N-terminal extension sequences for expression of recombinant therapeutic peptides

Abstract

The present invention relates to an N-terminal extension sequence for enhancing the expression of a recombinant therapeutic peptide. The present invention also relates to a method for expressing a recombinant therapeutic peptide at a high level using the N-terminal extension sequence. The present invention also provides nucleic acids, vectors and recombinant host cells for efficiently producing biologically active proteins, such as liratide.

Description

Technical Field

[0001] The present invention relates to an N-terminal extension sequence for high-level expression of a recombinant therapeutic peptide and a method for high-level expression of a recombinant therapeutic peptide using the N-terminal extension sequence. Background Art

[0002] Since the advent of insulin therapy in the 1920s, peptide therapeutics have played a significant role in medical practice. Currently, there are more than 60 approved peptide drugs on the market, and the number is expected to grow significantly.

[0003] Glucagon-like peptide-1 (GLP-1) is a 31-amino acid peptide hormone that is derived from tissue-specific post-translational processing of the original glucagon-like peptide. It is produced and secreted by the intestinal endocrine L cells and certain neurons in the nucleus tractus solitarius of the brainstem during feeding. Liraglutide is a derivative of human incretin (metabolic hormone). Glucagon-like peptide-1 (GLP-1) is used as a long-acting glucagon-like peptide-1 receptor agonist, binding to the same receptor as the endogenous metabolic hormone GLP-1 that stimulates insulin secretion.

[0004] Teriparatide is a recombinant protein form of parathyroid hormone, consisting of the first (N-terminal) 34 amino acids of parathyroid hormone, which is the biologically active portion of parathyroid hormone. It is a potent anabolic (bone-forming) agent used to treat certain forms of osteoporosis.

[0005] An expression plasmid is designed to contain regulatory sequences that act as enhancer and promoter regions and result in efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is to produce proteins efficiently, and this can be achieved by synthesizing large amounts of stable messenger RNA.

[0006] Expression vectors can be designed to exert strict control over expression, and proteins can be produced in large quantities only when necessary by using appropriate expression conditions. In the absence of strict control over gene expression, the protein can also be expressed constitutively.

[0007] US Patent No. 4,916,212 discloses a DNA sequence encoding a biosynthetic insulin precursor and a method for producing the insulin precursor and human insulin in yeast cells.

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

[0009] IN 201741024763A discloses a method for preparing liraglutide by expressing a synthetic oligonucleotide encoding liraglutide, which is operably linked to an oligonucleotide sequence of a signal peptide in a yeast cell.

[0010] WO 1998 / 008871A1 discloses GLP-1 derivatives and analogs thereof prepared by recombinant DNA technology.

[0011] WO 1998 / 008872A1 discloses GLP-2 derivatives prepared by recombinant DNA technology.

[0012] WO 1999 / 043708 A1 discloses incretin analogs (exendins) and GLP-1(7-C) derivatives prepared by recombinant DNA technology.

[0013] WO 2017 / 021819A1 discloses a method for preparing a peptide or protein or a derivative thereof by expressing a synthetic oligonucleotide encoding the desired protein or peptide as a ubiquitin fusion construct in a prokaryotic cell.

[0014] Avicenna J Med Biotech 2017;9(1):19-22 discloses overexpression of tripapatide (1-34), a recombinant biologically active portion of human parathyroid hormone (PTH), in Escherichia coli.

[0015] In an effort to increase the expression of recombinant therapeutic peptides several fold, the inventors of the present invention proposed a method for using a short N-terminal extension sequence, which is not disclosed in the above-mentioned prior art.

[0016] Purpose of the Invention

[0017] It is an object of the present invention to provide several-fold higher level expression of therapeutic peptides. Summary of the invention

[0018] The present invention provides an N-terminal extension sequence, a nucleic acid, a vector and a recombinant host cell for efficiently producing a bioactive peptide, such as liratide.

[0019] The present invention contemplates a multidimensional approach to obtaining high yields of peptides, such as liratide, in host cells by providing an expression construct in which a nucleic acid encoding liratide is operably fused to a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site and an N-terminal extension sequence (NE-3).

[0020] The present invention provides an N-terminal extension sequence (NE-3) as shown in SEQ ID NO: 1 to enhance the expression of therapeutic peptides in bacteria or yeast.

[0021] The present invention also provides an expression vector and a recombinant host cell for high-level expression of liratide, wherein the expression vector comprises a modified gene sequence encoding an N-terminal extension sequence NE-3, a modified gene sequence encoding a TEV (Tobacco Etch Virus) cleavage site, and a modified gene sequence encoding liratide. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A: Schematic representation of the expression cassette without the N-terminal extension sequence.

[0023] Figure 1 B: Schematic representation of the expression cassette with an N-terminal extension sequence (NE1).

[0024] Figure 1 C: Schematic representation of the expression cassette with an N-terminal extension sequence (NE3).

[0025] Figure 2A : Expression plasmid without N-terminal extension sequence.

[0026] Figure 2B : Expression plasmid with N-terminal extension sequence-1 (NE1).

[0027] Figure 2C : Expression plasmid with N-terminal extension sequence-3 (NE3).

[0028] Figure 3 : Liratide expression by ELISA.

[0029] Figure 4 : Cell dry weight of liratide.

[0030] Description of the Sequence Listing

[0031] SEQ ID NO: 1 (amino acid sequence of N-terminal extension sequence NE-3)

[0032] EEQAE

[0033] SEQ ID NO:2 (Amino acid sequence of modified TEV cleavage site)

[0034] ENLYFQ

[0035] SEQ ID NO:3 (amino acid sequence of liratide)

[0036] HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0037] SEQ ID NO:4 (nucleic acid sequence encoding N-terminal extension NE-3 sequence of Pichia pastoris)

[0038] gaagaacaagccgaa

[0039] SEQ ID NO: 5 (nucleic acid sequence encoding a modified TEV cleavage site of Pichia pastoris)

[0040] gagaacttgtacttccaa

[0041] SEQ ID NO:6 (Pichia pastoris nucleic acid sequence encoding liratide)

[0042] cacgctgagggtacttttacctctgacgtgtcctcttacttggagggtcaagctgccaaagagttcattgcctggttggttagaggtagaggttag

[0043] SEQ ID NO:7 (nucleic acid sequence encoding N-terminal extension NE-3 sequence of Corynebacterium glutamicum)

[0044] gaagaacaggcagaa

[0045] SEQ ID NO: 8 (nucleic acid sequence encoding a modified TEV cleavage site of Corynebacterium glutamicum)

[0046] gaaaacctgtacttccag

[0047] SEQ ID NO:9 (nucleic acid sequence encoding liratide of Corynebacterium glutamicum)

[0048] cacgcagaaggcacctttacctccgatgtgtcctcctacctggaaggccaggcagcaaaagaattcattgcatggctggttcgcggtcgcggttag

[0049] SEQ ID NO: 10 (nucleic acid sequence encoding N-terminal extension NE-3 sequence of Escherichia coli)

[0050] gaagaacaggcagaa

[0051] SEQ ID NO: 11 (nucleic acid sequence encoding a modified TEV cleavage site of Escherichia coli)

[0052] gaaaacctgtacttccag

[0053] SEQ ID NO: 12 (nucleic acid sequence encoding liratide from Escherichia coli)

[0054] catgcggaaggcaccttcaccagcgatgttagcagctacctggagggtcaggcggcgaaggaatttatcgcgtggctggttcgtggccgtggttaa

[0055] SEQ ID NO: 13 (nucleic acid sequence encoding N-terminal extension NE-3 sequence of Bacillus subtilis)

[0056] gaagaacaagccgaa

[0057] SEQ ID NO: 14 (nucleic acid sequence encoding a modified TEV cleavage site of Bacillus subtilis)

[0058] gagaacttgtacttccaa

[0059] SEQ ID NO: 15 (nucleic acid sequence encoding liratide from Bacillus subtilis)

[0060] cacgctgagggtacttttacctctgacgtgtcctcttacttggagggtcaagctgccaaagagttcattgcctggttggttagaggtagaggttag

[0061] SEQ ID NO: 16 (amino acid sequence of teriparatide)

[0062] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

[0063] SEQ ID NO: 17 (amino acid sequence of N-terminal extension sequence NE-1)

[0064] EEA

[0065] SEQ ID NO: 18 (nucleic acid sequence encoding N-terminal extension NE-1 sequence)

[0066] gaggaagcg

[0067] SEQ ID NO: 19 (a fusion protein comprising liratide operably fused to an N-terminal extension sequence NE-3 and a TEV cleavage site)

[0068] EEQAEENLYFQHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0069] SEQ ID NO: 20 (a fusion protein comprising liratide operably fused to an N-terminal extension sequence NE-1 and a TEV cleavage site)

[0070] EEAENLYFQHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0071] definition

[0072] Unless otherwise defined, 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 the vectors, host cells, methods and compositions, only representative examples are now described.

[0073] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value in that range is included in the methods and combinations. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the methods and combinations, subject to any specifically excluded limits in the stated ranges. If the stated range includes one or both limits, ranges excluding either or both limits are also included in the methods and combinations.

[0074] It should be understood that, for clarity, certain features of the methods described in the context of a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the methods and compositions described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. It should be further noted that a drafted claim may not include any optional elements. Therefore, this statement is intended to serve as a prior basis for the use of exclusive terms such as "alone", "only", etc. when reciting claim elements or using "negative" limitations.

[0075] It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other embodiment without departing from the scope or spirit of the present method. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0076] The term "host cell" includes individual cells or cell cultures, which may be or were the receptors of the object of the expression construct. Host cells include the offspring of individual host cells. Host cells for the purposes of the present invention refer to any bacterial strains of Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis that can be suitably used for the purposes of the present invention.

[0077] 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 present invention.

[0078] The term "expression vector" or "expression construct" refers to any vector, plasmid or vector designed to express an inserted nucleic acid sequence after transformation into a host.

[0079] The term "promoter" refers to a DNA sequence that defines the start point of transcription of a gene. The promoter sequence is usually located directly upstream or 5' of the transcription start site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription. Promoters can be constitutive or inducible promoters. A constitutive promoter is a promoter that allows continuous transcription of the associated gene because their expression is usually not restricted by environmental and developmental factors. Constitutive promoters are very useful tools in genetic engineering because they drive gene expression under inducer-free conditions and usually show better properties 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 a very powerful tool in genetic engineering because the expression of genes associated with them can be turned on or off at specific stages of development or growth of an organism or a specific tissue or cell.

[0080] 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 into a polypeptide product having the relevant biological activity. In addition, the expression process may involve further processing steps of the transcribed RNA product, such as splicing to remove introns, and / or post-translational processing of the polypeptide product.

[0081] The term "modified nucleic acid" as used herein refers to a nucleic acid encoding a modified liratide represented by SEQ ID NO: 19 or SEQ ID NO: 20 or a functionally equivalent variant thereof. Functional variants include any nucleic acid having substantial or significant sequence identity or similarity to SEQ ID NO: 19 or SEQ ID NO: 20 and retaining the biological activity of the protein.

[0082] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked to each other by peptide bonds or modified peptide bonds. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acids, as well as natural amino acid polymers, polymers containing modified residues, and non-natural amino acid polymers. "Polypeptide" refers to short chains (commonly referred to as peptides, oligopeptides or oligomers) and long 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, including proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of natural amino acids and peptide bonds or synthetic peptidomimetic structures. Therefore, "amino acid" or "peptide residue" used herein refers to natural amino acids and synthetic amino acids. "Amino acid" includes imino acid residues such as proline and hydroxyproline. The side chain may be in the (R) configuration or the (S) configuration.

[0083] The term "N-terminal extension sequence" 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 sequence comprises the amino acid sequence of SEQ ID NO:1. DETAILED DESCRIPTION

[0084] The present invention discloses an N-terminal extension sequence, a nucleic acid, a vector and a recombinant host cell for efficiently producing a bioactive peptide such as liratide.

[0085] The present invention contemplates a multidimensional method for obtaining high yields of recombinant liratide in host cells by providing an expression construct in which a nucleic acid encoding liratide is operably fused to a modified gene sequence encoding a TEV (tobacco etch virus) cleavage site and an N-terminal extension sequence (NE-3).

[0086] In one embodiment, the present invention relates to an N-terminal extension sequence (NE-3) as shown in SEQ ID NO: 1. The present invention relates to a method for enhancing the expression of a recombinant therapeutic peptide several fold in bacteria or yeast using a short N-terminal extension sequence as shown in SEQ ID NO: 1.

[0087] In another embodiment, a nucleic acid encoding the N-terminal extension sequence (NE-3) described in SEQ ID NO: 1 is also within the scope of the present invention.

[0088] Suitable host cells for expressing recombinant therapeutic peptides are selected from eukaryotic hosts, such as, but not limited to, yeasts including Pichia pastoris and Saccharomyces cerevisiae. Bacterial hosts can also be used, such as, but not limited to, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis.

[0089] The term therapeutic peptide includes peptides such as, but not limited to, liratide, teriparatide, exenatide, and the like.

[0090] Constitutive or inducible promoters known to those of skill in the art can be used in the expression cassettes of one or more embodiments of the present invention.

[0091] In another embodiment, the present invention provides an expression cassette comprising a promoter, a signal sequence, an N-terminal extension sequence (NE-3), a gene encoding liratide or teriparatide, a TEV cleavage site and a terminator.

[0092] In one embodiment, the present invention provides an N-terminal extension sequence as shown in SEQ ID NO: 1 to enhance the expression of therapeutic peptides in yeast, wherein the yeast is Pichia pastoris, Saccharomyces cerevisiae, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis.

[0093] The present invention also provides a TEV cleavage site having the amino acid sequence shown in SEQ ID NO:2.

[0094] In one embodiment, the present invention provides for enhancing the expression of liratide as shown in SEQ ID NO:3 and teriparatide as shown in SEQ ID NO:16 in bacteria or yeast using an N-terminal extension sequence as shown in SEQ ID NO:1.

[0095] Expression constructs known to those skilled in the art for expressing prokaryotic or eukaryotic proteins can be used in one or more embodiments of the present invention.

[0096] In one embodiment, the present invention also provides an expression construct for high-level expression of liratide, comprising:

[0097] 1. A modified gene sequence encoding an N-terminal extension sequence (NE3),

[0098] 2. A modified gene sequence encoding a TEV (tobacco etch virus) cleavage site, and

[0099] 3. A modified gene sequence encoding liratide.

[0100] In another embodiment, the present invention also provides an expression construct for high-level expression of liratide, comprising:

[0101] 1. The gene sequence encoding the N-terminal extension sequence (NE-3) as shown in SEQ ID NO: 4,

[0102] 2. The gene sequence encoding the TEV (tobacco etch virus) cleavage site as shown in SEQ ID NO: 5, and

[0103] 3. The gene sequence encoding liratide as shown in SEQ ID NO:6.

[0104] In another embodiment, the present invention also provides a method for expressing liratide as described in SEQ ID NO: 3 at a high level in Pichia pastoris, comprising:

[0105] 1. Construct a recombinant vector (expression construct) comprising the gene sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6,

[0106] 2. Transform the expression construct into Pichia pastoris,

[0107] 3. Evaluate and select clones,

[0108] 4. Ferment the selected clones.

[0109] 5. Isolate and purify liratide, and

[0110] 6. Cleavage of the N-terminal extension sequence from purified liratide.

[0111] In another embodiment, the present invention also provides an expression construct for high-level expression of liratide, comprising:

[0112] 1. The gene sequence encoding the N-terminal extension sequence as shown in SEQ ID NO: 7,

[0113] 2. The gene sequence encoding the TEV cleavage site as shown in SEQ ID NO: 8, and

[0114] 3. The gene sequence encoding liratide as shown in SEQ ID NO:9.

[0115] In one embodiment, the present invention also provides a method for expressing liratide as shown in SEQ ID NO: 3 at a high level in Corynebacterium glutamicum, comprising:

[0116] 1. Construct a recombinant vector (expression construct) comprising the gene sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9,

[0117] 2. Transform the expression construct into Corynebacterium glutamicum,

[0118] 3. Evaluate and select clones,

[0119] 4. Ferment the selected clones.

[0120] 5. Isolate and purify liratide, and

[0121] 6. Cleavage of the N-terminal extension sequence from purified liratide.

[0122] The present invention provides an expression construct for high-level expression of liratide, comprising:

[0123] 1. The gene sequence encoding the N-terminal extension sequence as shown in SEQ ID NO: 10,

[0124] 2. The gene sequence encoding the TEV cleavage site as shown in SEQ ID NO: 11, and

[0125] 3. The gene sequence encoding liratide as shown in SEQ ID NO: 12.

[0126] In one embodiment, the present invention also provides a method for high-level expression of liratide as shown in SEQ ID NO: 3 in Escherichia coli, comprising:

[0127] 1. Constructing a recombinant vector (expression construct) comprising the gene sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12,

[0128] 2. Transform the expression construct into Escherichia coli,

[0129] 3. Evaluate and select clones,

[0130] 4. Ferment the selected clones.

[0131] 5. Isolate and purify liratide, and

[0132] 6. Cleavage of the N-terminal extension sequence from purified liratide.

[0133] The present invention provides an expression construct for high-level expression of liratide, comprising:

[0134] 1. The gene sequence encoding the N-terminal extension sequence as shown in SEQ ID NO: 13,

[0135] 2. The gene sequence encoding the TEV cleavage site as shown in SEQ ID NO: 14, and

[0136] 3. The gene sequence encoding liratide as shown in SEQ ID NO:15.

[0137] In one embodiment, the present invention also provides a method for efficiently expressing liratide as shown in SEQ ID NO: 3 in Bacillus subtilis, comprising:

[0138] 1. Constructing a recombinant vector (expression construct) comprising the gene sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15,

[0139] 2. Transform the expression construct into Bacillus subtilis,

[0140] 3. Evaluation and selection of clones,

[0141] 4. Ferment the selected clones.

[0142] 5. Isolate and purify liratide, and

[0143] 6. Cleavage of the N-terminal extension sequence from purified liratide.

[0144] The present invention provides high-level expression of teriparatide as shown in SEQ ID NO: 16 in Corynebacterium glutamicum using an expression construct including an N-terminal extension sequence.

[0145] The present invention provides high-level expression of teriparatide as shown in SEQ ID NO: 16 in Pichia pastoris using an expression construct including an N-terminal extension sequence.

[0146] The present invention provides a method for expressing teriparatide as shown in SEQ ID NO: 16 at a high level in Corynebacterium glutamicum, comprising the following steps:

[0147] 1. Construction of recombinant vector (expression vector)

[0148] 2. Transform the expression construct into Corynebacterium glutamicum,

[0149] 3. Evaluate and select clones,

[0150] 4. Ferment the selected clones.

[0151] 5. Isolate and purify teriparatide, and

[0152] 6. Cleavage of the N-terminal extension sequence from purified teriparatide.

[0153] The present invention also provides an N-terminal extension sequence as shown in SEQ ID NO: 1 to enhance the expression of teriparatide in Pichia pastoris, comprising the following steps:

[0154] 1. Construct a recombinant vector (expression construct),

[0155] 2. Transform the constructed vector into Pichia pastoris.

[0156] 3. Evaluate and select clones,

[0157] 4. Ferment the selected clones.

[0158] 5. Isolate and purify teriparatide, and

[0159] 6. Cleavage of the N-terminal extension sequence from purified teriparatide.

[0160] In another embodiment, the present invention provides a modified liratide, wherein the liratide is operably fused to a TEV (tobacco etch virus) cleavage site and an N-terminal extension sequence (NE-3), and wherein the modified liratide is as shown in SEQ ID NO:19.

[0161] In another embodiment, the present invention provides a method for expressing liratide using the recombinant host cell of the present invention, wherein the fermentation process comprises:

[0162] a. Cultivating the recombinant host cells in BMGY medium for about 24 hours;

[0163] b. harvesting the recombinant host cells by centrifugation;

[0164] c. Resuspend the recombinant host cells in BMMY medium to an OD of 600nm is about 10;

[0165] d. Place the host cells in a shake flask incubator and culture at 30°C for about 24 hours;

[0166] e. Collect and purify the culture supernatant to obtain liratide.

[0167] Liraglutide is an analog of human GLP-1 and acts as a GLP-1 receptor agonist. Liraglutide is prepared by linking a C-16 fatty acid (palmitic acid) to the remaining lysine residue at position 26 of a peptide precursor (such as liraglutide shown in SEQ ID NO: 3) with a glutamic acid spacer.

[0168] In another embodiment, the present invention provides the preparation of liraglutide, comprising combining liraglutide produced according to the present invention with a palmitoylglutamate derivative, such as 1-methylpalmitoylglutamate, using methods known in the art.

[0169] In another embodiment, the present invention provides the preparation of liraglutide, comprising conjugating the liraglutide produced according to the present invention with a palmitoyl glutamate derivative using methods known in the art, wherein the derivative is methyl (1-methyl palmitoyl glutamate), ethyl (1-methyl palmitoyl glutamate), propyl (1-methyl palmitoyl glutamate), propan-2-yl (1-methyl palmitoyl glutamate), butyl (1-methyl palmitoyl glutamate), butan-2-yl (1-methyl palmitoyl glutamate), 2-methylpropan-1-yl (1-methyl palmitoyl glutamate), 2-methylpropan-2-yl (tert-butyl) (1-methyl palmitoyl glutamate), hexyl (1-methyl palmitoyl glutamate), etc. The conjugation reaction is carried out in the presence of a coupling agent. The coupling agent can be selected from DIC / 6-Cl-HOBt, DIC / HOBt, HBTU / HOBt / DIEA or DIC / Oxyma.

[0170] In another embodiment, the present invention provides a method for preparing liraglutide, comprising the following steps:

[0171] a. culturing the recombinant host cell of the present invention in a suitable culture medium to obtain liratide;

[0172] b. Converting liratide into liraglutide, wherein the method comprises conjugating the liratide obtained in step (a) with a palmitoyl glutamate derivative.

[0173] The above disclosure generally describes the present invention. A more complete understanding can be obtained through the following specific examples. This example is described only for the purpose of illustration and is not intended to limit the scope of the present invention. Although specific terms are used herein, the purpose of such terms is descriptive, not restrictive.

[0174] Example

[0175] Example 1: Modified nucleic acid for expressing liratide

[0176] In order to obtain optimal expression in Pichia pastoris, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis, the expression cassette encoding the liraglutide precursor peptide was modified. The modified open reading frame includes a nucleotide sequence encoding liraglutide, which is fused to a sequence encoding a TEV (tobacco etch virus) cleavage site and a sequence encoding an N-terminal extension sequence (NE-3 or NE-1). Preferred codons expressed in Pichia pastoris, Corynebacterium glutamicum, Escherichia coli and Bacillus subtilis are used to replace rare codons.

[0177] As a control, an open reading frame including a nucleotide sequence encoding liratide without any N-terminal extension sequence was prepared.

[0178] Expression in Pichia pastoris

[0179] In order to express in Pichia pastoris, the nucleotide sequence encoding liratide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension sequence were modified.

[0180] The nucleotide sequence encoding liratide is represented by SEQ ID NO: 6. The nucleotide sequence encoding the TEV cleavage site is represented by SEQ ID NO: 5. The nucleotide sequence encoding the N-terminal extension sequence (NE-3) is represented by SEQ ID NO: 4.

[0181] This modified open reading frame was artificially synthesized using the sequence for liratide, the TEV cleavage site sequence and the N-terminal extension sequence.

[0182] Figure 1 The modified open reading frame is shown, which includes the coding sequence without the N-terminal extension sequence ( Figure 1 A) and having N-terminal extension sequence-1 (NE1) (GagGaAgAgcG, Figure 1 B), N-terminal extension sequence-3 (NE3) (GaAgAgAgAgAgAgcGaA, Figure 1 C) liratide, and DNA of the TEV (tobacco etch virus) cleavage site sequence (GagAaAgtTactCtCaA) and signal sequence cassette.

[0183] The modified coding sequence of recombinant liratide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid includes an open reading frame and a promoter.

[0184] The plasmid vector map of pD912 is as follows: Figure 1 as shown in .

[0185] Expression in Corynebacterium glutamicum

[0186] For expression in Corynebacterium glutamicum, the nucleotide sequence encoding liratide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0187] The nucleotide sequence encoding liratide is represented by SEQ ID NO: 9. The nucleotide sequence encoding the 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.

[0188] This modified open reading frame was artificially synthesized using the Thermo Fisher Scientific Technique using the liratide sequence, the TEV cleavage site sequence, and the N-terminal extension sequence.

[0189] The modified coding sequence of recombinant liratide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid includes an open reading frame and a promoter.

[0190] Expression in Escherichia coli

[0191] For expression in Escherichia coli, the nucleotide sequence encoding liratide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0192] The nucleotide sequence encoding liratide is represented by SEQ ID NO: 12. The nucleotide sequence encoding the 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.

[0193] This modified open reading frame was artificially synthesized using a liratide sequence, a TEV cleavage site sequence, and an N-terminal extension sequence.

[0194] The modified coding sequence of recombinant liratide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid includes an open reading frame and a promoter.

[0195] Expression in Bacillus subtilis

[0196] For expression in Bacillus subtilis, the nucleotide sequence encoding liratide, the nucleotide sequence encoding the TEV cleavage site and the nucleotide sequence encoding the N-terminal extension were modified.

[0197] The nucleotide sequence encoding liratide is represented by SEQ ID NO: 15. The nucleotide sequence encoding the 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.

[0198] This modified open reading frame was artificially synthesized using a liratide sequence, a TEV cleavage site sequence, and an N-terminal extension sequence.

[0199] The modified coding sequence of recombinant liratide was cloned into the pD912 expression vector (Atum, USA). The recombinant plasmid includes an open reading frame and a promoter.

[0200] Confirmation of plasmid DNA linearization

[0201] The synthesized liratide DNA without N-terminal extension sequence, N-terminal extension sequence-1, N-terminal extension sequence-3 and plasmid pD912 was digested with EcoRI and BglII restriction endonucleases. The restriction enzyme fragments were ligated and transformed into Escherichia coli strains. Figure 2A )、N-terminal extension sequence-1( Figure 2B )、N-terminal extension sequence-3( Figure 2C ) were sequenced to confirm the liratide, N-terminal extension sequence and TEV cleavage site sequences. The sequence-confirmed plasmid DNA was linearized with Sac I enzyme.

[0202] Example 2: Development of recombinant host cells by transformation of recombinant plasmids

[0203] As described in the previous examples, the recombinant pD912 plasmid carrying the liratide precursor peptide gene fused to a signal peptide was used for the development of a recombinant host.

[0204] The plasmid was used to transform Pichia pastoris host cells (obtained from Atum, USA) by electroporation.

[0205] The transformed cells were inoculated on YPD agar (yeast peptone dextrose) plates containing 100 μg / ml zeocin. The transformed Pichia cells were cultured in 20 mL BMGY medium for 24 hours. The cells were collected by centrifugation and resuspended in 20 ml BMMY medium to an OD of 0. 600nm The cell suspension was cultured in a shaking flask incubator at 30°C for 24 hours.

[0206] Example 3: Analysis and evaluation of liratide expression

[0207] After 24 hours, the culture supernatant was collected, purified, and analyzed by ELISA using a monoclonal antibody specific for liratide ( Figure 3 ) to analyze the expression of liraglutide. In addition, the cell dry weight was measured using a moisture analyzer ( Figure 4 ).

[0208] Tables 1 and 2 provide a comparison determining the effectiveness of N-terminal extension sequences in increasing liratide production.

[0209] Table 1: Different N-terminal extension sequences and liratide expression compared to the control group without N-terminal extension sequence

[0210]

[0211] Table 2: Fold differences in expression of different N-terminal extension sequences and liratide compared to the control group without N-terminal extension sequence

[0212] Extension sequence clone Multiple Difference EEA 1 1.0 EEA 2 0.8 EEA 3 0.9 EEQAE 1 4.6 EEQAE 2 4.8 EEQAE 3 4.4

[0213] The above data clearly show that the N-terminal extension sequence can increase the expression of liratide by about 5 times compared with the control group and the known N-terminal extension sequence. Sequence Listing <110> Bio-E Ltd. <120> N-terminal extension sequences for expression of recombinant therapeutic peptides <130> IP40-220234 <150> IN201941009728 <151> 2019-09-13 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> Amino acid sequence of N-terminal extension sequence NE-3 <400> 1 Glu Glu Gln Ala Glu 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial <220> <223> Amino acid sequence of modified TEV cleavage site <400> 2 Glu Asn Leu Tyr Phe Gln 1 5 <210> 3 <211> 31 <212> PRT <213> Artificial <220> <223> Amino acid sequence of liratide <400> 3 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 <210> 4 <211> 15 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding N-terminal extension NE-3 sequence of Pichia pastoris <400> 4 gaagaacaag ccgaa 15 <210> 5 <211> 18 <212> DNA <213> Artificial <220> <223> Pichia pastoris nucleic acid sequence encoding a modified TEV cleavage site <400> 5 gagaacttgt acttccaa 18 <210> 6 <211> 96 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding liratide from Pichia pastoris <400> 6 cacgctgagg gtacttttac ctctgacgtg tcctcttact tggagggtca agctgccaaa 60 gagttcattg cctggttggt tagaggtaga ggttag 96 <210> 7 <211> 15 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding N-terminal extension NE-3 sequence of Corynebacterium glutamicum <400> 7 gaagaacagg cagaa 15 <210> 8 <211> 18 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding a modified TEV cleavage site of Corynebacterium glutamicum <400> 8 gaaaacctgt acttccag 18 <210> 9 <211> 96 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding liratide from Corynebacterium glutamicum <400> 9 cacgcagaag gcacctttac ctccgatgtg tcctcctacc tggaaggcca ggcagcaaaa 60 gaattcattg catggctggt tcgcggtcgc ggttag 96 <210> 10 <211> 15 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding N-terminal extension NE-3 sequence of Escherichia coli <400> 10 gaagaacagg cagaa 15 <210> 11 <211> 18 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding a modified TEV cleavage site in Escherichia coli <400> 11 gaaaacctgt acttccag 18 <210> 12 <211> 96 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding liratide from Escherichia coli <400> 12 catgcggaag gcaccttcac cagcgatgtt agcagctacc tggagggtca ggcggcgaag 60 gaatttatcg cgtggctggt tcgtggccgt ggttaa 96 <210> 13 <211> 15 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding N-terminal extension NE-3 sequence of Bacillus subtilis <400> 13 gaagaacaag ccgaa 15 <210> 14 <211> 18 <212> DNA <213> Artificial <220> <223> Bacillus subtilis nucleic acid sequence encoding a modified TEV cleavage site <400> 14 gagaacttgt acttccaa 18 <210> 15 <211> 96 <212> DNA <213> Artificial <220> <223> Nucleic acid sequence encoding liratide from Bacillus subtilis <400> 15 cacgctgagg gtacttttac ctctgacgtg tcctcttact tggagggtca agctgccaaa 60 gagttcattg cctggttggt tagaggtaga ggttag 96 <210> 16 <211> 34 <212> PRT <213> Artificial <220> <223> Amino acid sequence of teriparatide <400> 16 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe <210> 17 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of N-terminal extension sequence NE-1 <400> 17 Glu Glu Ala 1 <210> 18 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding the N-terminal extension NE-1 sequence <400> 18 gaggaagcg 9 <210> 19 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Comprising an N-terminal extension sequence NE-3 and a TEV cleavage site operably fused to Liratide fusion protein <400> 19 Glu Glu Gln Ala Glu Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr 1 5 10 15 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu 20 25 30 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 35 40 <210> 20 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Comprising an N-terminal extension sequence NE-1 and a TEV cleavage site operably fused to Liratide fusion protein <400> 20 Glu Glu Ala Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr 1 5 10 15 Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile 20 25 30 Ala Trp Leu Val Arg Gly Arg Gly 35 40

Claims

1. A vector for recombinant expression of liratide in yeast, comprising: a. A modified gene sequence encoding an N-terminal extension sequence, wherein the modified gene sequence is represented by SEQ ID NO: 4; b. a modified gene sequence encoding a tobacco etch virus cleavage site, the modified gene sequence being represented by SEQ ID NO: 5; and c. A modified gene sequence encoding liratide, wherein the modified gene sequence is represented by SEQ ID NO:

6.

2. A recombinant host cell comprising the vector according to claim 1, wherein the recombinant host cell is selected from Pichia pastoris.

3. A modified liratide represented by the amino acid sequence of SEQ ID NO: 19, wherein the liratide is operably fused to a tobacco etch virus cleavage site and an N-terminal extension sequence.

4. A method for expressing liratide using the recombinant host cell according to claim 2, wherein the fermentation process of the method comprises: a. culturing the recombinant host cell in BMGY medium for 24 hours; b. harvesting the recombinant host cells by centrifugation; c. Resuspend the recombinant host cells in BMMY medium to an OD of 600nm is 10; d. placing the recombinant host cells in a shake flask incubator at 30° C. for 24 hours, and e. Harvest and purify the culture supernatant to obtain liratide.

5. A method for preparing liraglutide, comprising the following steps: a. culturing the recombinant host cell according to claim 2 in a suitable culture medium to obtain liratide; b. Converting liratide into liraglutide, wherein the method comprises conjugating the liratide obtained in step (a) with a palmitoyl glutamate derivative.

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