N-Terminal Extension Sequences for Expression of Recombinant Therapeutic Peptides

Abstract

The present invention relates to N-terminal extension sequences utilized to enhance expression of recombinant therapeutic peptides. The present invention also relates to processes for high-level expression of recombinant therapeutic peptides using said N-terminal extension sequences. The present invention also provides nucleic acids, vectors, and recombinant host cells for the efficient production of biologically active proteins such as lyrapeptides.

Description

[Technical field]

[0001] The present invention relates to an N-terminal extension sequence for high-level expression of recombinant therapeutic peptides.The present invention also relates to a process for high-level expression of recombinant therapeutic peptides using said N-terminal extension sequence. [Background technology]

[0002] Peptide therapeutics have played a prominent role in medical practice since the advent of insulin therapy in the 1920s. To date, there are more than 60 approved peptide drugs on the market, and that number is predicted to increase significantly.

[0003] Glucagon-like peptide 1 (GLP-1) is a 31 amino acid long peptide hormone derived from tissue-specific post-translational processing of the proglucagon peptide. It is produced and secreted by enteroendocrine L-cells in the intestine and by specific neurons in the nucleus of the solitary tract in the brainstem in response to food ingestion. Liraglutide is a derivative of the human incretin (metabolic hormone), glucagon-like peptide-1 (GLP-1), used as a long-acting glucagon-like peptide-1 receptor agonist, binding to the same receptor as the endogenous metabolic hormone GLP-1, which stimulates insulin secretion.

[0004] Teriparatide is a recombinant protein form of parathyroid hormone consisting of the first (N-terminal) 34 amino acids, which are the bioactive portion of the hormone. It is an effective anabolic (promotes bone formation) drug used to treat some forms of osteoporosis.

[0005] Expression plasmids contain regulatory sequences that act as enhancer and promoter regions and are engineered to direct the efficient transcription of genes carried on the expression vector. The goal of a properly designed expression vector is the efficient production of proteins, which can be achieved by synthesizing significant amounts of stable messenger RNA.

[0006] It is possible to design expression vectors that exert tight control of expression, such that the protein is produced in large amounts only when needed through the use of appropriate expression conditions In the absence of tight control of gene expression, the protein may be expressed constitutively.

[0007] US Pat. No. 4,916,212 discloses DNA sequences encoding biosynthetic insulin precursors and processes for preparing the insulin precursor and human insulin in yeast cells.

[0008] US Patent No. 7,572,884 discloses a method for preparing recombinant Lirapeptide, a precursor of Liraglutide, in Saccharomyces cerevisiae.

[0009] Indian Patent No. 201741024763A discloses a process for preparation of liraglutide by expression of a synthetic oligonucleotide encoding a liraglutide peptide operably linked to an oligonucleotide sequence of a signal peptide in yeast cells.

[0010] WO 1998 / 008871 discloses GLP-1 derivatives and analogues thereof prepared using recombinant DNA techniques. WO 1998 / 008872 discloses GLP-2 derivatives prepared using recombinant DNA techniques.

[0011] WO 1999 / 043708 discloses derivatives of exendin and GLP-1(7-C) prepared using recombinant DNA techniques. WO 2017 / 021819 discloses a process for preparing a peptide or protein or derivative thereof by expression of synthetic oligonucleotides encoding the desired protein or peptide in prokaryotic cells as a ubiquitin fusion construct.

[0012] Avicenna J Med Biotech 2017;9(1):19-22 discloses overexpression of teriparatide (1-34), a recombinant bioactive portion of human parathyroid hormone (PTH), in Eschericha coli.

[0013] The present inventors, in an effort to enhance the expression of recombinant therapeutic peptides several fold, have devised the use of short N-terminal extension sequences not disclosed in the prior art mentioned above. Summary of the Invention [Problem to be solved by the invention]

[0014] It is an object of the present invention to provide several-fold higher level expression of therapeutic peptides. [Means for solving the problem]

[0015] The present invention provides N-terminal extensions, nucleic acids, vectors and recombinant host cells for the efficient production of biologically active peptides such as lyrapeptides. The present invention contemplates a multi-dimensional approach to achieve high yields of peptides such as lirapeptide in host cells by providing an expression construct in which a nucleic acid encoding lirapeptide is operably fused to a modified gene sequence encoding a TEV (Tobacco Etch virus) cleavage site and an N-terminal extension (NE-3).

[0016] The present invention provides an N-terminal extension sequence (NE-3) as set forth in SEQ ID NO:1 for enhancing expression of a therapeutic peptide in bacteria or yeast. The present invention also provides an expression vector and a recombinant host cell for high-level expression of lyrapeptide, wherein the expression vector comprises a modified gene sequence encoding the N-terminal extension sequence NE-3, a modified gene sequence encoding a TEV (tobacco etch virus) cleavage site, and a modified gene sequence encoding lyrapeptide. [Brief description of the drawings]

[0017] [Figure 1] Figure 1A: Schematic diagram of an expression cassette without an N-terminal extension, Figure 1B: Schematic diagram of an expression cassette with an N-terminal extension (NE1), Figure 1C: Schematic diagram of an expression cassette with an N-terminal extension (NE3). [Figure 2A] FIG. 2A: Expression plasmid without N-terminal extension. [Figure 2B] FIG. 2B: Expression plasmid with N-terminal extension 1 (NE1). [Figure 2C] FIG. 2C: Expression plasmid with N-terminal extension 3 (NE3). [Diagram 3] Figure 3: Lirapeptide expression by ELISA. [Figure 4] FIG. 4: Dry cell weight of Lirapeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Description of sequence listing SEQ ID NO:1 (amino acid sequence of N-terminal extension sequence NE-3) EEQAE SEQ ID NO:2 (amino acid sequence of modified TEV cleavage site) ENLYFQ SEQ ID NO:3 (amino acid sequence of Lyrapeptide) HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG SEQ ID NO: 4 (nucleic acid sequence encoding the N-terminal extension NE-3 sequence of Pichia pastoris) gaagaacaagccgaa SEQ ID NO:5 (nucleic acid sequence encoding a modified TEV cleavage site in Pichia pastoris) gagaacttgtacttccaa SEQ ID NO:6 (nucleic acid sequence encoding the relaxeptide of Pichia pastoris) cacgctgagggtacttttacctctgacgtgtcctcttacttggagggtcaagctgccaaagagttcattgcctggttggttagaggtagaggttag SEQ ID NO: 7 (nucleic acid sequence encoding the N-terminal extension NE-3 sequence of Corynebacterium glutamicum) gaagaacaggcagaa SEQ ID NO:8 (nucleic acid sequence encoding a modified TEV cleavage site of Corynebacterium glutamicum) gaaaacctgtacttccag SEQ ID NO:9 (nucleic acid sequence encoding the relax peptide of Corynebacterium glutamicum) cacgcagaaggcacctttacctccgatgtgtcctcctacctggaaggccaggcagcaaaagaattcattgcatggctggt tcgcggtcgcggttag SEQ ID NO:10 (nucleic acid sequence encoding the N-terminal extension NE-3 sequence of E. coli) gaagaacaggcagaa SEQ ID NO:11 (nucleic acid sequence encoding a modified TEV cleavage site in E. coli) gaaaacctgtacttccag SEQ ID NO:12 (nucleic acid sequence encoding the E. coli relax peptide) catgcggaaggcaccttcaccagcgatgttagcagctacctggagggtcaggcggcgaaggaatttatcgcgtggctggttcgtggccgtggttaa SEQ ID NO:13 (nucleic acid sequence encoding the N-terminal extension NE-3 sequence of Bacillus subtilis) gaagaacaagccgaa SEQ ID NO:14 (nucleic acid sequence encoding a modified TEV cleavage site in Bacillus subtilis) gagaacttgtacttccaa SEQ ID NO:15 (nucleic acid sequence encoding the relaxeptide of Bacillus subtilis) cacgctgagggtacttttacctctgacgtgtcctcttacttggagggtcaagctgccaaagagttcattgcctggttggttagaggtagaggttag SEQ ID NO: 16 (Amino acid sequence of teriparatide) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF SEQ ID NO: 17 (amino acid sequence of N-terminal extension sequence NE-1) EEA SEQ ID NO: 18 (Nucleic acid sequence encoding the N-terminal extension NE-1 sequence) gaggaagcg SEQ ID NO:19 (fusion protein containing the N-terminal extension sequence NE-3 and the lyrapeptide operably fused to a TEV cleavage site) EEQAEENLYFQHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG SEQ ID NO:20 (fusion protein containing the N-terminal extension sequence NE-1 and the lyrapeptide operably fused to a TEV cleavage site) EEAENLYFQHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0019] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the method belongs. Although any vectors, host cells, methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of vectors, host cells, methods and compositions, representative examples are described below.

[0020] When a range of values ​​is provided, it is understood that values ​​between the upper and lower limits of that range, and any other stated or intervening values ​​within that stated range, are encompassed by the methods and compositions. The upper and lower limits of these smaller ranges may each be independently included in the smaller ranges, and are also encompassed by the methods and compositions, subject to any limit specifically excluded in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the methods and compositions.

[0021] It is recognized that certain features of the methods that are described for clarity in the context of separate embodiments may also be provided in combination in one embodiment. Conversely, various features of the methods and compositions that are described for brevity in the context of one embodiment may also be provided separately or in any suitable subcombination. It is noted that in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. Thus, this description is intended to serve as a precedent basis for the use of exclusive language such as "solely," "only," and the like in connection with the description of claim elements or the use of "negative" constraints.

[0022] As will be apparent to one of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features which may be readily distinguished or combined with the features of any of the other embodiments without departing from the scope or spirit of the method. Any method recited may be carried out in the order of events recited or in any other order which is logically possible.

[0023] The term "host cell" includes an individual cell or cell culture that can be or has been the recipient for the subject of an expression construct. A host cell includes the progeny of a single host cell. For purposes of the present invention, a host cell refers to any strain of Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis that can be suitably used for the purposes of the invention.

[0024] The term "recombinant strain" or "recombinant host cell" refers to a host cell that has been transfected or transformed with an expression construct or vector of the invention. The term "expression vector" or "expression construct" refers to any vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence after transformation into a host.

[0025] The term "promoter" refers to a DNA sequence that defines where transcription of a gene begins. Promoter sequences are generally located immediately upstream or at the 5' end of the transcription start site. RNA polymerase and necessary transcription factors bind to the promoter sequence and initiate transcription. Promoters can be either constitutive or inducible. Constitutive promoters are promoters that allow for continuous transcription of the associated gene because their expression is not usually conditioned by environmental and developmental factors. Constitutive promoters are very useful tools in genetic engineering because they drive gene expression under conditions without inducers and often exhibit better characteristics than the commonly used inducible promoters. Inducible promoters are promoters that are induced by the presence or absence of biotic or abiotic and chemical or physical factors. Inducible promoters are very powerful tools in genetic engineering because the expression of a gene operably linked to them can be turned on or off at specific stages of development or growth of an organism, or in specific tissues or cells.

[0026] The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases, a DNA sequence, including a coding sequence, is transcribed to form a messenger RNA (mRNA). The messenger RNA is then translated to form a polypeptide product having a relevant biological activity. The process of expression may also include further processing steps on the RNA product of transcription, such as splicing to remove introns, and / or post-translational processing steps of the polypeptide product.

[0027] The term "modified nucleic acid" is used herein to refer to a nucleic acid encoding a modified lyrapeptide represented by SEQ ID NO: 19 or 20, or a functionally equivalent variant thereof. Functional variants include any nucleic acid that has a substantial or significant amount of sequence identity or similarity to SEQ ID NO: 19 or 20 and retains the biological activity of the protein.

[0028] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked together by peptide bonds or modified peptide bonds. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers containing modified residues, and non-naturally occurring amino acid polymers. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids. Similarly, "protein" refers to at least two covalently linked amino acids, and includes proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus, as used herein, "amino acid" or "peptide residue" refers to both naturally occurring and synthetic amino acids. "Amino acid" includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or (S) configuration.

[0029] The term "N-terminal extension" refers to a peptide or polypeptide sequence that is removably linked to the N-terminal amino acid of a desired polypeptide. In a preferred embodiment, the N-terminal extension comprises the amino acid sequence of SEQ ID NO:1.

[0030] The present invention discloses N-terminal extensions, nucleic acids, vectors and recombinant host cells for the efficient production of biologically active peptides such as lyrapeptides. The present invention contemplates a multi-dimensional approach to achieve high yields of recombinant lirapeptide in a host cell by providing an expression construct in which a nucleic acid encoding lirapeptide is operably fused to a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site and an N-terminal extension (NE-3).

[0031] In one embodiment, the invention relates to the N-terminal extension sequence (NE-3) as set forth in SEQ ID NO: 1. The invention relates to a process for enhancing the expression of recombinant therapeutic peptides several-fold in bacteria or yeast using the short N-terminal extension sequence as set forth in SEQ ID NO: 1.

[0032] In another embodiment, a nucleic acid encoding the N-terminal extension sequence (NE-3) set forth in SEQ ID NO:1 is also encompassed within the scope of the present invention. Suitable host cells for the expression of recombinant therapeutic peptides are selected from eukaryotic hosts such as yeasts, including but not limited to Pichia pastoris and Saccharomyces cerevisiae. Bacterial hosts, including but not limited to Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis, may also be used.

[0033] The term therapeutic peptide includes, but is not limited to, peptides such as lyrapeptide, teriparatide, exenatide, and the like. Constitutive or inducible promoters known to those skilled in the art may be used in the expression cassette of one or more embodiments of the present invention.

[0034] In another embodiment, the present invention provides an expression cassette comprising a promoter, a signal sequence, an N-terminal extension (NE-3), a gene encoding lyrapeptide or teriparatide, a TEV cleavage site and a terminator.

[0035] In one embodiment, the present invention provides an N-terminal extension sequence as set forth in SEQ ID NO: 1 for enhancing expression of a therapeutic peptide in yeast, the yeast being Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis.

[0036] The present invention also provides a TEV cleavage site having the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the invention provides enhanced expression of the lirapeptide set forth in SEQ ID NO:3 and teriparatide set forth in SEQ ID NO:16 in bacteria or yeast using the N-terminal extension sequence set forth in SEQ ID NO:1.

[0037] Expression constructs known to those of skill in the art for the expression of prokaryotic or eukaryotic proteins may be used in one or more embodiments of the present invention. In one embodiment, the present invention provides 1. A modified gene sequence encoding an N-terminal extension sequence (NE-3), 2. A modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site, and 3. Modified gene sequence encoding the lyrapeptide Also provided is an expression construct for high level expression of a lyrapeptide comprising:

[0038] In another embodiment, the present invention provides 1. A gene sequence encoding the N-terminal extension sequence (NE3) set forth in SEQ ID NO: 4; 2. A gene sequence encoding a TEV (tobacco etch virus) cleavage site as set forth in SEQ ID NO:5, and 3. Gene sequence encoding the lyrapeptide set forth in SEQ ID NO:6 Also provided is an expression construct for high level expression of the lyrapeptide, comprising:

[0039] In another embodiment, the present invention provides 1. Construction of recombinant vectors (expression constructs) containing the gene sequences set forth in SEQ ID NOs: 4, 5 and 6; 2. Transformation of Expression Constructs into Pichia pastoris 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of lyrapeptide, and 6. Cleavage of the N-terminal extension sequence from the purified lyrapeptide Also provided is a method for high level expression of the lirapeptide set forth in SEQ ID NO:3 in Pichia pastoris comprising:

[0040] In another embodiment, the present invention provides 1. A gene sequence encoding the N-terminal extension sequence set forth in SEQ ID NO: 7, 2. A gene sequence encoding a TEV cleavage site as set forth in SEQ ID NO:8, and 3. Gene sequence encoding the lyrapeptide set forth in SEQ ID NO:9 Also provided is an expression construct for high level expression of a lyrapeptide comprising:

[0041] In one embodiment, the present invention provides 1. Construction of recombinant vectors (expression constructs) containing the gene sequences set forth in SEQ ID NOs: 7, 8 and 9; 2. Transformation of Expression Constructs into Corynebacterium glutamicum 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of lyrapeptide, and 6. Cleavage of the N-terminal extension sequence from the purified lyrapeptide Also provided is a method for high level expression of the lyrapeptide set forth in SEQ ID NO:3 in Corynebacterium glutamicum, comprising:

[0042] The present invention relates to 1. A gene sequence encoding the N-terminal extension sequence set forth in SEQ ID NO: 10, 2. A gene sequence encoding the TEV cleavage site set forth in SEQ ID NO: 11, and 3. Gene sequence encoding the lyrapeptide set forth in SEQ ID NO: 12 The present invention provides an expression construct for high level expression of lyrapeptide, comprising:

[0043] In one embodiment, the present invention provides 1. Construction of recombinant vectors (expression constructs) containing the gene sequences set forth in SEQ ID NOs: 10, 11 and 12; 2. Transformation of the expression construct into E. coli; 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of lyrapeptide, and 6. Cleavage of the N-terminal extension sequence from the purified lyrapeptide Also provided is a method for high-level expression of the lyrapeptide set forth in SEQ ID NO:3 in E. coli, comprising:

[0044] The present invention relates to 1. A gene sequence encoding the N-terminal extension sequence set forth in SEQ ID NO: 13, 2. A gene sequence encoding the TEV cleavage site set forth in SEQ ID NO: 14, and 3. Gene sequence encoding the lyrapeptide set forth in SEQ ID NO: 15 The present invention provides an expression construct for high level expression of lyrapeptide, comprising:

[0045] In one embodiment, the present invention provides 1. Construction of recombinant vectors (expression constructs) containing the gene sequences set forth in SEQ ID NOs: 13, 14 and 15; 2. Transformation of Expression Constructs into Bacillus subtilis 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of lyrapeptide, and 6. Cleavage of the N-terminal extension sequence from the purified lyrapeptide Also provided is a method for high level expression of the lyrapeptide set forth in SEQ ID NO:3 in Bacillus subtilis, comprising:

[0046] The present invention provides high level expression of teriparatide, set forth in SEQ ID NO:16, in Corynebacterium glutamicum using an expression construct that includes an N-terminal extension sequence. The present invention provides high level expression of teriparatide, as set forth in SEQ ID NO:16, in Pichia pastoris using an expression construct that includes an N-terminal extension sequence.

[0047] The present invention relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: 1. Construction of recombinant vectors (expression constructs); 2. Transformation of Expression Constructs into Corynebacterium glutamicum 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of teriparatide, and 6. Cleavage of the N-terminal extension sequence from purified teriparatide The present invention provides high-level expression of teriparatide as set forth in SEQ ID NO: 16 in Corynebacterium glutamicum, comprising:

[0048] The present invention relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: 1. Construction of recombinant vectors (expression constructs); 2. Transformation of the Constructed Vector into Pichia pastoris 3. Evaluation of clones and their selection; 4. Subjecting the selected clones to a fermentation process; 5. Isolation and purification of teriparatide, and 6. Cleavage of the N-terminal extension sequence from purified teriparatide Also provided is an N-terminal extension sequence as set forth in SEQ ID NO:1 for enhancing expression of teriparatide in Pichia pastoris, comprising:

[0049] In another embodiment, the present invention provides a modified lyrapeptide operably fused to a TEV (Tobacco Etch Virus) cleavage site and an N-terminal extension sequence (NE-3), as set forth in SEQ ID NO:19.

[0050] In another embodiment, the present invention provides a method for expressing lirapeptide using a recombinant host cell of the present invention, the fermentation process comprising: a. culturing the recombinant host cells in BMGY medium for about 24 hours; b. harvesting the recombinant host cells by centrifugation; c.OD 600nm resuspending the recombinant host cells in BMMY medium to a culture volume of about 10; d. incubating the host cells in a shaking incubator at 30° C. for about 24 hours; e. harvesting and purifying the culture supernatant to obtain the lyrapeptide; The present invention provides a method comprising:

[0051] Liraglutide is an analogue of human GLP-1 and acts as a GLP-1 receptor agonist. Liraglutide is made by adding a C-16 fatty acid (palmitic acid) with a glutamic acid spacer to the remaining lysine residue at position 26 of the peptide precursor (the lirapeptide set forth in SEQ ID NO: 3).

[0052] In another embodiment, the present invention provides for the preparation of liraglutide comprising the conjugation of a palmityl glutamic acid derivative, such as 1-methyl palmityl glutamic acid, with a lirapeptide produced as per the present invention, using methods known to those skilled in the art.

[0053] In another embodiment, the present invention provides the preparation of liraglutide comprising the conjugation of palmityl glutamic acid derivatives with the lirapeptide produced as in the present invention, using methods known to those skilled in the art, the derivatives being methyl (1-methylpalmityl glutamic acid), ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop-1-yl, 2-methyl-prop-2-yl (t-butyl), hexyl, etc. This conjugation reaction is carried out in the presence of a coupling reagent. The coupling agent may be selected from the group of DIC / 6-Cl-HOBt, DIC / HOBt, HBTU / HOBt / DIEA or DIC / Oxyma.

[0054] In another embodiment, the present invention provides a method for preparing liraglutide, comprising the steps of: a. culturing a recombinant host cell of the present invention in an appropriate culture medium to obtain the lyrapeptide; b. converting the lirapeptide into liraglutide, wherein the method comprises conjugating the lirapeptide obtained in step (a) with a palmityl glutamic acid derivative.

[0055] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific example. This example is provided for illustrative purposes only and is not intended to limit the scope of the present invention. Although specific terms are utilized herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES

[0056] Example 1: Modified Nucleic Acids for Expression of Lyrapeptide An expression cassette encoding the liraglutide precursor peptide was modified for optimal expression in Pichia pastoris, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis. The modified open reading frame containing the nucleotide sequence encoding the liraglutide peptide was fused to a sequence encoding a TEV (Tobacco Etch Virus) cleavage site and a sequence encoding an N-terminal extension (NE-3 or NE-1). Codons preferred for expression in Pichia pastoris, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis were used instead of rare codons.

[0057] As a control, an open reading frame containing a nucleotide sequence encoding the lyrapeptide without any N-terminal extension was prepared. For expression in Pichia pastoris For expression in Pichia pastoris, the nucleotide sequence encoding the lyrapeptide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0058] The nucleotide sequence encoding the lyrapeptide is represented by SEQ ID NO: 6. The nucleotide sequence TEV cleavage site is represented by SEQ ID NO: 5. The nucleotide sequence encoding the N-terminal extension (NE-3) is represented by SEQ ID NO: 4.

[0059] This modified open reading frame was artificially synthesized using the sequence of the lyrapeptide, the sequence of the TEV cleavage site and the sequence of the N-terminal extension. The modified open reading frames containing DNA encoding the lyrapeptide without an N-terminal extension (Figure 1A) as well as with N-terminal extension-1 (NE1) (GAGGAAGCG - Figure 1B) and with N-terminal extension-3 (NE3) (GAAGAACAAGCCGAA - Figure 1C) in addition to the TEV (Tobacco Etch Virus) cleavage sequence (GAGAACTTGTACTTCCAA) and signal sequence cassette are represented in Figure 1.

[0060] The modified sequence encoding the recombinant lyrapeptide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid contains an open reading frame and a promoter.

[0061] The vector map of pD912 is presented in FIG. Expression in Corynebacterium glutamicum For expression in Corynebacterium glutamicum, the nucleotide sequence encoding the lyrapeptide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0062] The nucleotide sequence encoding the lyrapeptide is represented by SEQ ID NO: 9. The nucleotide sequence TEV cleavage site is represented by SEQ ID NO: 8. The nucleotide sequence encoding the N-terminal extension (NE-3) is represented by SEQ ID NO: 7.

[0063] This modified open reading frame was artificially synthesized using Thermo Fisher Scientific technology, utilizing the sequence of the lyrapeptide, the sequence of the TEV cleavage site and the sequence of the N-terminal extension.

[0064] The modified sequence encoding the recombinant lyrapeptide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid contains an open reading frame and a promoter. For expression in E. coli For expression in E. coli, the nucleotide sequence encoding the lyrapeptide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0065] The nucleotide sequence encoding the lyrapeptide is represented by SEQ ID NO: 12. The nucleotide sequence TEV cleavage site is represented by SEQ ID NO: 11. The nucleotide sequence encoding the N-terminal extension (NE-3) is represented by SEQ ID NO: 10.

[0066] This modified open reading frame was artificially synthesized using the sequence of the lyrapeptide, the sequence of the TEV cleavage site and the sequence of the N-terminal extension. The modified sequence encoding the recombinant lyrapeptide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid contains an open reading frame and a promoter. For expression in Bacillus subtilis For expression in Bacillus subtilis, the nucleotide sequence encoding the lyrapeptide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0067] The nucleotide sequence encoding the lyrapeptide is represented by SEQ ID NO: 15. The nucleotide sequence TEV cleavage site is represented by SEQ ID NO: 14. The nucleotide sequence encoding the N-terminal extension (NE-3) is represented by SEQ ID NO: 13.

[0068] This modified open reading frame was artificially synthesized using the sequence of the lyrapeptide, the sequence of the TEV cleavage site and the sequence of the N-terminal extension. The modified sequence encoding the recombinant lyrapeptide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid contains an open reading frame and a promoter.

[0069] Confirmation of plasmid DNA linearization The synthetic DNA encoding the lirapeptide without, with, and with N-terminal extension-1 and with N-terminal extension-3 as well as the plasmid pD912 were digested with EcoRI and BglII restriction enzymes. The restriction digested fragments were ligated and transformed into an E. coli strain. The resulting plasmids containing the lirapeptide expression cassettes without (Figure 2A), with (Figure 2B), and with (Figure 2C) N-terminal extension-1 and N-terminal extension-3 were sequenced to confirm the lirapeptide, N-terminal extension and TEV cleavage sequences. The sequence-confirmed plasmid DNA was linearized with SacI enzyme.

[0070] Example 2: Development of recombinant host cells by transformation with recombinant plasmids The recombinant pD912 plasmid described in the previous example, carrying the gene for liraglutide precursor peptide fused to a signal peptide, was used for the development of recombinant hosts.

[0071] Pichia pastoris host cells (obtained from Atum, USA) were transformed with the plasmids by electroporation. The transformed cells were plated on YPD (yeast peptone dextrose) agar plates containing 100 μg / mL Zeocin. The transformed Pichia pastoris cells were grown in 20 mL of BMGY medium for 24 h. The cells were harvested by centrifugation and the OD 600nm The cells were resuspended in 20 mL of BMMY medium to a concentration of 10. The cell suspension was incubated at 30° C. in a shaking incubator for 24 hours.

[0072] Example 3: Analysis and evaluation of Lyrapeptide expression After 24 h, culture supernatants were harvested, purified, and analyzed for lyrapeptide expression by ELISA using a monoclonal antibody specific for lyrapeptide (Figure 3). In addition, dry cell weight was measured using a moisture analyzer (Figure 4).

[0073] Tables 1 and 2 provide a comparison establishing the effectiveness of N-terminal extension in improving the yield of lyrapeptides.

[0074] [Table 1]

[0075] [Table 2]

[0076] The above data clearly show that the N-terminal extension can improve the expression of the lyrapeptide by about 5-fold compared to the control and known N-terminal extensions. In one aspect, the present invention may be as follows. [Aspect 1] An N-terminal extension comprising the amino acid sequence of SEQ ID NO:1. [Aspect 2] A nucleic acid encoding the N-terminal extension described in aspect 1. [Aspect 3] The nucleic acid according to aspect 2, selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13. [Aspect 4] A vector comprising the modified nucleic acid described in aspect 3. [Aspect 5] The vector described in Aspect 4, which is pD912. [Aspect 6] The vector described in Aspect 4, comprising a modified TEV cleavage site selected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:14. [Aspect 7] a. A modified gene sequence encoding an N-terminal extension sequence (NE-3) selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 13; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site selected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:14; and c. A modified gene sequence encoding a lyrapeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:15. A vector for recombinant expression of a lyrapeptide comprising:

[0023]

[0024]

[0025]

[0026]

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[0029] a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO:4; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:5; and c. A modified gene sequence encoding the lyrapeptide comprising the nucleotide sequence of SEQ ID NO:6 8. The vector of embodiment 7, comprising:

[0023]

[0024]

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[0028]

[0029]

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[0029]

[0031] a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO:7; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:8; and c. A modified gene sequence encoding the lyrapeptide comprising the nucleotide sequence of SEQ ID NO:9 8. The vector of embodiment 7, comprising: [Aspect 10] Modified for high-level expression of lyrapeptide in E. coli, a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO: 10; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:11; and c. The vector of embodiment 7, comprising a modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO:12.

[0023]

[0024]

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[0029] a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO: 13; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO: 14; and c. The vector of embodiment 7, comprising a modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO: 15. [Aspect 12] A recombinant host cell comprising the vector described in aspect 7. [Aspect 13] A recombinant host cell according to aspect 12, which is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. [Aspect 14] A modified lyrapeptide comprising the amino acid sequence of SEQ ID NO: 19, which is operably fused to a TEV (tobacco etch virus) cleavage site and an N-terminal extension sequence (NE-3). [Aspect 15] A method for expressing a lyrapeptide using the recombinant host cell according to aspect 12, comprising the steps of: a. culturing the recombinant host cells in BMGY medium for about 24 hours; b. harvesting the recombinant host cells by centrifugation; c.OD 600nm resuspending the recombinant host cells in BMMY medium to a culture volume of about 10; d. incubating the host cells in a shaking incubator at 30° C. for about 24 hours; e. harvesting and purifying the culture supernatant to obtain the lyrapeptide; The above method. [Aspect 16] A method for preparing liraglutide, comprising: c. culturing the recombinant host cell of embodiment 12 in a suitable culture medium to obtain the lyrapeptide; d. A step of converting the lirapeptide into liraglutide, wherein the method comprises conjugation of the lirapeptide obtained in step (a) with a palmityl glutamic acid derivative.

Claims

1. An N-terminal extension peptide as shown in the amino acid sequence of SEQ ID NO:

1.

2. A nucleic acid encoding the peptide of claim 1.

3. The nucleic acid of claim 2, as set forth in SEQ ID NO:

4.

4. A vector comprising the nucleic acid of claim 3.

5. The vector of claim 4, which is pD912.

6. The vector of claim 4, comprising a modified TEV cleavage site as shown in SEQ ID NO:

5.

7. a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) selected from the group consisting of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:13; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site selected from the group consisting of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:14; and c. A modified gene sequence encoding a lyrapeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:

15. A vector for recombinant expression of a lyrapeptide comprising:

8. engineered for high-level expression of lyrapeptide in Pichia pastoris; a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO:4; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:5; and c. A modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO:6 The vector of claim 7 .

9. engineered for high-level expression of lyrapeptide in Corynebacterium glutamicum; a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO:7; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:8; and c. A modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO:9 The vector of claim 7 .

10. It has been engineered for high-level expression of lyrapeptide in E. coli, a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO: 10; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO:11; and c) The vector of claim 7, comprising a modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO:

12.

11. engineered for high-level expression of lyrapeptide in Bacillus subtilis; a. a modified gene sequence encoding an N-terminal extension sequence (NE-3) comprising the nucleotide sequence of SEQ ID NO: 13; b. a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site comprising the nucleotide sequence of SEQ ID NO: 14; and c) The vector of claim 7, comprising a modified gene sequence encoding a lyrapeptide comprising the nucleotide sequence of SEQ ID NO:

15.

12. A recombinant host cell comprising the vector of claim 7.

13. 13. The recombinant host cell of claim 12, selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis.

14. A modified lyrapeptide comprising the amino acid sequence of SEQ ID NO:19, operably fused to a TEV (Tobacco Etch Virus) cleavage site and an N-terminal extension sequence (NE-3).

15. 13. A method for expressing a lyrapeptide using the recombinant host cell of claim 12, comprising the steps of: a. culturing the recombinant host cells in BMGY medium for about 24 hours; b. harvesting the recombinant host cells by centrifugation; c.OD ６００ｎｍ resuspending the recombinant host cells in BMMY medium to a concentration of about 10; d. Incubating the recombinant host cells in a shaking incubator at 30° C. for about 24 hours; e. harvesting and purifying the culture supernatant to obtain the lyrapeptide; The above method.

16. A method for preparing liraglutide, comprising: c. Cultivating the recombinant host cell of claim 12 in an appropriate culture medium to obtain the lyrapeptide; d. converting the lirapeptide into liraglutide, the method comprising conjugating the lirapeptide obtained in step (a) with a palmityl glutamic acid derivative; The palmityl glutamic acid derivative is methyl (1-methyl palmityl glutamic acid), ethyl (1-ethyl palmityl glutamic acid), propyl (1-propyl palmityl glutamic acid), prop-2-yl (1-prop-2-yl palmityl glutamic acid), butyl (1-butyl palmityl glutamic acid), but-2-yl (1-but-2-yl palmityl glutamic acid), 2-methyl prop-1-yl (2-methyl prop-1-yl palmityl glutamic acid), 2-methyl-prop-2-yl (t-butyl) (2-methyl-prop-2-yl (t-butyl) palmityl glutamic acid), or hexyl (1-hexyl palmityl glutamic acid). The above method.

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