K); and/or
  • the histidine (H) at position 29 is mutated to arginine (R) or lysine (K).
  • the mutation in the mutant lysyl-tRNA synthetase is selected from the group consisting of R19H, R19K, H29R, H29K, or a combination thereof.
  • the mutant lysyl-tRNA synthetase further includes a mutation at a site selected from the following group: isoleucine (I) at position 26, threonine (T) at position 122 , Leucine (L) at position 309, Cysteine at position 348 (C), Tyrosine at position 384 (Y), or a combination thereof.
  • the mutation site of the mutant lysyl-tRNA synthetase further includes isoleucine (I) at position 26; preferably, isoleucine (I) at position 26 Mutation to valine (V).
  • the mutation site of the mutant lysyl-tRNA synthetase further includes threonine (T) at position 122; preferably, the mutation of threonine (T) at position 122 is Tryptophan (S).
  • the mutation site of the mutant lysyl-tRNA synthetase further includes the 309th leucine (L); preferably, the 309th leucine (L) is mutated to Alanine (A).
  • the mutation site of the mutant lysyl-tRNA synthetase further includes the 348th cysteine (C); preferably, the 348th cysteine (C) Mutation to tryptophan (S).
  • the mutation site of the mutant lysyl-tRNA synthetase further includes the 384th tyrosine (Y); preferably, the 384th tyrosine (Y) is mutated to Phenylalanine (F).
  • the mutant lysyl-tRNA synthetase further includes a mutation selected from the following group: I26V, T122S, L309A, C348S, Y384F, or a combination thereof.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19H and H29R.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19K and H29R.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19H and H29K.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19K and H29K.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19H, I26V and H29R.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19H, H29R, T122S and Y384F.
  • the mutant lysyl-tRNA synthetase includes a mutation selected from the group consisting of R19H, H29R, L309A and C348S.
  • mutant lysyl-tRNA synthetase except for the mutation (such as the 19th and/or 29th, and optionally the 26th, 122, 309, 348 and/or 384th ), the remaining amino acids 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 lysyl-tRNA synthetase.
  • the amino acid sequence of the mutant lysyl-tRNA synthetase has at least 70%, preferably at least 75%, 80%, 85% compared with SEQ ID NO. 1 or SEQ ID NO.: 2. %, 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • mutant lysyl-tRNA synthetase is formed by mutation of the wild-type lysyl-tRNA synthetase shown in SEQ ID NO.: 1 or SEQ ID NO.: 2.
  • mutant lysyl-tRNA synthetase is selected from the following group:
  • a polypeptide whose amino acid sequence is SEQ ID NO.: 3-8; or
  • amino acid sequence of the mutant lysyl-tRNA synthetase is shown in any one of SEQ ID NO.: 3-8.
  • the mutant lysyl-tRNA synthetase is a non-natural protein.
  • mutant lysyl-tRNA synthetase is used to introduce lysine derivatives into the target protein.
  • mutant lysyl-tRNA synthetase has the following characteristics:
  • the second aspect of the present invention provides an isolated polynucleotide, which encodes the mutant lysyl-tRNA synthetase of the first aspect of the present invention.
  • polynucleotide encodes a polypeptide shown in any one of SEQ ID NO.: 3-8.
  • 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, and tag sequences (such as 6His) flanking the ORF of the mutant lysyl-tRNA synthetase. , Or a combination thereof.
  • auxiliary elements selected from the group consisting of signal peptides, secretory peptides, and tag sequences (such as 6His) flanking the ORF of the mutant lysyl-tRNA synthetase. , Or a combination thereof.
  • 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 mutant lysyl-tRNA synthetase.
  • the fourth aspect of the present invention provides a host cell which contains the vector according to the third aspect of the present invention, or its genome integrates the polynucleotide according to the second 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:
  • a target nucleic acid sequence encoding a target protein comprising a codon recognized by the orthogonal tRNA at a position for introducing a lysine derivative.
  • the host cell further comprises:
  • the lysine derivative is a substrate of the mutant lysyl-tRNA synthetase.
  • the lysine derivative is selected from the following group: alkynyloxycarbonyl lysine derivative, BOC-lysine (tert-butoxycarbonyl-L-lysine) derivative, fat Acylated lysine, or a combination thereof.
  • the structure of the alkynyloxycarbonyl lysine is as shown in formula I:
  • n 0-8.
  • the orthogonal tRNA is suppressor tRNA, preferably amber suppressor tRNA.
  • the encoding nucleic acid sequence of the orthogonal tRNA is shown in SEQ ID NO.:9.
  • the codon recognized by the orthogonal tRNA is an amber codon.
  • the host cell is a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli.
  • an expression system in the fifth aspect of the present invention, includes:
  • a target nucleic acid sequence encoding a target protein comprising a codon recognized by the orthogonal tRNA at a position for introducing a lysine derivative.
  • the expression system further includes:
  • the lysine derivative is selected from the following group: alkynyloxycarbonyl lysine derivative, BOC-lysine (tert-butoxycarbonyl-L-lysine) derivative, fat Acylated lysine, or a combination thereof.
  • the structure of the alkynyloxycarbonyl lysine is as shown in formula I:
  • n 0-8.
  • the orthogonal tRNA is suppressor tRNA, preferably amber suppressor tRNA.
  • nucleic acid sequence of the orthogonal tRNA is shown in SEQ ID NO.:9.
  • the codon recognized by the orthogonal tRNA is an amber codon.
  • the expression system is a cell or a cell extract.
  • the expression system is used to introduce alkynyloxycarbonyl lysine into a target protein or prepare a target protein containing unnatural amino acids.
  • the sixth aspect of the present invention provides a method for introducing unnatural amino acids into a target protein, including the steps:
  • the target protein is expressed in the host cell or expression system.
  • 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 a lysine derivative.
  • the method includes the steps:
  • the cell or cell extract is cultured to introduce the lysine derivative into the target protein through the orthogonal tRNA synthetase and orthogonal tRNA pair .
  • the seventh aspect of the present invention provides a kit containing (a) a container, and (b) the mutant lysyl-tRNA synthesis according to the first aspect of the present invention in the container Enzyme or the polynucleotide of the second aspect of the invention.
  • the kit includes:
  • a third nucleic acid sequence which contains a third expression cassette for expressing the target protein, and the coding sequence of the target protein contains the orthogonal tRNA at a position for introducing a lysine derivative Recognized codon.
  • the kit further includes a cell extract.
  • nucleic acid sequences are located in the same or different containers.
  • each nucleic acid sequence is located on a different vector
  • a translation system in an eighth aspect of the present invention, includes:
  • the ninth aspect of the present invention provides the mutant lysyl-tRNA synthetase according to the first aspect of the present invention, or the host cell according to the fourth aspect of the present invention, or the expression according to the fifth aspect of the present invention
  • the system, or the use of the kit according to the seventh aspect of the present invention is used to incorporate unnatural amino acids into a target protein or to prepare a target protein containing unnatural amino acids.
  • the tenth aspect of the present invention provides a method for preparing a target protein containing unnatural amino acids, including the steps:
  • the target protein is expressed in the host cell or expression system.
  • the eleventh aspect of the present invention provides a method for producing the mutant lysyl-tRNA synthetase according to the first aspect of the present invention, comprising the steps of: (i) culturing the host cell according to the fourth aspect of the present invention, Thus, the mutant lysyl-tRNA synthetase is expressed.
  • the twelfth aspect of the present invention provides an enzyme preparation containing the mutant lysyl-tRNA synthetase according to 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 map of plasmid pEvol-pylRs(R19H, H29R)-pylT.
  • the mutant-type lysyl-tRNA synthetase of the present invention can significantly increase the insertion amount of unnatural amino acids and the amount of target protein containing unnatural amino acids.
  • 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 (e.g., 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 are within the scope well known to those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitutions
  • 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.: 3-8.
  • the mutant protein of the present invention generally has higher homology (identity).
  • the mutant protein is The homology of the sequence shown in SEQ ID NO.: 1 or SEQ ID NO.: 2 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, more preferably at least 98 %, preferably at least 99%.
  • 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 mutant lysyl-tRNA synthetase may include the polynucleotide encoding the mutant lysyl-tRNA synthetase of the present invention, or may also include additional coding and/or non-coding sequences Of polynucleotides.
  • 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.
  • 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:
  • polynucleotide (or variant) of the present invention encoding the mutant protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • 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 an appropriate 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.
  • the present invention mainly has the following advantages:
  • the present invention found for the first time that the 19th arginine (R) and/or the 29th position corresponding to the wild-type lysyl-tRNA synthetase (such as SEQ ID NO.: 1 or 2) Histidine (H) mutation, the resulting mutant lysyl-tRNA synthetase can introduce lysine derivatives with large functional groups into proteins.
  • the mutant-type lysyl-tRNA synthetase of the present invention can significantly increase the insertion amount of unnatural amino acids and the amount of target protein containing unnatural amino acids.
  • 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.
  • the present invention also found that the mutation of one or more amino acids at other positions of lysyl-tRNA synthetase, such as the 26th, 122, 309, 348 and/or 384 alleles, can further increase the amount of unnatural amino acid insertion , The amount of target protein containing unnatural amino acids and/or the stability of the target protein.
  • amino acid sequence 1 (SEQ ID NO.:1) of the wild-type lysyl-tRNA synthetase pylRs, cloned into the expression vector plasmid pEvol-pBpF (purchased from NTCC, chloramphenicol resistant) downstream of the araBAD promoter SpeI -SalI site, 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.: 9) of the tRNA (pylTcua) of lysyl-tRNA synthetase was inserted by PCR.
  • This plasmid was named pEvol-pylRs-pylT.
  • the mutation R19H was introduced to obtain the mutant lysyl-tRNA synthetase pylRs whose amino acid sequence is shown in SEQ ID NO.:7 (R19H).
  • the mutation H29R was introduced to obtain the mutant lysyl-tRNA synthetase pylRs whose amino acid sequence is shown in SEQ ID NO.:8 (H29R).
  • mutant lysyl-tRNA synthetase pylRs According to the amino acid sequence of wild-type lysyl-tRNA synthetase pylRs (SEQ ID NO.: 1), mutations R19H, H29R were introduced, and the amino acid sequence of mutant lysyl-tRNA was synthesized as shown in SEQ ID NO.: 6 Enzyme pylRs (R19H, H29R). According to the amino acid sequence of wild-type lysyl-tRNA synthetase pylRs (SEQ ID NO.:1), the mutations R19H, I26V, H29R are introduced, and the mutant lysyl-amino acid sequence shown in SEQ ID NO.: 3 is obtained.
  • tRNA synthetase pylRs (R19H, I26V, H29R). According to the amino acid sequence of wild-type lysyl-tRNA synthetase pylRs (SEQ ID NO.: 1), the mutations R19H, H29R, T122S, Y384F are introduced, and the mutant lysine whose amino acid sequence is shown in SEQ ID NO.: 4 Acyl-tRNA synthetase pylRs (R19H, H29R, T122S, Y384F).
  • 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 obtained plasmid was named pEvol-pylRs(R19H)-pylT;
  • 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 to 10 times, and perform conventional folding for 16 to 30 hours at 4 to 8° C. and pH 10.5 to 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 SDS-polyacrylamide gel electrophoresis purity reached 90%.
  • the obtained Boc-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.
  • 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%.
  • Enzyme Human insulin production (mg/L fermentation broth) pylRs 360 R19H 440 H29R 400 R19H,H29R 450 R19H,I26V,H29R 580 R19H, H29R, T122S, Y384F 700
  • Plasmid pEvol-pylRs-pylT, plasmid pEvol-pylRs(R19H)-pylT, plasmid pEvol-pylRs(H29R)-pylT, plasmid pEvol-pylRs(R19H, H29R)-pylT, plasmid pEvol-pylRs(R19H, I26V, H29R) )-pylT and plasmid pEvol-pylRs (R19H, H29R, L309A, C348S)-pylT were co-transformed with the insulin fusion protein expression vector pBAD-INS (plasmid constructed by our company, kana resistance) by chemical method (CaCl 2 method) In large E.coli Top10 competent cells (competent cells purchased from Thermo Company), the transformed cells were cultured in LB agar medium (10g/m
  • L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar were incubated overnight at 37°C.
  • 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.
  • 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

提供了一种突变型赖氨酰-tRNA合成酶。与野生型赖氨酰-tRNA合成酶相比，本发明的突变型赖氨酰-tRNA合成酶可提高非天然氨基酸的插入量和含有非天然氨基酸的目的蛋白量，还可以提高目的蛋白的稳定性，使其不容易断裂。

Description

在蛋白中高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 技术领域
本发明涉及生物技术领域，具体地涉及在蛋白中高效引入赖氨酸衍生物的氨酰基—tRNA合成酶。
背景技术
将蛋白质中的希望的位置的氨基酸残基用通常的蛋白质合成所涉及的20种类以外的氨基酸(非天然氨基酸)替换而得到的非天然氨基酸整合蛋白质(全蛋白质)，能够成为用于蛋白质的结构功能解析的有效手段。使用源自各种生物种类的氨基酰-tRNA合成酶(aaRS)/tRNA对，已经合成了30种类以上的全蛋白质。历史最长并且应用于许多有用非天然氨基酸导入的体系为酪氨酰-tRNA合成酶(TyrRS)突变体与经琥珀抑制基因化tRNA Tyr对。就该方法而言，成为关键的是其正交的关系，即，真细菌、与古细菌和真核生物这两个组中的aaRS在各自的组内将tRNA氨基酰化，但不能将其它组的tRNA氨基酰化。
另一方面，源自马氏甲烷八叠球菌(Methanosarcina mazei)的吡咯赖氨酰-tRNA合成酶(PylRS)和琥珀抑制基因tRNAPyl，在大肠杆菌细胞内作为具有正交性的aaRS/tRNA对而发挥功能。吡咯赖氨酸(Pyrrolysine)是在侧链上具有大体积甲基吡咯啉部分的赖氨酸衍生物。野生型PylRS能够在大肠杆菌内使Nε-Boc-L-赖氨酸与tRNAPyl结合。但是，由于LysRS对赖氨酸的识别严密，因此，至今为止，很难将具有各种大小、形状的官能团的赖氨酸衍生物位点特异性地导入蛋白质。
因此，本领域需要对野生型赖氨酰-tRNA合成酶进行改造，开发在蛋白中高效引入赖氨酸衍生物的氨酰基-tRNA合成酶。
发明内容
本发明的目的是提供一种在蛋白中高效引入赖氨酸衍生物的氨酰基-tRNA合成酶及方法。
本发明的第一方面，提供了一种突变型赖氨酰-tRNA合成酶，所述突变型赖氨酰-tRNA合成酶在对应于野生型赖氨酰-tRNA合成酶的氨基酸序列中第19位精氨酸(R)和/或第29位组氨酸(H)发生突变。
在另一优选例中，所述野生型赖氨酰-tRNA合成酶来自产甲烷古细菌的马氏甲烷八叠球菌(Methanosarcina mazei)、巴氏甲烷八叠球菌(Methanosarcina barkeri)或噬乙酸甲烷八叠球菌(Methanosarcina acetivorans)。
在另一优选例中，所述野生型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述野生型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述第19位精氨酸(R)突变为组氨酸(H)或赖氨酸(K)；和/或
第29位组氨酸(H)突变为精氨酸(R)或赖氨酸(K)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶中的突变选自下组：R19H、R19K、H29R、H29K、或其组合。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶还包括选自下组的位点发生突变：第26位异亮氨酸(I)、第122位苏氨酸(T)、第309位亮氨酸(L)、第348位半胱氨酸(C)、第384位酪氨酸(Y)、或其组合。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的突变位点还包括第26位异亮氨酸(I)；优选地，所述第26位异亮氨酸(I)突变为缬氨酸(V)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的突变位点还包括第122位苏氨酸(T)；优选地，所述第122位苏氨酸(T)突变为色氨酸(S)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的突变位点还包括第309位亮氨酸(L)；优选地，所述第309位亮氨酸(L)突变为丙氨酸(A)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的突变位点还包括第348位半胱氨酸(C)；优选地，所述第348位半胱氨酸(C)突变为色氨酸(S)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的突变位点还包括第384位酪氨酸(Y)；优选地，所述第384位酪氨酸(Y)突变为苯丙氨酸(F)。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶还包括选自下组的突变：I26V、T122S、L309A、C348S、Y384F、或其组合。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19H和H29R。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19K和H29R。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19H和H29K。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19K和H29K。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19H、I26V和H29R。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19H、H29R、T122S和Y384F。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶包括选自下组的突变：R19H、H29R、L309A和C348S。
在另一优选例中，所述的突变型赖氨酰-tRNA合成酶除所述突变(如第19和/或29位，以及任选的第26、122、309、348和/或384位)外，其余氨基酸与SEQ ID NO.:1或SEQ ID NO.:2所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的突变蛋白仍具有赖氨酰-tRNA合成酶的活性。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的氨基酸序列与SEQ ID NO.1或SEQ ID NO.:2相比具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶为SEQ ID NO.:1或SEQ ID NO.:2所示的野生型赖氨酰-tRNA合成酶经突变形成的。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶选自下组：
(1)氨基酸序列如SEQ ID NO.:3-8任一所示的多肽；或
(2)将SEQ ID NO.:3-8任一所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(1)所述多肽功能的由SEQ ID NO.:3-8任一所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:3-8任一所示。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶为非天然蛋白。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶用于将赖氨酸衍生物引入目 的蛋白中。
在另一优选例中，所述突变型赖氨酰-tRNA合成酶具有以下特征：
与野生型赖氨酰-tRNA合成酶相比，能将大官能团的赖氨酸衍生物导入蛋白中。
本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的突变型赖氨酰-tRNA合成酶。
在另一优选例中，所述多核苷酸编码如SEQ ID NO.:3-8任一所示多肽。
在另一优选例中，所述的多核苷酸包括DNA序列、RNA序列、或其组合。
在另一优选例中，所述的多核苷酸在突变型赖氨酰-tRNA合成酶的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
本发明的第三方面，提供了一种载体，所述载体含有如本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
在另一优选例中，所述载体选自下组：pET、pCW、pUC、pPIC9k、pMA5、或其组合。
在另一优选例中，所述载体为pEvol载体和/或pBAD载体。
在另一优选例中，所述载体用于表达突变型赖氨酰-tRNA合成酶。
本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述宿主细胞选自下组：原核细胞、真核细胞、或其组合。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞、植物细胞或动物细胞(如哺乳动物细胞)。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述的宿主细胞为大肠杆菌Top10、或BL21。
在另一优选例中，所述宿主细胞包含：
(a)本发明第一方面所述的突变型赖氨酰-tRNA合成酶；和
(b)在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合的 正交tRNA；以及任选地
(c)编码目的蛋白的目的核酸序列，所述目的核酸序列在用于引入赖氨酸衍生物的位置上包含由所述正交tRNA识别的密码子。
在另一优选例中，所述宿主细胞还包含：
(d)赖氨酸衍生物。
在另一优选例中，所述赖氨酸衍生物是所述突变型赖氨酰-tRNA合成酶的底物。
在另一优选例中，所述赖氨酸衍生物选自下组：炔基氧羰基赖氨酸衍生物、BOC-赖氨酸(叔丁氧羰基-L-赖氨酸)衍生物、脂肪酰化赖氨酸、或其组合。
在另一优选例中，所述炔基氧羰基赖氨酸的结构如下式I所示：
Figure PCTCN2020080034-appb-000001
其中，n为0-8。
在另一优选例中，所述正交tRNA为抑制基因tRNA，较佳地为琥珀抑制型tRNA。
在另一优选例中，所述正交tRNA的编码核酸序列如SEQ ID NO.:9所示。
Figure PCTCN2020080034-appb-000002
在另一优选例中，所述正交tRNA识别的密码子为琥珀密码子。
在另一优选例中，所述宿主细胞为原核细胞或真核细胞，较佳地为大肠杆菌。
本发明的第五方面，提供了一种表达系统，所述表达系统包括：
(a)本发明第一方面所述的突变型赖氨酰-tRNA合成酶；和
(b)在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合的正交tRNA；以及任选地
(c)编码目的蛋白的目的核酸序列，所述目的核酸序列在用于引入赖氨酸衍生物的位置上包含由所述正交tRNA识别的密码子。
在另一优选例中，所述表达系统还包括：
(d)赖氨酸衍生物。
在另一优选例中，所述赖氨酸衍生物选自下组：炔基氧羰基赖氨酸衍生物、BOC-赖氨酸(叔丁氧羰基-L-赖氨酸)衍生物、脂肪酰化赖氨酸、或其组合。
在另一优选例中，所述炔基氧羰基赖氨酸的结构如下式I所示：
Figure PCTCN2020080034-appb-000003
其中，n为0-8。
在另一优选例中，所述正交tRNA为抑制基因tRNA，较佳地为琥珀抑制型tRNA。
在另一优选例中，所述正交tRNA的核酸序列如SEQ ID NO.:9所示。
在另一优选例中，所述正交tRNA识别的密码子为琥珀密码子。
在另一优选例中，所述表达系统为细胞或细胞提取液。
在另一优选例中，所述表达系统用于将炔基氧羰基赖氨酸引入到目的蛋白中或制备含非天然氨基酸的目的蛋白。
本发明的第六方面，提供了一种在目的蛋白中引入非天然氨基酸的方法，包括步骤：
(1)提供本发明第四方面所述的宿主细胞或本发明第五方面所述的表达系统；和
(2)在赖氨酸衍生物的存在下，在所述宿主细胞或表达系统中表达目的蛋白。
在另一优选例中，所述目的蛋白选自下组：人胰岛素前体蛋白、赖脯胰岛素的前体蛋白、甘精胰岛素的前体蛋白、甲状旁腺素、可的瑞琳、降血钙素、比伐卢定、胰高血糖素样肽及其衍生物艾塞那肽和利拉鲁肽、齐考诺肽、舍莫瑞林、生长瑞林、分泌素、替度鲁肽、水蛭素、生长激素、生长因子、生长激素释放因子、促肾上腺皮质激素、释放因子、德舍瑞林、去氨加压素、依降钙素、胰高血糖素、亮丙瑞林、促黄体激素释放激素、生长激素抑制素、促甲状腺激素释放激素、曲普瑞林、血管活性肠肽、干扰素、甲状旁腺激素、BH3肽、淀粉样变肽、或上述肽的片段、或其组合。
在另一优选例中，所述非天然氨基酸为赖氨酸衍生物。
在另一优选例中，所述方法包括步骤：
(1)提供表达本发明第一方面所述的突变型赖氨酰-tRNA合成酶、正交tRNA、目的核酸序列的细胞或细胞提取液；和
(2)在赖氨酸衍生物的存在下，培养所述细胞或细胞提取液，从而通过所述正交tRNA合成酶和正交tRNA对将所述赖氨酸衍生物引入所述目的蛋白中。
本发明的第七方面，提供了一种试剂盒，所述试剂盒含有(a)容器，以及(b)位于所述容器内的本发明第一方面所述的突变型赖氨酰-tRNA合成酶或本发明第二方面所述的多核苷酸。
在另一优选例中，所述试剂盒包括：
(i)第一核酸序列，所述第一核酸序列含有用于表达所述突变型赖氨酰-tRNA合成酶的第一表达盒；和/或
(ii)第二核酸序列，所述第二核酸序列含有用于表达正交tRNA的第二表达盒，其中所述正交tRNA在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合；和/或
(iii)第三核酸序列，所述第三核酸序列含有用于表达目的蛋白的第三表达盒，目的蛋白的编码序列在用于引入赖氨酸衍生物的位置上包含由所述正交tRNA识别的密码子。
在另一优选例中，所述试剂盒还包含细胞提取液。
在另一优选例中，所述的各核酸序列位于相同或不同的容器内。
在另一优选例中，所述的各核酸序列位于不同的载体上
本发明的第八方面，提供了一种翻译系统，所述翻译系统包括：
(a)本发明第一方面所述的突变型赖氨酰-tRNA合成酶；
(b)在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合的正交tRNA；以及
(c)赖氨酸衍生物。
本发明的第九方面，提供了如本发明第一方面所述的突变型赖氨酰-tRNA合成酶、或本发明第四方面所述的宿主细胞、或本发明第五方面所述的表达系统、或本发明第七方面所述的试剂盒的用途，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。
本发明的第十方面，提供了一种含非天然氨基酸的目的蛋白的制备方法，包括步骤：
(1)提供本发明第四方面所述的宿主细胞或本发明第五方面所述的表达系统；和
(2)在赖氨酸衍生物的存在下，在所述宿主细胞或表达系统中表达目的蛋白。
本发明的第十一方面，提供了一种产生本发明第一方面所述突变型赖氨酰-tRNA合成酶的方法，包括步骤：(i)培养本发明第四方面所述的宿主细胞，从而表达出突变型赖氨酰-tRNA合成酶。
本发明的第十二方面，提供了一种酶制剂，所述酶制剂含有本发明第一方面所述的突变型赖氨酰-tRNA合成酶。
在另一优选例中，所述药物制剂的剂型包括：冻干制剂、液体制剂、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了质粒pEvol-pylRs(R19H,H29R)-pylT图谱。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，意外地获得一种突变型赖氨酰-tRNA合成酶。相比野生型赖氨酰-tRNA合成酶，本发明突变型赖氨酰-tRNA合成酶可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白量。此外，本发明突变型赖氨酰-tRNA合成酶还可以提高目的蛋白的稳定性，使其不容易断裂。在此基础上，发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文 所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性(或同源性)通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
在本文中，“氨酰-tRNA合成酶”、“赖氨酰-tRNA合成酶”可互换使用。
野生型赖氨酰-tRNA合成酶
如本文所用，“野生型赖氨酰-tRNA合成酶”是指天然存在的、未经过人工改造的氨酰-tRNA合成酶，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型赖氨酰-tRNA合成酶的来源没有特别限制，一种优选的来源为产甲烷古细菌的马氏甲烷八叠球菌(Methanosarcina mazei)、巴氏甲烷八叠球菌(Methanosarcina barkeri)和噬乙酸甲烷八叠球菌(Methanosarcina acetivorans)等取得，但不限于此。
在本发明的一个优选例中，所述野生型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:1所示。
Figure PCTCN2020080034-appb-000004
在本发明的一个优选例中，所述野生型赖氨酰-tRNA合成酶的氨基酸序列如SEQ ID NO.:2所示。
Figure PCTCN2020080034-appb-000005
Figure PCTCN2020080034-appb-000006
突变型赖氨酰-tRNA合成酶
如本文所用，术语“突变蛋白”、“本发明突变蛋白”、“本发明突变的氨酰-tRNA合成酶”、“突变型赖氨酰-tRNA合成酶”、“突变体酶”、“氨酰-tRNA合成酶的突变体”可互换使用，均指非天然存在的突变的氨酰-tRNA合成酶，且所述突变的氨酰-tRNA合成酶为对SEQ ID NO.:1或SEQ ID NO.:2所示多肽进行人工改造的蛋白。具体地，所述突变的氨酰-tRNA合成酶如本发明第一方面所述。
应理解，本发明突变型赖氨酰-tRNA合成酶中的氨基酸编号基于野生型赖氨酰-tRNA合成酶(优选地，SEQ ID NO.:1或SEQ ID NO.:2)作出。当某一具体突变蛋白与SEQ ID NO.:1或SEQ ID NO.:2所示序列的同源性达到80％或以上时，突变蛋白的氨基酸编号可能会有相对于SEQ ID NO.:1或SEQ ID NO.:2的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似糖基转移酶活性的突变蛋白不在本发明突变蛋白的范围内。
本发明突变蛋白是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述突变蛋白的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述突变蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白 半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
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
PylRS的氨基酸底物的识别与催化活性功能域的立体结构相关，野生型PylRS能够活化的赖氨酸衍生物的大小有限，具有大官能团的赖氨酸衍生物就不能导入到蛋白质中，因此，通过对PylRS位点的突变，避免结合底物的空间位阻，或突变氨 基酸与底物氨基酸或者是主链部分发生的相互作用，来提高效果。
优选地，所述的突变蛋白如SEQ ID NO.:3-8中任一所示。
Figure PCTCN2020080034-appb-000007
Figure PCTCN2020080034-appb-000008
应理解，本发明突变蛋白与SEQ ID NO.:1或SEQ ID NO.:2所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变蛋白与SEQ ID NO.:1或SEQ ID NO.:2所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，更佳地至少为98％，最佳地至少为99％。
此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
术语“编码突变型赖氨酰-tRNA合成酶的多核苷酸”可以是包括编码本发明突变型赖氨酰-tRNA合成酶的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或突变蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的突变蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的突变蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅 读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如人参细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温 度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
与现有技术相比，本发明主要具有以下优点：
经大量筛选和改造，本发明首次发现了在对应于野生型赖氨酰-tRNA合成酶(如SEQ ID NO.:1或2)中第19位精氨酸(R)和/或第29位组氨酸(H)突变，得到的突变型赖氨酰-tRNA合成酶能够将大官能团的赖氨酸衍生物导入蛋白中。
相比野生型赖氨酰-tRNA合成酶，本发明突变型赖氨酰-tRNA合成酶可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白量。此外，本发明突变型赖氨酰-tRNA合成酶还可以提高目的蛋白的稳定性，使其不容易断裂。
本发明还发现对赖氨酰-tRNA合成酶的其他位点如第26、122、309、348和/或384位等位点的一个或几个氨基酸进行突变，可进一步提高非天然氨基酸插入量、含有非天然氨基酸的目的蛋白量和/或目的蛋白的稳定性。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1 突变体的构建
根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列1(SEQ ID NO.:1)，克隆至表达载体质粒pEvol-pBpF(购自NTCC公司，氯霉素抗性)的araBAD启动子下游SpeI-SalI位点，其中SpeI酶切位点通过PCR方法增加，SalI位点为载体本身具有。保留表达载体质粒pEvol-pBpF原有的谷氨酰胺启动子glnS。在表达载体质粒 pEvol-pBpF的proK启动子下游，以PCR方法插入赖氨酰-tRNA合成酶的tRNA(pylTcua)的DNA序列(SEQ ID NO.:9)。该质粒命名为pEvol-pylRs-pylT。
根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变R19H，得到氨基酸序列如SEQ ID NO.:7所示的突变型赖氨酰-tRNA合成酶pylRs(R19H)。根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变H29R，得到氨基酸序列如SEQ ID NO.:8所示的突变型赖氨酰-tRNA合成酶pylRs(H29R)。根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变R19H,H29R，得到氨基酸序列如SEQ ID NO.:6所示的突变型赖氨酰-tRNA合成酶pylRs(R19H,H29R)。根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变R19H,I26V,H29R，得到氨基酸序列如SEQ ID NO.:3所示的突变型赖氨酰-tRNA合成酶pylRs(R19H,I26V,H29R)。根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变R19H,H29R,T122S,Y384F，得到氨基酸序列如SEQ ID NO.:4所示的突变型赖氨酰-tRNA合成酶pylRs(R19H,H29R,T122S,Y384F)。根据野生型赖氨酰-tRNA合成酶pylRs的氨基酸序列(SEQ ID NO.:1)，引入突变R19H,H29R,L309A,C348S，得到氨基酸序列如SEQ ID NO.:5所示的突变型赖氨酰-tRNA合成酶pylRs(R19H,H29R,L309A,C348S)。
以质粒pEvol-pylRs-pylT为模板，设计引物，见SEQ ID NO.:10和SEQ ID NO.:11，引入定点突变R19H。将PCR产物经DpnI处理，以核酸电泳进行分离，以琼脂糖凝胶DNA回收试剂盒提取，使用T4DNA Ligase连接，以化学法(CaCl 2法)转化大E.coli Top10感受态细胞中，将所述转化的细胞培养在含有氯霉素的LB琼脂培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl，1.5％琼脂)上37℃培养过夜。挑取单个活菌落，在含有氯霉素的液体LB培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl)中37℃220rpm培养过夜。以质粒小量提取试剂盒提取质粒，得到的质粒命名为pEvol-pylRs(R19H)-pylT；
以质粒pEvol-pylRs-pylT为模板，设计引物，见SEQ ID NO.:12和SEQ ID NO.:13，引入定点突变H29R，得到的质粒命名为pEvol-pylRs(H29R)-pylT；
以质粒pEvol-pylRs(R19H)-pylT为模板，设计引物，见SEQ ID NO.:12和SEQ ID NO.:13，引入定点突变H29R，得到的质粒命名为pEvol-pylRs(R19H,H29R)-pylT。质粒图谱见图1。
5’-ATACCGGCACCATCCACAAG-3’(SEQ ID NO.:10)
5’-GGCTCATCCACAGACCAGTT-3’(SEQ ID NO.:11)
5’-GCGAGGTTTCCCGTAGCAAA-3’(SEQ ID NO.:12)
5’-GGTGTTTGATCTTGTGGATGGT-3’(SEQ ID NO.:13)
利用上述方法另分别构建pEvol-pylRs(R19H,I26V,H29R)-pylT、质粒pEvol-pylRs(R19H,H29R,T122S,Y384F)-pylT和质粒pEvol-pylRs(R19H,H29R,L309A,C348S)-pylT。
实施例2 菌种构建和叔丁氧羰基(Boc)修饰融合蛋白的高密度表达
将质粒pEvol-pylRs-pylT、质粒pEvol-pylRs(R19H)-pylT、质粒pEvol-pylRs(H29R)-pylT、质粒pEvol-pylRs(R19H,H29R)-pylT、质粒pEvol-pylRs(R19H,I26V,H29R)-pylT和质粒pEvol-pylRs(R19H,H29R,T122S,Y384F)-pylT分别与胰岛素融合蛋白表达载体pBAD-INS(卡那抗性)以化学法(CaCl 2法)共转化到E.coli Top10感受态细胞中(感受态细胞购买自Thermo公司)，将所述转化的细胞培养在含有25μg/mL卡那霉素和17μg/mL氯霉素的LB琼脂培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl，1.5％琼脂)上37℃培养过夜。挑取单个活菌落，在含有25μg/mL卡那霉素和17μg/mL氯霉素的液体LB培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl)中37℃220rpm培养过夜。加入终浓度20％甘油保存菌种。
将各菌种接种于液体LB培养基中37℃、220rpm培养过夜，以1％(v/v)接种罐发酵培养基(12g/L酵母蛋白胨，24g/L酵母浸粉，4mL/L甘油，12.8g/L磷酸氢二钠，3g/L磷酸二氢钾，0.3‰消泡剂)，在35(±3)℃，200～1000rpm，空气流量2～6L/min的条件下培养。培养3～10h后，以步进速率流加含甘油和酵母蛋白胨的补料培养基，持续至发酵结束。培养至OD 600达到25～80时，加入终浓度0.25％L-ara和终浓度5mM Boc-Lys进行诱导。继续培养至OD 600达到180～220时，放罐。然后离心(5000rpm，30min，25℃)收集。以SDS-聚丙烯酰胺电泳对各菌种全细胞中含有Boc修饰赖氨酸的融合蛋白表达情况进行检测。
融合蛋白是以不可溶的“包涵体”形式表达。为释放包涵体，用高压均质机将大肠杆菌细胞破碎。通过10000g离心法去除核酸、细胞碎片和可溶性蛋白。用纯水洗涤含有融合蛋白的包涵体，所得的包涵体沉淀用作折叠的原材料。
不同突变酶的融合蛋白表达量如下表：
酶 Boc赖氨酸的融合蛋白表达量(g/L发酵液)
pylRs 7.9
R19H 8.9
H29R 8.6
R19H,H29R 9.4
R19H,I26V,H29R 11.4
R19H,H29R,T122S,Y384F 14.0
为了使融合蛋白重折叠，将包涵体溶解于pH 10.5并含有2～10mM巯基乙醇的7.5M脲溶液中，使溶解后总蛋白的浓度为10～25mg/mL。将样品稀释5～10倍，在4～8℃，pH为10.5～11.7的条件下进行常规折叠16～30小时。于18～25℃下,pH值维持在8.0～9.5，用胰蛋白酶和羧肽酶B将融合蛋白酶解10～20小时，然后加入0.45M硫酸铵终止酶解反应。反相HPLC分析结果表明，该酶解步骤的收率高于90％。胰蛋白酶与羧肽酶B酶解后获得的胰岛素类似物被命名为BOC-赖氨酸胰岛素。Boc-赖氨酸胰岛素在上述条件下不能被酶解。通过膜过滤澄清样品，以0.45mM硫酸铵作为缓冲液，经疏水层析初纯化，SDS-聚丙烯酰胺凝胶电泳纯度达90％。并且对获得的Boc-人胰岛素进行MALDI-TOF质谱分析，结果检测出其分子量与理论分子量5907.7Da相符合。经疏水层析洗脱收集样品，加入盐酸进行Boc-人胰岛素脱保护反应，加入氢氧化钠溶液控制pH为2.8～3.2以终止反应，再经两步高压反相层析，重组人胰岛素的得率高于85％。
不同突变酶的重组人胰岛素表达量如下表：
酶 人胰岛素的产量(mg/L发酵液)
pylRs 360
R19H 440
H29R 400
R19H,H29R 450
R19H,I26V,H29R 580
R19H,H29R,T122S,Y384F 700
结果表明，利用本发明的突变酶制备含有Boc-赖氨酸的目的蛋白，可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白量。
实施例3 菌种构建和丁炔氧羰基修饰融合蛋白的高密度表达
将质粒pEvol-pylRs-pylT、质粒pEvol-pylRs(R19H)-pylT、质粒pEvol-pylRs(H29R)-pylT、质粒pEvol-pylRs(R19H,H29R)-pylT、质粒 pEvol-pylRs(R19H,I26V,H29R)-pylT和质粒pEvol-pylRs(R19H,H29R,L309A,C348S)-pylT分别与胰岛素融合蛋白表达载体pBAD-INS(质粒由本公司构建，卡那抗性)以化学法(CaCl 2法)共转化大E.coli Top10感受态细胞中(感受态细胞购买自Thermo公司)，将所述转化的细胞培养在含有25μg/mL卡那霉素和17μg/mL氯霉素的LB琼脂培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl，1.5％琼脂)上37℃培养过夜。挑取单个活菌落，在含有25μg/mL卡那霉素和17μg/mL氯霉素的液体LB培养基(10g/L酵母蛋白胨，5g/L酵母浸粉，10g/L NaCl)中37℃220rpm培养过夜。加入终浓度20％甘油保存菌种。
将各菌种接种于液体LB培养基中37℃220rpm培养过夜，以1％(v/v)接种罐发酵培养基(12g/L酵母蛋白胨，24g/L酵母浸粉，4mL/L甘油，12.8g/L磷酸氢二钠，3g/L磷酸二氢钾，0.3‰消泡剂)，在35(±3)℃，200～1000rpm，空气流量2～6L/min的条件下培养。培养3～10h后，以步进速率流加含甘油和酵母蛋白胨的补料培养基，持续至发酵结束。培养至OD 600达到25～80时，加入终浓度0.25％L-ara和终浓度5mM丁炔氧羰基-Lys进行诱导。继续培养至OD 600达到180～220时，放罐。然后离心(5000rpm，30min，25℃)收集。以SDS-聚丙烯酰胺电泳对各菌种全细胞中含有丁炔氧羰基修饰赖氨酸的融合蛋白表达情况进行检测。
酶 丁炔氧羰基赖氨酸的融合蛋白表达量(g/L发酵液)
pylRs 4.5
H29R 5.0
R19H 5.2
R19H,H29R 6.8
R19H,I26V,H29R 6.7
R19H,H29R,L309A,C348S 8.4
融合蛋白是以不可溶的“包涵体”形式表达。为释放包涵体，用高压均质机将大肠杆菌细胞破碎。通过10000g离心法去除核酸、细胞碎片和可溶性蛋白。用纯水洗涤含有融合蛋白的包涵体，所得的包涵体沉淀用作折叠的原材料。为了使融合蛋白重折叠，将包涵体溶解于pH 10.5并含有2～10mM巯基乙醇的7.5M脲溶液中，使溶解后总蛋白的浓度为10～25mg/mL。将样品稀释5～10倍，在4～8℃，pH为10.5～11.7的条件下进行常规折叠16～30小时。于18～25℃下,pH值维持在8.0～9.5，用胰蛋白酶和羧肽酶B将融合蛋白酶解10～20小时，然后加入0.45M硫酸铵终止酶解反应。反相HPLC分析结果表明，该酶解步骤的收率高于90％。胰 蛋白酶与羧肽酶B酶解后获得的胰岛素类似物被命名为丁炔氧羰基-赖氨酸胰岛素。丁炔氧羰基-赖氨酸胰岛素在上述条件下不能被酶解。通过膜过滤澄清样品，以0.45mM硫酸铵作为缓冲液，经疏水层析初纯化，SDS-聚丙烯酰胺凝胶电泳纯度达90％。并且对获得的丁炔氧羰基-人胰岛素进行MALDI-TOF质谱分析，结果检测出其分子量与理论分子量5907.7Da相符合。
酶 丁炔氧羰基人胰岛素的产量(mg/L发酵液)
pylRs 530
H29R 590
R19H 610
R19H,H29R 810
R19H,I26V,H29R 790
R19H,H29R,L309A,C348S 1000
结果表明，利用本发明的突变酶制备丁炔氧羰基修饰的目的蛋白，可显著提高非天然氨基酸插入量和含有非天然氨基酸的目的蛋白量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种突变型赖氨酰-tRNA合成酶，其特征在于，所述突变型赖氨酰-tRNA合成酶在对应于野生型赖氨酰-tRNA合成酶的氨基酸序列中第19位精氨酸(R)和/或第29位组氨酸(H)发生突变。
  2. 如权利要求1所述的突变型赖氨酰-tRNA合成酶，其特征在于，所述突变型赖氨酰-tRNA合成酶还包括选自下组的位点发生突变：第26位异亮氨酸(I)、第122位苏氨酸(T)、第309位亮氨酸(L)、第348位半胱氨酸(C)、第384位酪氨酸(Y)、或其组合。
  3. 如权利要求1所述的突变型赖氨酰-tRNA合成酶，其特征在于，所述突变型赖氨酰-tRNA合成酶选自下组：
    (1)氨基酸序列如SEQ ID NO.:3-8任一所示的多肽；或
    (2)将SEQ ID NO.:3-8任一所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-8个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(1)所述多肽功能的由SEQ ID NO.:3-8任一所示氨基酸序列的多肽衍生的多肽。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1所述的突变型赖氨酰-tRNA合成酶。
  5. 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。
  6. 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。
  7. 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所述的突变型赖氨酰-tRNA合成酶；和
    (b)在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合的正交tRNA；以及任选地
    (c)编码目的蛋白的目的核酸序列，所述目的核酸序列在用于引入赖氨酸衍生物的位置上包含由所述正交tRNA识别的密码子。
  8. 一种表达系统，其特征在于，所述表达系统包括：
    (a)权利要求1所述的突变型赖氨酰-tRNA合成酶；和
    (b)在所述突变型赖氨酰-tRNA合成酶的存在下能够与赖氨酸衍生物结合的正交tRNA；以及任选地
    (c)编码目的蛋白的目的核酸序列，所述目的核酸序列在用于引入赖氨酸衍生物的位置上包含由所述正交tRNA识别的密码子。
  9. 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所述的突变型赖氨酰-tRNA合成酶、或权利要求4所述的多核苷酸、或权利要求5所述的载体。
  10. 如权利要求1所述的突变型赖氨酰-tRNA合成酶、或权利要求6所述的宿主细胞、或权利要求8所述的表达系统、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。
PCT/CN2020/080034 2019-03-19 2020-03-18 在蛋白中高效引入赖氨酸衍生物的氨酰基—tRNA合成酶 WO2020187269A1 (zh)

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BR112021018681A BR112021018681A2 (pt) 2019-03-19 2020-03-18 Aminoacil-trna sintetase para introdução eficiente de derivado de lisina em proteína
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