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WO2019154311A1 - 一种密码子优化的人胰岛素类似物前体基因和信号肽基因 - Google Patents

一种密码子优化的人胰岛素类似物前体基因和信号肽基因 Download PDF

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WO2019154311A1
WO2019154311A1 PCT/CN2019/074384 CN2019074384W WO2019154311A1 WO 2019154311 A1 WO2019154311 A1 WO 2019154311A1 CN 2019074384 W CN2019074384 W CN 2019074384W WO 2019154311 A1 WO2019154311 A1 WO 2019154311A1
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Prior art keywords
nucleic acid
acid molecule
seq
human insulin
molecule encoding
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PCT/CN2019/074384
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English (en)
French (fr)
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王菲菲
陈磊
王宏伟
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江苏恒瑞医药股份有限公司
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Application filed by 江苏恒瑞医药股份有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to EP19751736.0A priority Critical patent/EP3750998A4/en
Priority to KR1020207020915A priority patent/KR20200119237A/ko
Priority to CA3086618A priority patent/CA3086618A1/en
Priority to US16/967,117 priority patent/US20210032307A1/en
Priority to RU2020128190A priority patent/RU2020128190A/ru
Priority to MX2020007929A priority patent/MX2020007929A/es
Priority to CN201980004477.8A priority patent/CN111094572B/zh
Priority to JP2020541883A priority patent/JP2021513330A/ja
Priority to BR112020015238-2A priority patent/BR112020015238A2/pt
Priority to AU2019218315A priority patent/AU2019218315A1/en
Publication of WO2019154311A1 publication Critical patent/WO2019154311A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present invention relates to a codon-optimized human insulin analog precursor gene and a codon-optimized ⁇ -factor signal peptide gene, and provides a method for expressing the human insulin analog precursor gene .
  • Human insulin is a polypeptide consisting of 51 amino acids and contains two chains, A and B, respectively.
  • the main effect of insulin is to regulate glucose metabolism.
  • insulin intervention is the most direct and effective method as an alternative or supplementary treatment.
  • Insulin also promotes fat synthesis, inhibits fat breakdown, and reduces ketone body formation. It is also used to correct various symptoms of insulin-related ketosis and acidosis.
  • Insulin was first obtained from the pancreas of pigs, cattle and other animals, but these products are different from human insulin, so they are immunogenic.
  • Eli Lilly and Danish Novo Nordisk developed the technology of recombinant human insulin production.
  • human insulin and its analogues became the mainstream of the industry.
  • the short duration of action of human insulin makes it necessary for patients to inject frequently, which is extremely inconvenient. Therefore, efforts have been made to obtain insulin analogs and derivatives thereof that can act on the human body for a longer period of time.
  • the use of an acylating group to modify human insulin or an analog thereof is an effective method for increasing the half-life thereof.
  • WO2018024186 discloses a human insulin analog in which the B29 position is substituted with a long chain fatty acid and the amino acid at position B30 is deleted, and the structure and biological activity of the human insulin analog are disclosed.
  • WO9507931 discloses an insulin analog having a B29 position linked to a tetradecanoyl side chain, a B30 amino acid deletion, and a preparation thereof.
  • WO2005012347 discloses a human insulin analog in which the B29 position is substituted by a glutamic acid and a long chain fatty acid, and the amino acid at position B30 is deleted.
  • the commonly used expression systems for expressing human insulin and its analogues are mainly Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, in which E. coli is expressed in the form of inclusion bodies, which undergo cleavage and renaturation of inclusion bodies, and the process is cumbersome and The yield is low, and S. cerevisiae and Pichia pastoris have the advantages of simple operation, easy cultivation, foreign protein modification, and secretory expression, but the secretion efficiency of Saccharomyces cerevisiae is low and the expression strain is not stable enough, compared to Pichia pastoris.
  • Industrial production of recombinant proteins uses a broader expression system.
  • the fermentation yield in the production process is a key factor in controlling the production cost. Due to the large demand in the insulin market, Novo Nordisk, an important producer, uses yeast to express its production scale when the size of the can reaches several tens of tons, which is very demanding on the plant and equipment. High and high cost, so increasing the fermentation yield of human insulin and its analogues is of great significance to industrial production.
  • the genetic code is a triplet code and one codon consists of three adjacent bases on the messenger ribonucleic acid (mRNA).
  • mRNA messenger ribonucleic acid
  • the nucleotide sequence thereof is also derived from Saccharomyces cerevisiae, and the ⁇ -factor signal peptide nucleotide sequence optimized for Pichia pastoris has not been reported so far.
  • codon optimization of insulin precursors such as the human insulin precursor gene sequence optimized by Gurramkonda et al. and its expression in the expression of simple fed-batch technique to high-level secretory production of Insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.
  • WO1998028429 discloses a gene sequence expressing a precursor of a human insulin analog, the amino acid sequence encoding the insulin precursor of the gene is EEGEPK-B(1-29)-AAK-A(1-21), wherein EEGEPK is an N-terminal extension of the insulin precursor, called a spacer peptide or a leader peptide, which protects the N-terminus of the insulin precursor from the hydrolysis of yeast protease.
  • B (1-29) is the human insulin B chain lacking B30 threonine
  • a (1-21) is the human insulin A chain amino acid sequence
  • AAK is the connecting B chain A peptide linked to the A chain, also known as a C peptide.
  • the present invention combines the Pichia pastoris codon preference to optimize the insulin analog precursor gene and the ⁇ -factor signal peptide gene for secretion expression in Pichia pastoris. And as a control with the human insulin analog precursor gene in the prior art, the results show that the codon-optimized gene expression of the human insulin analog precursor in the present invention is nearly doubled, which can greatly reduce the late human insulin. The cost of industrial production of its analogues.
  • PS is a nucleic acid molecule encoding a processing site, a is 0 or 1;
  • SP is a nucleic acid molecule encoding a signal peptide, b is 0 or 1;
  • LS is a nucleic acid molecule encoding a spacer peptide, c is 0 or 1;
  • GE is a nucleic acid molecule encoding a polypeptide of interest
  • P'S is a nucleic acid molecule encoding a processing site, and d is 0 or 1.
  • PS is a nucleic acid molecule encoding a processing site, a is 0 or 1;
  • SP is a nucleic acid molecule encoding a signal peptide, b is 1;
  • LS is a nucleic acid molecule encoding a spacer peptide, c is 1;
  • GE is a nucleic acid molecule encoding a polypeptide of interest
  • P'S is a nucleic acid molecule encoding a processing site, and d is 0 or 1.
  • the nucleic acid molecule encoding the signal peptide comprises the sequence set forth in SEQ ID NO:1.
  • the polypeptide of interest is a human insulin analog precursor polypeptide;
  • the nucleic acid molecule encoding a human insulin analog precursor polypeptide comprises the sequence set forth in SEQ ID NO:3.
  • nucleic acid sequence of the nucleic acid molecule (SP) encoding the signal peptide is set forth in SEQ ID NO: 1
  • amino acid sequence is set forth in SEQ ID NO: 2:
  • the nucleic acid molecule (GE) encoding the polypeptide of interest may be a nucleic acid molecule encoding a precursor of a human insulin analog, which may be human insulin depleted at the B30 position.
  • the nucleic acid molecule sequence of the human insulin analog precursor is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4:
  • position 88-96 of the nucleic acid molecule encoding a precursor of a human insulin analog is a nucleic acid molecule encoding a linker peptide (also referred to as a C-peptide), which may be substituted, including but not limited to the following Sequence: GCCGCTAAG, GCTGCCAAG, GCTGCTAAA, GCCGCCAAG.
  • sequence of the nucleic acid molecule (LS) encoding the spacer peptide is set forth in SEQ ID NO: 5:
  • the PS and/or P'S is a nucleic acid molecule encoding a restriction site
  • PS is a nucleic acid molecule encoding an EcoR I cleavage site
  • P'S is a nucleic acid molecule encoding a Not I cleavage site.
  • a nucleic acid molecule capable of expressing a precursor of a human insulin analog comprising a nucleic acid molecule encoding a spacer peptide and a nucleic acid molecule encoding a precursor of a human insulin analog, capable of expressing a human after recombination with a vector comprising a signal peptide Insulin analog precursor.
  • the human insulin analog precursor nucleic acid sequence encoded by the human insulin analog precursor nucleic acid molecule is as follows:
  • EGEPK SEQ ID NO: 16
  • B(1-29) may be a human insulin B chain lacking the B30-threonine
  • A(1-21) may be the human insulin A chain amino acid sequence
  • AAK is a linker peptide linking the B chain and the A chain, also referred to as C peptide.
  • sequence of the human insulin analog precursor nucleic acid molecule can be as set forth in SEQ ID NO: 6, and the amino acid sequence is set forth in SEQ ID NO:7:
  • a nucleic acid molecule capable of expressing a precursor of a human insulin analog comprising a signal peptide sequence, a spacer peptide sequence, and a sequence encoding a precursor of a human insulin analog, capable of and without a signal, is provided
  • the peptide carrier is recombinantly expressed to express a human insulin analog precursor.
  • nucleic acid sequence of the nucleic acid molecule that expresses the human insulin analog precursor is set forth in SEQ ID NO: 8
  • the encoded amino acid sequence is set forth in SEQ ID NO:9:
  • a nucleic acid molecule that expresses a precursor of a human insulin analog can also be ligated to a cleavage site sequence, preferably an EcoR I cleavage site and a Not I cleavage site.
  • a vector capable of expression in eukaryotic or prokaryotic cells is capable of secreting expression of a human insulin analog precursor in a prokaryotic or eukaryotic cell.
  • a host cell is also provided, preferably a yeast, more preferably Pichia, which is capable of secreting a human insulin analog precursor.
  • a method of making a human insulin analog comprising the use of a nucleic acid molecule, vector, and/or host cell as described above.
  • the method may further comprise the following steps:
  • nucleic acid molecule encoding a precursor of a human insulin analog can be exemplified by SEQ ID NO: 6, the enzyme-cutting insulin precursor Methods well known to those skilled in the art are employed.
  • step 1) comprises expressing a human insulin analog precursor with an expression vector comprising a signal peptide sequence, said signal peptide sequence being as set forth in SEQ ID NO: 1.
  • the human insulin analog is a B30 deleted human insulin, the human insulin analog further substituted with an acylating group.
  • the B30 deleted human insulin is substituted with an acylating group at the lysine at position B29.
  • the substituted product of the above substitution is lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) des (B30) human insulin.
  • Codon optimization refers to the use of host cell preference codon rules, the use of preferred codons and avoidance of low utilization or rare codons to synthesize genes.
  • Control 1 is an encoding nucleic acid molecule of "EEGEPK” (GAAGAAGGTGAACCAAAG shown under double underline) is linked to the encoding nucleic acid molecule of the insulin precursor gene in patent WO1998028429, as shown in SEQ ID NO: 10 below:
  • Control 2 is an "EEGEPK” encoding nucleic acid molecule (shown underlined) linked to the encoded nucleic acid molecule of the optimized insulin precursor gene in the literature of Gurramkonda et al. (Microbial Cell Factories, 2010, 9:31), as follows SEQ ID NO: 11:
  • IP-S is a nucleic acid molecule corresponding to a codon optimized insulin precursor gene.
  • ⁇ -factor is a nucleic acid molecule corresponding to the ⁇ -factor signal peptide gene carried by the pPIC9K expression vector provided by Invitrogen, which is derived from Saccharomyces cerevisiae.
  • ⁇ -Factor-S is a nucleic acid molecule corresponding to a codon-optimized ⁇ -factor signal peptide gene.
  • a “vector” includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, including, but not limited to, a plasmid and a viral vector. Certain vectors are capable of autonomous replication in the host cell into which they are introduced, while other vectors can be integrated into the genome of the host cell upon introduction into the host cell and thus replicated along with the host genome. In addition, certain vectors are capable of directing expression of genes operably linked thereto, such vectors are referred to herein as “recombinant expression vectors" (or simply “expression vectors”), and typical vectors are well known in the art.
  • target polypeptide is a polypeptide that can be expressed in yeast, including but not limited to enzymes, antibodies, interferons, insulin, interleukins, and the like, and variants, precursors, intermediates thereof, such as may be insulin precursors.
  • Cell and “host cell” are used interchangeably.
  • Polynucleotide molecule “nucleic acid molecule” are used interchangeably and the sequence may be a DNA sequence.
  • the vector, host bacteria and culture medium used in the examples of the present invention were purchased from Invitrogen, wherein the Pichia pastoris expression vector pPIC9K contains an alcohol oxidase AOX1 promoter, which can be induced by methanol, and the vector also contains an ⁇ -factor signal peptide sequence. It can secrete and express foreign protein; Pichia pastoris expression vector pPIC3.5K contains alcohol oxidase AOX1 promoter, which can be induced by methanol.
  • the vector does not contain ⁇ -factor signal peptide sequence;
  • the host strain is Pichia pastoris GS115 strain; Based on the media formulation provided by the Pichia pastoris manual.
  • Control 1 SEQ ID NO: 10
  • Control 2 SEQ ID NO: 11
  • IP-S SEQ ID NO: 6
  • the point was synthesized by Nanjing Kingsray Biotech Co., Ltd., and the synthesized nucleic acid molecule sequence was ligated to the T vector.
  • the T vector carrying the insulin precursor nucleic acid molecule and the expression vector pPIC9K were digested with the endonuclease EcoR I and Not I, and then the target fragment and the vector fragment were separately recovered by a gel recovery kit, and the enzyme was digested with T4 ligase. The target fragment after digestion and purification was ligated to the vector pPIC9K after digestion.
  • Example 2 Pichia pastoris recombinant strain expresses insulin precursor
  • Example 1 The three recombinant expression vectors constructed in Example 1 were transformed into Pichia pastoris GS115, and the recombinant strains expressing Control 1 and Control 2 were used as control strains, and the recombinant strain expressing IP-S was used as an experimental strain.
  • Table 1 shows that the amount of insulin precursor expressed by the optimized insulin precursor gene is increased by 1.8 to 2.25 times compared with the two control groups. It can be seen that the optimized insulin precursor gene has a better expression effect and its expression. The yield of insulin precursors is significantly increased.
  • ⁇ -factor SEQ ID NO: 12
  • ⁇ -factor-S SEQ ID NO: 1
  • SEQ ID NO: 13 SEQ ID NO: 8
  • the synthesized nucleic acid molecule is ligated to the T vector.
  • ⁇ -factor SEQ ID NO: 12
  • ⁇ -factor-S SEQ ID NO: 1
  • SEQ ID NO: 14 SEQ ID NO: 15
  • the synthesis is carried out, and the 5' and 3' ends of the synthesized nucleic acid molecule carry EcoR I and Not I cleavage sites, respectively, and the synthesized nucleic acid molecule is ligated to the T vector.
  • T vector and the expression vector pPIC3.5K were digested with the endonucleases EcoR I and Not I, and then the target fragment and the vector fragment were separately recovered by a gel recovery kit, and the target fragment was digested with T4 ligase.
  • the vector pPIC3.5K after restriction enzyme digestion was separately ligated.
  • the above-mentioned ligation solution was transformed into E. coli TOP10 competent cells, and the resistant plate containing ampicillin was applied. After the culture, the cloned plasmid was picked and cloned, and the two recombinant expression vectors were finally verified.
  • An insulin precursor gene that expresses a different nucleotide sequence is fused with an ⁇ -factor signal peptide and an ⁇ -factor-S signal peptide.
  • Example 4 Insulin precursor before and after optimization of expression of Pichia pastoris recombinant bacteria
  • the recombinant expression vector constructed in Example 3 was separately transformed into Pichia pastoris GS115.
  • the cells were collected and resuspended in 50 mL of BMGY medium, and cultured overnight at 30 ° C with a constant temperature shaker at 250 rpm until the OD 600 was about 30.
  • the cells were collected by centrifugation at 1500 rpm for 5 minutes and resuspended in 25 mL of BMMY medium.
  • the recombinant strain expressing the control 1 gene of the fusion ⁇ -factor was used as a control strain, and the insulin precursor production of the other strains was converted into a percentage relative to the yield of the control strain, as shown in Table 2.
  • the data in Table 2 shows that only the signal peptide-optimized nucleic acid molecule sequence expresses the amount of insulin precursor increased by a factor of 1.5, and only the insulin precursor gene-optimized nucleic acid molecule sequence expresses the amount of the insulin precursor to 2.25 times, while the signal peptide
  • the amount of the insulin precursor expressed by the nucleic acid molecule sequence optimized simultaneously with the insulin precursor gene was increased to 2.75 times. Taken together, codon optimization increases the expression of insulin precursors to 1.5-2.75 times.

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Abstract

提供一种密码子优化的人胰岛素类似物前体基因和信号肽基因的核酸分子。该核酸分子包含一种融合胰岛素类似物前体的编码核酸分子和一种酵母分泌信号肽α-因子的编码核酸分子。该核酸分子使得胰岛素类似物前体在毕赤酵母中的表达量提高，降低人胰岛素类似物的生产成本。

Description

一种密码子优化的人胰岛素类似物前体基因和信号肽基因 技术领域
本发明涉及一种密码子优化的人胰岛素类似物前体基因和一种密码子优化的α-因子(α-factor)信号肽基因，并提供了所述人胰岛素类似物前体基因的表达方法。
背景技术
人胰岛素是由51个氨基酸组成的多肽，包含两条链，分别为A链和B链。胰岛素的主要药效是调节糖代谢，在糖尿病治疗中，胰岛素干预作为替代或补充治疗是最直接有效的方法。胰岛素还有促进脂肪合成、抑制脂肪分解、减少酮体生成的作用，所以亦被用来纠正胰岛素相关酮症和酸血症的各种症状。
胰岛素最早一直由猪、牛等动物胰腺提取而得，但这些产品与人胰岛素结构有所不同，故存在免疫原性。上世纪八十年代初、中期美国礼来和丹麦诺和诺德公司相继开发出基因重组人胰岛素生产技术，从此基因工程表达人胰岛素及其类似物成为工业主流手段。但是人胰岛素作用时间较短，使得病人必须频繁注射，极为不便。因此，人们致力于获得一些能够更长时间作用于人体的胰岛素类似物及其衍生物。其中，使用酰化基团修饰人胰岛素或其类似物，是一种提高其半衰期的有效方法。WO2018024186公开了一种B29位被长链脂肪酸取代、B30位氨基酸缺失的人胰岛素类似物，公开了该人胰岛素类似物的结构和生物活性。WO9507931公开了一种B29位连接一个十四酰基侧链、B30位氨基酸缺失的胰岛素类似物及其制剂。WO2005012347公开了一种B29位被一个谷氨酸和长链脂肪酸取代、B30位氨基酸缺失的人胰岛素类似物。目前常用的表达人胰岛素及其类似物的表达系统主要有是大肠杆菌、酿酒酵母和巴斯德毕赤酵母，其中大肠杆菌表达为包涵体形式，需经过包涵体裂解和复性，工艺繁琐且得率低，酿酒酵母与毕赤酵母有着操作简易、易于培养、对外源蛋白修饰、可以分泌表达等遗传表达优点，但酿酒酵母分泌效率低、表达菌株不够稳定，相比之下毕赤酵母是工业生产重组蛋白应用更广泛的表达系统。工业上，生产过程中发酵产量是控制生产成本的关键因素，由于胰岛素市场需求量大，作为重要生产商的诺和诺德利用酵母表达生产时罐子规模达到几十吨，对厂房、设备要求很高，成本也高，所以提高人胰岛素及其类似物的发酵产量对工业生产有着重要意义。
遗传密码子是三联体密码,一个密码子由信使核糖核酸(mRNA)上相邻的三个碱基组成。遗传密码共有64种，但不同的生物，甚至同种生物不同的蛋白质编码基因，对不同密码子使用频率并不相同，具有一定的偏爱性。外源基因 的密码子主要在翻译水平上影响基因的表达，很多文献已经证明密码子的优化对于在毕赤酵母中提高外源蛋白的表达量卓有成效。毕赤酵母表达外源基因分为胞内表达和分泌表达两种，后者需要信号肽来引导外源基因表达产物的分泌，目前最普遍使用的信号肽为来源于酿酒酵母α-因子信号肽，其核苷酸序列也来源于酿酒酵母，目前还没有报道针对毕赤酵母优化的α-因子信号肽核苷酸序列。胰岛素前体密码子优化的相关文献很多，如Gurramkonda等人优化的人胰岛素前体基因序列并在毕赤酵母的表达研究(Gurramkonda et al.Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.Microbial Cell Factories,2010,9:31)，专利WO1998028429公开了表达人胰岛素类似物前体的基因序列，该基因编码的胰岛素前体氨基酸序列是EEGEPK-B(1-29)-AAK-A(1-21)，其中EEGEPK为胰岛素前体N端延伸，称为间隔肽或前导肽，可以保护胰岛素前体的N端免于酵母蛋白酶的水解作用，并且能提高胰岛素前体的表达效率；B(1-29)是缺失B30位苏氨酸的人胰岛素B链；A(1-21)是人胰岛素A链氨基酸序列；AAK为连接B链和A链的连接肽，也称为C肽。
为了进一步提升人胰岛素及其类似物前体的产量，本发明结合毕赤酵母密码子偏好性，优化了胰岛素类似物前体基因和α-因子信号肽基因，在毕赤酵母中进行分泌表达，并与现有技术中的人胰岛素类似物前体基因作为对照，结果表明，本发明中经过密码子优化后的基因表达人胰岛素类似物前体量提高了近2倍，能够大大降低后期人胰岛素及其类似物工业生产的成本。
发明内容
本发明的一些实施方案中提供了核酸分子，其包含如下结构：
5′—(PS) a—(SP) b—(LS) c—GE—(P′S) d—3′，其中，
PS是编码加工位点的核酸分子，a是0或1；
SP是编码信号肽的核酸分子，b是0或1；
LS是编码间隔肽的核酸分子，c是0或1；
GE是编码目标多肽的核酸分子；
P′S是编码加工位点的核酸分子，d是0或1。
一些实施方案中提供了核酸分子，其包含如下结构：
5′—(PS) a—(SP) b—(LS) c—GE—(P′S) d—3′，其中，
PS是编码加工位点的核酸分子，a是0或1；
SP是编码信号肽的核酸分子，b是1；
LS是编码间隔肽的核酸分子，c是1；
GE是编码目标多肽的核酸分子；
P′S是编码加工位点的核酸分子，d是0或1。
在一些实施方案中，编码信号肽的核酸分子包含如SEQ ID NO:1所示序列。
在一些实施方案中，目标多肽是人胰岛素类似物前体多肽；编码人胰岛素类似物前体多肽的核酸分子包含如SEQ ID NO:3所示序列。
在一些实施方案中，编码信号肽的核酸分子(SP)的核酸序列如SEQ ID NO:1所示，氨基酸序列如SEQ ID NO:2所示：
Figure PCTCN2019074384-appb-000001
在一些实施方案中，编码目标多肽的核酸分子(GE)可以是编码人胰岛素类似物前体的核酸分子，其可以是B30位苏氨酸缺失的人胰岛素。人胰岛素类似物前体的核酸分子序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示：
Figure PCTCN2019074384-appb-000002
在一些实施方案中，编码人胰岛素类似物前体的核酸分子的88-96位(即GCTGCTAAG)为编码连接肽(也称C肽)的核酸分子，其可以被替代，包括但不限于如下的序列：GCCGCTAAG，GCTGCCAAG，GCTGCTAAA，GCCGCCAAG。
在一些实施方案中，编码间隔肽的核酸分子(LS)的序列如SEQ ID NO:5所示：
Figure PCTCN2019074384-appb-000003
Figure PCTCN2019074384-appb-000004
在一些实施方案中，PS和/或P′S是编码酶切位点的核酸分子；
优选的，PS是编码EcoR I酶切位点的核酸分子，和/或P′S是编码Not I酶切位点的核酸分子。
在一些实施方案中，提供能够表达人胰岛素类似物前体的核酸分子，其包含编码间隔肽的核酸分子和编码人胰岛素类似物前体的核酸分子，能够与包含信号肽的载体重组后表达人胰岛素类似物前体。
在一些实施方案中，人胰岛素类似物前体核酸分子编码的人胰岛素类似物前体氨基酸序列如下：
EEGEPK-B(1-29)-AAK-A(1-21)
其中，“EEGEPK(SEQ ID NO：16)”可以是胰岛素前体N端延伸，称为间隔肽或前导肽；“B(1-29)”可以是缺失B30位苏氨酸的人胰岛素B链；“A(1-21)”可以是人胰岛素A链氨基酸序列，“AAK”为连接B链和A链的连接肽，也称为C肽。
在一些实施方案中，人胰岛素类似物前体核酸分子的序列可以如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:7所示：
Figure PCTCN2019074384-appb-000005
在一些实施方案中，提供另一种能够表达人胰岛素类似物前体的核酸分子，该序列中包含信号肽序列、间隔肽序列和编码人胰岛素类似物前体的序列，其能够与不包含信号肽的载体重组后表达人胰岛素类似物前体。
在一些实施方案中，表达人胰岛素类似物前体的核酸分子的核酸序列如SEQ ID NO:8所示，编码的氨基酸序列如SEQ ID NO:9所示：
Figure PCTCN2019074384-appb-000006
Figure PCTCN2019074384-appb-000007
在一些实施方案中，表达人胰岛素类似物前体的核酸分子还可以连接酶切位点序列，所述酶切位点优选EcoR I酶切位点和Not I酶切位点。
在一些实施方案中，还提供了一种能够在真核或原核细胞中表达的载体，其能够在原核或真核细胞中分泌表达人胰岛素类似物前体。
在一些实施方案中，还提供了一种宿主细胞，所述的宿主细胞优选为酵母菌，更优选为毕赤酵母，其能够分泌表达人胰岛素类似物前体。
在一些实施方案中，还提供一种制备人胰岛素类似物的方法，包括使用如前所述的核酸分子，载体，和/或宿主细胞。
所述的方法还可以包含如下步骤：
1)通过编码人胰岛素类似物前体的核酸分子在真核细胞中表达人胰岛素类似物前体；
2)通过酶切处理人胰岛素类似物前体得到人胰岛素类似物，所述的编码人胰岛素类似物前体的核酸分子可以如SEQ ID NO:6所示，所述的酶切处理胰岛素前体采用本领域技术人员公知的方法。
在一些实施方案中，步骤1)中包含用表达载体表达人胰岛素类似物前体，所述的表达载体包含信号肽序列，所述的信号肽序列可以如SEQ ID NO:1所示。
在一些实施方案中，所述的人胰岛素类似物为B30缺失的人胰岛素，所述的人胰岛素类似物进一步经过酰化基团的取代。
在一些实施方案中，所述的B30缺失的人胰岛素在B29位的赖氨酸处被酰化基团取代。
优选的，上述取代的取代产物为赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))des(B30)人胰岛素。
缩写和术语
“密码子优化”是指利用宿主细胞偏爱密码子规则，使用偏爱密码子并避 免使用利用率低或稀有密码子来合成基因。
“对照1”是“EEGEPK”的编码核酸分子(双下划线所示的GAAGAAGGTGAACCAAAG)与专利WO1998028429中的胰岛素前体基因的编码核酸分子相连，如下SEQ ID NO:10所示：
Figure PCTCN2019074384-appb-000008
“对照2”是“EEGEPK”的编码核酸分子(双下划线所示)与Gurramkonda等人的文献(Microbial Cell Factories,2010,9:31)中经优化的胰岛素前体基因的编码核酸分子相连，如下SEQ ID NO:11所示：
Figure PCTCN2019074384-appb-000009
“IP-S”是经密码子优化的胰岛素前体基因对应的核酸分子。
“α-因子”是Invitrogen公司提供的pPIC9K表达载体带有的α-因子信号肽基因对应的核酸分子，来源于酿酒酵母。
“α-因子-S”是经密码子优化的α-因子信号肽基因对应的核酸分子。
“载体”包括核酸分子，其能够运输它连接的另一核酸，包括但不局限于质粒和病毒载体。某些载体能够在其被引入的宿主细胞中自主复制，而其它载体可在引入宿主细胞时整合到宿主细胞的基因组中，并因此与所述宿主基因组一起复制。此外，某些载体能够指导有效连接到它们的基因的表达，此类载体此处称作“重组表达载体”(或简单地“表达载体”)，并且典型载体为本领域熟知。
“目标多肽”是能够在酵母中表达的多肽，包括但不限于酶、抗体、干扰素、胰岛素、白介素等及其变体、前体、中间体，例如可以是胰岛素前体。
“细胞”、“宿主细胞”可互换使用。
“多核苷酸分子”、“核酸分子”可互换使用，其序列可以是DNA序列。
具体实施方式
以下结合实施例用于进一步描述本发明，但并非限制本发明的范围。
本发明实施例中所用的载体、宿主菌和培养基均购自Invitrogen公司，其中毕赤酵母表达载体pPIC9K含有醇氧化酶AOX1启动子，可受甲醇诱导，该载体还含有α-因子信号肽序列，能够分泌表达外源蛋白；毕赤酵母表达载体pPIC3.5K含有醇氧化酶AOX1启动子，可受甲醇诱导，该载体不含有α-因子信号肽序列；宿主菌为毕赤酵母GS115菌株；培养基为毕赤酵母手册提供的培养基配方。
实施例1构建胰岛素前体重组表达载体
将对照1(SEQ ID NO:10)、对照2(SEQ ID NO:11)、IP-S(SEQ ID NO:6)的5′端和3′端都分别加入EcoR I和Not I酶切位点，由南京金斯瑞生物科技公司进行合成，并将合成的核酸分子序列连接到T载体。
Figure PCTCN2019074384-appb-000010
用内切酶EcoR I和Not I将上述带有胰岛素前体核酸分子的T载体和表达载体pPIC9K进行双酶切，之后采用胶回收试剂盒分别回收目标片段和载体片段，用T4连接酶将酶切纯化后的目标片段分别与酶切纯化后的载体pPIC9K进行连接。
将上述连接液分别转化到大肠杆菌TOP10感受态细胞，涂布含氨苄青霉素的抗性平板，培养后，挑取克隆菌提取质粒，双酶切验证，通过验证最终得到三种重组表达载体，分别含有对照1、对照2、IP-S的序列。
实施例2毕赤酵母重组菌表达胰岛素前体
将实施例1中构建的三种重组表达载体分别转化到毕赤酵母GS115中，表达对照1和对照2的重组菌为对照菌株，表达IP-S的重组菌为实验菌株。
将三种重组菌的菌落接种至5mL的YPD培养基中，30℃恒温摇床250rpm振荡培养至OD 600＝10左右(16-18小时)。收集并重悬细胞于50mL BMGY培养基中，30℃恒温摇床250rpm振荡培养过夜，至OD 600为30左右。1500rpm离心5分钟收集细胞，用25mL BMMY培养基重悬。在培养基中加入1/200体积的甲醇(终浓度为0.5％)，30℃恒温摇床250rpm振荡培养96小时，其中每24小时补加1/200体积的甲醇。表达结束后，10,000rpm离心取上清，通过HPLC检测上清液中胰岛素前体的产量，换算为相对于对照菌株胰岛素前体表达量的百分比，胰岛素前体的表达量百分比如表1所示。
表1
菌株 载体 所用信号肽 表达基因 产量百分比(％)
对照菌 pPIC9K α-因子 对照1 100
对照菌 pPIC9K α-因子 对照2 125
实验菌 pPIC9K α-因子 IP-S 225
表1的数据显示，经过优化的胰岛素前体基因表达的胰岛素前体量与两个对照组相比提高到1.8到2.25倍，可见经过优化的胰岛素前体基因有更好的表达效果，其表达的胰岛素前体的产量显著提高。
实施例3构建融合了不同α-因子的胰岛素前体基因的重组表达载体
在IP-S前分别加入α-因子(SEQ ID NO:12)和α-因子-S(SEQ ID NO:1)，融合后的核酸分子序列分别如SEQ ID NO:13和SEQ ID NO:8所示，进行合成，合成后的核酸分子的5′端和3′端分别带有EcoR I和Not I酶切位点，并将合成的核酸分子连接到T载体。
Figure PCTCN2019074384-appb-000011
Figure PCTCN2019074384-appb-000012
将对照1前分别加入α-因子(SEQ ID NO:12)和α-因子-S(SEQ ID NO：1)，融合后的核酸分子序列分别如SEQ ID NO:14和SEQ ID NO:15所示，进行合成，合成后的核酸分子5′端和3′端分别带有EcoR I和Not I酶切位点，并将合成的核酸分子连接到T载体。
Figure PCTCN2019074384-appb-000013
Figure PCTCN2019074384-appb-000014
用内切酶EcoR I和Not I将上述T载体和表达载体pPIC3.5K进行双酶切，之后采用胶回收试剂盒分别回收目标片段和载体片段，用T4连接酶将酶切纯化后的目标片段分别与酶切纯化后的载体pPIC3.5K进行连接。
将上述连接液分别转化到大肠杆菌TOP10感受态细胞，涂布含氨苄青霉素的抗性平板，培养后，挑取克隆菌提取质粒，双酶切验证，通过验证最终得到四种重组表达载体，分别融合了α-因子信号肽和α-因子-S信号肽，表达不同核苷酸序列的胰岛素前体基因。
实施例4毕赤酵母重组菌表达优化前后的胰岛素前体
将实施例3中构建的重组表达载体分别转化到毕赤酵母GS115中。
将重组菌菌落接种至5mL的YPD培养基中，30℃恒温摇床250rpm振荡培养至OD 600＝10左右(16-18小时)。收集并重悬细胞于50mL BMGY培养基中，30℃恒温摇床250rpm振荡培养过夜，至OD 600为30左右。1500rpm离心5分钟收集细胞，用25mL BMMY培养基重悬。在培养基中加入1/200体积的甲醇(终浓度为0.5％)，30℃恒温摇床250rpm振荡培养96小时，其中每24小时补加1/200体积的甲醇。表达结束后，10,000rpm离心取上清，通过HPLC检测上清液中胰岛素前体的产量。
将表达融合α-因子的对照1基因的重组菌作为对照菌，将其他菌株的胰岛素前体产量换算为相对于对照菌株产量的百分比，如表2所示。
表2
菌株 载体 所用信号肽 表达基因 产量百分比(％)
对照菌 pPIC3.5K α-因子 对照1 100
实验菌 pPIC3.5K α-因子-S 对照1 150
实验菌 pPIC3.5K α-因子 IP-S 225
实验菌 pPIC3.5K α-因子-S IP-S 275
表2的数据显示，仅信号肽优化的核酸分子序列表达胰岛素前体的量提高到1.5倍，仅胰岛素前体基因优化的核酸分子序列表达胰岛素前体的量提高到2.25倍，而经信号肽和胰岛素前体基因同时优化的核酸分子序列表达胰岛素前体量提高到2.75倍。综合以上，密码子优化使得胰岛素前体的表达量增加到1.5-2.75倍。

Claims (13)

  1. 核酸分子，其包含具有以下通式的分子或结构：
    5′—(PS) a—(SP) b—(LS) c—GE—(P′S) d—3′，
    其中，
    PS是编码加工位点的核酸分子，a是0或1；
    SP是编码信号肽的核酸分子，b是1；
    LS是编码间隔肽的核酸分子，c是0或1；
    GE是编码目标多肽的核酸分子；
    P′S是编码加工位点的核酸分子，d是0或1；
    所述编码信号肽的核酸分子包含如SEQ ID NO:1所示序列。
  2. 核酸分子，其包含具有以下通式的分子或结构：
    5′—(PS) a—(SP) b—(LS) c—GE—(P′S) d—3′，
    其中，
    PS是编码加工位点的核酸分子，a是0或1；
    SP是编码信号肽的核酸分子，b是0或1；
    LS是编码间隔肽的核酸分子，c是0或1；
    GE是编码人胰岛素类似物前体多肽的核酸分子；
    P′S是编码加工位点的核酸分子，d是0或1；
    所述编码人胰岛素类似物前体多肽的核酸分子包含如SEQ ID NO:3所示序列。
  3. 根据权利要求1所述的核酸分子，其中所述目标多肽是人胰岛素类似物前体，编码所述人胰岛素类似物前体的核酸分子包含编码SEQ ID NO:4所示氨基酸序列的核酸分子，优选的，包含如SEQ ID NO:3所示核酸序列。
  4. 根据权利要求2所述的核酸分子，其中编码所述信号肽的核酸分子包含编码SEQ ID NO:2所示氨基酸序列的核酸分子，优选的，包含如SEQ ID NO：1或SEQ ID NO:12所示核酸序列。
  5. 根据前述权利要求2-4中任一项所述的核酸分子，所述编码人胰岛素类似物前体的核酸分子在相当于SEQ ID NO:3的第88-96位被替换，优选的，被GCCGCTAAG、GCTGCCAAG、GCTGCTAAA或GCCGCCAAG替换。
  6. 根据前述权利要求中任一项所述的核酸分子，所述间隔肽的氨基酸序列 包含EEGEPK(Glu-Glu-Gly-Glu-Pro-Lys)，优选的，编码间隔肽的核酸分子包含如SEQ ID NO:5所示序列。
  7. 根据前述权利要求中任一项所述的核酸分子，其包含如SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15所示序列中的任意一个；或为SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15所示序列中的任意一个。
  8. 根据前述权利要求中任一项所述的核酸分子，所述加工位点是连接酶切位点，优选的，PS是编码EcoR I酶切位点的核酸分子和/或P′S是编码Not I酶切位点的核酸分子。
  9. 载体，其包含如前任一项权利要求所述的核酸分子，优选的，其是真核细胞表达载体或原核细胞表达载体。
  10. 宿主细胞，其含有权利要求1-8任一项所述的核酸分子和/或权利要求9所述的载体，优选的，其为酵母菌；更优选的，其为毕赤酵母。
  11. 制备人胰岛素类似物的方法，包括使用权利要求1-8任一项所述的核酸分子，权利要求9所述的载体，和/或权利要求10所述的宿主细胞。
  12. 根据权利要求11所述的方法，其还包括如下步骤：
    1)表达人胰岛素类似物前体；
    2)酶切步骤1)获得的人胰岛素类似物前体，得到人胰岛素类似物。
  13. 根据权利要求11或12所述的方法，所述的人胰岛素类似物为B30缺失的人胰岛素，和/或进一步经过酰化基团的取代，
    优选的，所述取代是B29位的赖氨酸被取代；
    更优选的，所述取代的产物为赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))des(B30)人胰岛素。
PCT/CN2019/074384 2018-02-09 2019-02-01 一种密码子优化的人胰岛素类似物前体基因和信号肽基因 WO2019154311A1 (zh)

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