WO2020187271A1 - Introduction d'acides aminés non naturels dans des protéines à l'aide d'un système plasmidique double - Google Patents

Introduction d'acides aminés non naturels dans des protéines à l'aide d'un système plasmidique double Download PDF

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WO2020187271A1
WO2020187271A1 PCT/CN2020/080039 CN2020080039W WO2020187271A1 WO 2020187271 A1 WO2020187271 A1 WO 2020187271A1 CN 2020080039 W CN2020080039 W CN 2020080039W WO 2020187271 A1 WO2020187271 A1 WO 2020187271A1
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plasmid
trna synthetase
amino acid
protein
expression cassette
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PCT/CN2020/080039
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English (en)
Chinese (zh)
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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 CN202080023302.4A priority Critical patent/CN113631712A/zh
Publication of WO2020187271A1 publication Critical patent/WO2020187271A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention belongs to the technical field of biomedical engineering. Specifically, the present invention relates to the use of a two-plasmid system to introduce unnatural amino acids into proteins.
  • unnatural amino acids play an important role in the process of various proteins performing their physiological and pathological functions.
  • modification of unnatural amino acids may not only enhance the efficacy of peptide drugs and reduce drug toxicity, but also because of the incorporation of unnatural amino acids, peptide drugs greatly reduce immunogenicity and reduce immune rejection.
  • proteases may no longer recognize polypeptides that incorporate unnatural amino acids, allowing the drug to remain in the body for a longer time without being degraded, thereby prolonging the half-life of the drug, and eliminating the drawbacks of continuous injection and administration of peptide drugs;
  • peptide drugs "carry" other chemical attachments, leading to the emergence of new methods of disease treatment.
  • modification of peptide drugs most of them are only modified by chemical synthesis.
  • the purpose of the present invention is to provide a method for efficiently introducing non-natural amino acids into polypeptide drugs.
  • the first aspect of the present invention provides a double-plasmid system, which comprises:
  • a first plasmid said first plasmid containing a first expression cassette for expressing a target protein, said first expression cassette containing a first coding sequence encoding a target protein, said first coding sequence containing a Introduce predetermined natural codons for modified amino acids, the natural codons being UAG (amber), UAA (ochre), or UGA (opal); and
  • a second plasmid which contains a second expression cassette for expressing aminoacyl-tRNA synthetase
  • the system also contains a third expression cassette for encoding an artificial tRNA, wherein the artificial tRNA contains an anticodon corresponding to the natural codon, and the third expression cassette is located in the first plasmid and/or In the second plasmid;
  • 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 natural 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 structure of the alkynyloxycarbonyl lysine is as shown in formula I:
  • n 0-8.
  • the third expression cassette is located in a second plasmid.
  • the second expression cassette contains a second coding sequence encoding an aminoacyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase is a wild-type aminoacyl-tRNA synthetase or a mutant aminoacyl-tRNA synthetase.
  • aminoacyl-tRNA synthetase is lysyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase is a mutant lysyl-tRNA synthetase.
  • the mutant lysyl-tRNA synthetase has the 19th arginine (R) and/or the 29th group in the amino acid sequence corresponding to the wild-type lysyl-tRNA synthetase
  • the amino acid (H) is 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.: 3-9; or
  • amino acid sequence of the mutant lysyl-tRNA synthetase is shown in any one of SEQ ID NO.: 3-9.
  • 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 coding nucleic acid sequence of the artificial tRNA is shown in SEQ ID NO.: 10.
  • 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.
  • first plasmid and/or the second plasmid further comprise one or more promoters, and the promoters are operably linked to the first coding sequence, enhancer, transcription termination signal, multiple The adenylation sequence, the origin of replication, selectable markers, nucleic acid restriction sites, and/or homologous recombination sites are linked.
  • 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 resistance gene is selected from the following group: AmpR chloramphenicol resistance gene (CmR), kanamycin resistance gene (KanaR), tetracycline resistance gene (TetR), or a combination thereof .
  • 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, a ribosome binding site RBS, the first coding sequence, a terminator or a tag sequence in order from 5'-3'.
  • 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 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 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 a promoter (araBAD), a ribosome binding site RBS, the second coding sequence, and a terminator (rrnB) in order from 5'-3'.
  • araBAD promoter
  • RBS ribosome binding site
  • rrnB terminator
  • 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, a ribosome binding site RBS, an artificial tRNA coding sequence, a terminator or a tag sequence in order from 5'-3'.
  • the second aspect of the present invention provides a host cell or cell extract containing the dual plasmid system of the first 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 third aspect of the present invention provides a kit, which contains (a) a container, and (b) located in the container:
  • a first plasmid said first plasmid containing a first expression cassette for expressing a target protein, said first expression cassette containing a first coding sequence encoding a target protein, said first coding sequence containing a Introducing predetermined natural codons for modified amino acids, the natural codons being UAG (amber), UAA (ochre), or UGA (opal); and
  • a second plasmid which contains a second expression cassette for expressing aminoacyl-tRNA synthetase
  • the kit also contains a third expression cassette for encoding artificial tRNA, wherein the artificial tRNA contains an anticodon corresponding to the natural codon, and the third expression cassette is located in the first plasmid and/ Or in the second plasmid;
  • 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 kit further includes a cell extract.
  • first plasmid and the second plasmid are located in the same or different containers.
  • the natural codons are UAG (amber) or UGA (opal) codons.
  • 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 third expression cassette is located in a second plasmid.
  • the second expression cassette contains a second coding sequence encoding an aminoacyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase is a wild-type aminoacyl-tRNA synthetase or a mutant aminoacyl-tRNA synthetase.
  • aminoacyl-tRNA synthetase is lysyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase is a mutant lysyl-tRNA synthetase.
  • 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, amyloid peptide, or the above Fragments of peptides, or combinations thereof.
  • first plasmid and/or the second plasmid further comprise one or more promoters, and the promoters are operably linked to the first coding sequence, enhancer, transcription termination signal, multiple The adenylation sequence, the origin of replication, the selectable marker, the nucleic acid restriction site, and/or the homologous recombination site are linked.
  • the fourth aspect of the present invention provides the dual plasmid system according to the first aspect of the present invention, or the host cell or cell extract according to the second aspect of the present invention, or the kit according to the third aspect of the present invention. Uses: used to prepare proteins containing predetermined modified amino acids.
  • 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 fifth aspect of the present invention provides a method for preparing a protein containing a predetermined modified amino acid, the method comprising the steps:
  • 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 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 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.
  • Figure 1 shows the map of plasmid pBAD-A1-u4-u5-TEV-R-MiniINS.
  • Figure 2 shows the plasmid pEvol-pylRs(R19K, H29K, T122S, Y384F)-pylT map.
  • Figure 3 shows the expression of wild-type lysyl-tRNA synthetase pylRs (SEQ ID NO.:1) and mutant lysyl-tRNA synthetase pylRs (R19K, H29K, T122S, Y384F) on the Boc-modified fusion protein .
  • Figure 3a The expression of Boc modified GFP-TEV-R-MiniINS fusion protein by two enzymes. Lane 1, strain 1, wild type pylRs; Lane 2, strain 3, mutant pylRs (R19K, H29K, T122S, Y384F); M is the protein standard plasmid standard.
  • FIG. 3b The expression of Boc modified A1-u4-u5-TEV-R-MiniINS fusion protein by two enzymes.
  • M is the protein standard plasmid standard; lane 1, strain 2, wild-type pylRs; lane 2, strain 4, mutant pylRs (R19K, H29K, T122S, Y384F).
  • the present inventors unexpectedly discovered for the first time that unnatural amino acids can be directly introduced into proteins through the dual plasmid system of the present application, with low cost, high yield, and low environmental pollution.
  • this application also unexpectedly obtained a mutant lysyl-tRNA synthetase.
  • the mutant lysyl-tRNA synthetase of the present invention can 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” means that the value can vary from the recited value by no more than 1%.
  • 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
  • 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 coding sequence of the protein of interest can be incorporated into the vector.
  • Vectors can be used to replicate nucleic acids in compatible host cells.
  • the vector can be recovered from the host cell.
  • the vector may be an expression vector for expressing the nucleic acid sequence of interest in a compatible host cell.
  • the coding sequence of the protein of interest is operably linked to a control sequence (e.g., a promoter or enhancer) capable of providing expression of the coding sequence of the protein of interest in the host cell.
  • control sequence e.g., a promoter or enhancer
  • 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.
  • expression vectors containing the DNA sequence encoding the protein of the present invention and appropriate transcription/translation control signals preferably commercially available vectors: bacterial plasmids, phages, yeast plasmids, plant cell viruses, Mammalian cell viruses such as adenovirus, retrovirus or other vectors.
  • bacterial plasmids bacterial plasmids
  • phages phages
  • yeast plasmids preferably commercially available vectors: bacterial plasmids, phages, yeast plasmids, plant cell viruses, Mammalian cell viruses such as adenovirus, retrovirus or other vectors.
  • Mammalian cell viruses such as adenovirus, retrovirus or other vectors.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters include Escherichia coli lac or trp promoter; lambda phage PL promoter: eukaryotic promoters include CMV 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 their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, usually about 10-300bp, acting on promoters to enhance gene transcription. Such as adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the present invention also provides a recombinant vector, which comprises the target protein of the present invention, a mutant lysyl-tRNA synthetase gene, and an optional tRNA DNA sequence.
  • the recombinant vector contains a multiple cloning site or at least one restriction site downstream of the promoter. When the target gene needs to be expressed, the target gene is ligated into a suitable multiple cloning site or restriction site, so that the target gene and the promoter can be operably connected.
  • the recombinant vector includes a promoter, a target gene and a terminator in the 5'to 3'direction. If necessary, the recombinant vector can also include the following elements: protein purification tag; 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; selection marker (antibiotic resistance gene, fluorescent protein, etc.) ; Enhancer; or operator.
  • the methods for preparing recombinant vectors are well known to those of ordinary skill in the art.
  • the expression vector can be a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vectors.
  • the expression vector can be pET, pCW, pUC, pPIC9k, pMA5 or other vectors. In short, as long as it can replicate and stabilize in the host, any plasmid and vector can be used.
  • Those of ordinary skill in the art can use well-known methods to construct a vector containing the promoter of the present invention and/or the target gene sequence. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the expression vector of the present invention can be used to transform an appropriate host cell to enable the host to transcribe the target RNA or express the target protein.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells , Preferably, rape, tobacco, soybean; insect cells Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells.
  • the expression host can be Escherichia coli, Bacillus subtilis, Pichia pastoris, Streptomyces or other host cells.
  • Those of ordinary skill in the art know how to select an appropriate vector and host cell. 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), it can be treated with the CaCl 2 method or electroporation method.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • This method is realized by growing or culturing the host cell by a method well known to those skilled in the art.
  • microbial cells are usually at 0-100°C, preferably 10-60°C, and oxygen is also required.
  • the medium contains carbon sources, such as glucose; nitrogen sources, usually in the form of organic nitrogen, such as yeast extract, amino acids; salts, such as ammonium sulfate, trace elements such as iron and magnesium salts; and vitamins if necessary.
  • the pH of the medium can be maintained at a fixed value, that is, controlled or not controlled during the cultivation period.
  • Cultivation can be carried out in the form of batch culture, semi-discontinuous culture or continuous culture. After incubation, the cells are collected, broken or used directly.
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc.
  • the transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain transgenic plants.
  • the obtained transformants can be cultured by conventional methods to express the target protein 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 double plasmid system, which comprises:
  • a first plasmid said first plasmid containing a first expression cassette for expressing a target protein, said first expression cassette containing a first coding sequence encoding a target protein, said first coding sequence containing a Introduce predetermined natural codons for modified amino acids, the natural codons being UAG (amber), UAA (ochre), or UGA (opal); and
  • a second plasmid which contains a second expression cassette for expressing aminoacyl-tRNA synthetase
  • the system also contains a third expression cassette for encoding an artificial tRNA, wherein the artificial tRNA contains an anticodon corresponding to the natural codon, and the third expression cassette is located in the first plasmid and/or In the second plasmid;
  • 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 natural 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 third expression cassette is located in a second plasmid.
  • the second expression cassette contains a second coding sequence encoding an aminoacyl-tRNA synthetase.
  • 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 resistance gene is selected from the following group: AmpR chloramphenicol resistance gene (CmR), kanamycin resistance gene (KanaR), tetracycline resistance gene (TetR), or a combination thereof .
  • 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, a ribosome binding site RBS, the first coding sequence, a terminator or a tag sequence in order from 5'-3'.
  • the second plasmid is a pEvol-pBpF vector.
  • the second plasmid further contains a resistance gene, a repressor protein gene (araC), a promoter gene (araBAD), a tag sequence, 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 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 a promoter (araBAD), a ribosome binding site RBS, the second coding sequence, and a terminator (rrnB) in order from 5'-3'.
  • araBAD promoter
  • RBS ribosome binding site
  • rrnB terminator
  • 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, a ribosome binding site RBS, an artificial tRNA coding sequence, a terminator or a tag sequence in order from 5'-3'.
  • wild-type lysyl-tRNA synthetase and “wild-type enzyme pylRs” refer to a naturally-occurring aminoacyl-tRNA synthetase that has not been artificially modified, and its nucleotides can be obtained by genetic engineering technology. Obtained, such as genome sequencing, polymerase chain reaction (PCR), etc., whose 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.: 3-9.
  • 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 directly introduces unnatural amino acids into the protein synthesis process through the double plasmid system, with low cost, high yield and little environmental pollution.
  • the mutant lysyl-tRNA synthetase of the present invention can 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. By mutating a partial sequence of lysyl-tRNA synthetase, it can promote its soluble expression, which is more conducive to the separation and purification of target proteins with different expression forms.
  • the DNA of A1-u4-u5-TEV-R-MiniINS was synthesized
  • the sequence (SEQ ID NO.: 12) was cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD/HisA (purchased from NTCC, kanamycin resistance).
  • the original tag gene (6*His) of the expression vector plasmid pBAD/His A is not retained.
  • A1-u4-u5-TEV-R-MiniINS amino acid sequence 1 (SEQ ID NO.: 12)
  • the sequence 1 was cut from the cloning vector pUC57-A1-u4-u5-TEV-R-MiniINS with restriction enzymes NcoI and XhoI, and the expression vector plasmid pBAD/His A was cut with NcoI and XhoI at the same time, and the nucleic acid Separated by electrophoresis, extracted with agarose gel DNA recovery kit, ligated with T4 DNA Ligase, and transformed into E. coli Top10 competent cells by chemical method (CaCl2 method), and the transformed cells were cultured in E. coli Top10 competent cells.
  • the DNA sequence of pylRs was synthesized and cloned into the expression vector plasmid pEvol-pBpF (purchased from NTCC, chloramphenicol resistance) SpeI-SalI site downstream of the araBAD promoter, 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.: 10) of the tRNA (pylTcua) of lysyl-tRNA synthetase was inserted by PCR.
  • This plasmid was named pEvol-pylRs-pylT.
  • the sequence 6 was cut from the cloning vector pUC57-pylRs (R19K, H29K, T122S, Y384F) with restriction enzymes SpeI and SalI, and the plasmid pEvol-pylRs-pylT was cut with SpeI and SalI (the target DNA fragment is Among them, the 4.3kb large fragment) was separated by nucleic acid electrophoresis, extracted with agarose gel DNA recovery kit, connected with T4 DNA Ligase, and transformed into large E.coli Top10 competent cells by chemical method (CaCl 2 method).
  • the transformed cells were cultured on LB agar medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar) containing chloramphenicol at 37°C overnight.
  • LB agar medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl, 1.5% agar) containing chloramphenicol at 37°C overnight.
  • 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(R19K, H29K, T122S, Y384F)-pylT.
  • the plasmid map is shown in Figure 2.
  • the DNA sequence of GFP-TEV-R-MiniINS (SEQ ID NO.: 14) ), cloned into the expression vector plasmid pBAD/His A, the obtained plasmid was named pBAD-GFP-TEV-R-MiniINS, and the method was the same as that described in Example 1.
  • 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 the 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 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%.
  • 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). And according to the codon preference of E. coli, the DNA sequence of pylRs(R19H) was synthesized.
  • 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). And according to the codon preference of E. coli, the DNA sequence of pylRs(H29R) was synthesized.
  • the DNA sequence of pylRs (R19K, H29K, T122S, Y384F) was replaced with that of pylRs (R19H), the DNA sequence of pylRs (H29R), the DNA sequence of pylRs (R19H, H29R), The DNA sequence of pylRs (R19H, I26V, H29R), the DNA sequence of pylRs (R19H, H29R, T122S, Y384F) and the DNA sequence of pylRs (R19H, H29R, L309A, C348S) were constructed to obtain plasmid pEvol-pylRs (R19H).
  • 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 the 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 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%.
  • each strain in liquid LB medium at 37°C 220rpm overnight, and inoculate the tank fermentation medium (12g/L yeast peptone, 24g/L yeast extract powder, 4mL/L glycerol, 12.8) at 1% (v/v) g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.3 ⁇ defoamer), culture at 35( ⁇ 3)°C, 200 ⁇ 1000rpm, air flow 2 ⁇ 6L/min. After culturing for 3-10 hours, feed the feed medium containing glycerol and yeast peptone at a stepping rate until the end of the fermentation.
  • tank fermentation medium (12g/L yeast peptone, 24g/L yeast extract powder, 4mL/L glycerol, 12.8) at 1% (v/v) g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.3 ⁇ defoamer
  • 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

L'invention concerne un système à double plasmide comprenant un premier plasmide pour une cassette d'expression pour exprimer une protéine cible contenant un acide aminé modifié prédéterminé, un second plasmide pour une cassette d'expression pour exprimer l'aminoacyl-ARNt synthétase, et une troisième cassette d'expression dans le premier plasmide ou le second plasmide pour coder l'ARNt artificiel. L'invention concerne également une cellule hôte ou un liquide d'extrait cellulaire contenant le système plasmidique double, un kit et une utilisation correspondant, ainsi qu'un procédé de préparation d'un acide aminé modifié prédéterminé.
PCT/CN2020/080039 2019-03-19 2020-03-18 Introduction d'acides aminés non naturels dans des protéines à l'aide d'un système plasmidique double WO2020187271A1 (fr)

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