WO2004106525A1 - Proteine de fusion pouvant etre exprimee tres efficacement et son procede de production - Google Patents

Proteine de fusion pouvant etre exprimee tres efficacement et son procede de production Download PDF

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Publication number
WO2004106525A1
WO2004106525A1 PCT/CN2003/000426 CN0300426W WO2004106525A1 WO 2004106525 A1 WO2004106525 A1 WO 2004106525A1 CN 0300426 W CN0300426 W CN 0300426W WO 2004106525 A1 WO2004106525 A1 WO 2004106525A1
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Prior art keywords
peptide
fusion protein
protein
amino acids
expression
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PCT/CN2003/000426
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English (en)
French (fr)
Inventor
Yi Lu
Xin Gao
Dafu Cui
Youshang Zhang
Yangbin Huang
Jiuru Sun
Jiang Li
Jian Fei
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Shanghai Centre Of Research & Development Of New Drugs
Shanghai Newsummit Biopharma Co.,Ltd
Shanghai Yizhong Biotechnology Co., Ltd
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Application filed by Shanghai Centre Of Research & Development Of New Drugs, Shanghai Newsummit Biopharma Co.,Ltd, Shanghai Yizhong Biotechnology Co., Ltd filed Critical Shanghai Centre Of Research & Development Of New Drugs
Priority to CNB038017652A priority Critical patent/CN1298742C/zh
Priority to US10/559,059 priority patent/US7795384B2/en
Priority to AU2003244053A priority patent/AU2003244053A1/en
Priority to PCT/CN2003/000426 priority patent/WO2004106525A1/zh
Publication of WO2004106525A1 publication Critical patent/WO2004106525A1/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
    • 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
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • Fusion protein suitable for efficient expression and production method thereof
  • the invention relates to the field of genetic engineering. More specifically, the present invention relates to a fusion protein suitable for efficient expression and a method for producing the same.
  • the fusion protein is particularly suitable for efficient production of short peptides containing no lysine. Background technique
  • Pichia and E. coli expression systems are suitable for the expression of molecular weights of about 5 to 200KD.
  • molecular weights of about 5 to 200KD.
  • short peptides with a molecular weight of 1 to 5KD either they cannot be expressed or they can be expressed, but usually there is a problem of low expression.
  • the molecular weight of common molecular weight standards is 14 to 65KD.
  • the molecular weight of the short peptide is outside the detection range (the concentration of the SDS-PAGE electrophoresis gel is up to about 20%, and the linear range of the molecular weight detected at this concentration is 10 to 40KD), so it is easy to run out of the gel and be lost, and it is not accurate Test results.
  • diabetic nephropathy is one of the common chronic complications of diabetic patients and has become a leading cause of end-stage renal disease (ESRD). About 35% of people with type 1 diabetes and 15% of people with type 2 diabetes will eventually develop diabetic nephropathy.
  • Proinsulin C peptide is a 31-amino acid peptide secreted by islet P cells. Studies have shown that proinsulin C peptide has the effect of improving long-term complications of diabetes, including diabetic nephropathy. In addition, studies have shown that C peptide alone or in combination with insulin can reduce the accumulation of glomerular extracellular matrix leading to mesangial expansion.
  • C-peptide is its main mechanism for the treatment of diabetic microangiopathy, including diabetic nephropathy. Therefore, in the case of insulin moderately controlling glucose metabolism, combined with c-peptide therapy can significantly improve the occurrence and development of diabetic nephropathy. In view of the large number of patients with diabetes, there is a great demand for short peptide drugs such as C peptide.
  • Multiple copies are an effective means to increase the expression level of short peptides. Constructing multiple copies of genes can express large molecular weight fusion proteins, achieving high-efficiency expression and easy detection. ,
  • a common method is to fuse multiple copies of short peptides with larger molecular weight peptides (about 10-20KD), so that the expressed fusion protein can stably exist in the host cell.
  • the disadvantage of this method is that the required short peptides account for a small proportion of the fusion protein, so the efficiency is not high enough.
  • the fusion protein is further digested and purified during purification, and the process is more complicated.
  • Another ideal method is to add shorter upstream and downstream sequences upstream and downstream of multiple copies of the short peptide, which can significantly increase the proportion of short peptides in the fusion protein.
  • most of the fusion proteins currently constructed by this method are unstable and it is difficult to obtain a large number of expression products.
  • the object of the present invention is to provide a method for efficiently and stably expressing multi-copy short peptides.
  • Another object of the present invention is to provide related fusion proteins, vectors and host cells.
  • a fusion protein is provided, and its structural formula is:
  • C1 and C2 are each independently a short peptide of 20-40 amino acids in length and containing no Lys in the sequence;
  • B is AGSK (SEQ ID NO: 5);
  • D is a downstream peptide of 3 to 15 amino acids in length and the first 3 amino acids are AGS; n is an integer of 3 to 30.
  • C1 and C2 are the same, and more preferably C1 and C2 are both proinsulin C peptide.
  • the C1 and C2 are different, and the lengths of the two are 8-10 amino acids different.
  • the C1 and C2 are selected from the group consisting of: proinsulin C peptide, ⁇ -human atrial natriuretic peptide, and pre-HB1 S1 antigen peptide.
  • the fusion protein has an amino acid sequence shown in SEQ ID NO: 2 or 3.
  • a DNA molecule is provided, which encodes the above-mentioned fusion protein of the present invention.
  • an expression vector is provided, which contains the aforementioned DNA molecule of the present invention.
  • a host cell is provided, which contains the expression vector of the present invention, or integrates the DNA molecule of the present invention into its genome.
  • the host cell is Pichia or E. coli.
  • a method for producing a fusion protein comprising the steps of-( a ) culturing the host cell according to the present invention so as to express the fusion protein;
  • a method for producing a short peptide including the steps ⁇
  • the C1 and C2 are the same and both are proinsulin C peptide.
  • FIG. 1 Construction of Pichia pastoris expressing human proinsulin c-peptide fusion protein.
  • FIG. 1 Electrophoresis of Pichia pastoris expressing human proinsulin C peptide fusion protein SDS-PAG.E.
  • Lane a molecular weight standards (96KD, 66KD, 43KD, 31KD, 20.1KD, 14.4KD) from top to bottom;
  • b blank control;
  • c culture supernatant (C peptide fusion protein).
  • FIG. 3 SDS-PAGE electrophoresis of E. coli expressing human proinsulin C peptide fusion protein (shake flask identification). Lanes a, b, c, and d respectively indicate that the induction time is 0 hours, 1 hour, 2 hours, and 3 hours (wherein the target protein content is: 0%, 11. 47%, 11. 88%, 12. 36%) ; E: molecular weight standard (from top to bottom molecular weight of 96KD, 66KD, 43KD, 31KD, 20. 1KD, 14. 4KD);
  • FIG. 4 SDS-PAGE electrophoresis of human proinsulin C peptide fusion protein fermented by Pichia pastoris.
  • Lane a molecular weight standards (96KD, 66KD, 43KD, 31KD, 20. KD 14.4KD from top to bottom);
  • b Before induction;
  • c-g induction 8, 16, 24, 32, 48hr.
  • FIG. 5 SDS-PAGE electrophoresis diagram of E. coli fermentation expressing human proinsulin C peptide fusion protein. Lanes a, b, c, and d respectively indicate that the induction time is 0 hours, 1 hour, 2 hours, and 3 hours (wherein the target protein content is: 0%, 24. 87%, 27. 89%, 30. 33%) ; E: molecular weight standard (96KD, 66KD, 43KD, 31KD, 20. 1KD, 14. KD) from top to bottom 0
  • Figure 6 shows the results of HPLC analysis of C peptide after digestion.
  • Figure 7 shows the results of HPLC analysis of the purified C peptide.
  • the inventors found that the structure of 1-1 ⁇ (8- € 2-13 ⁇ 411-0) formed by adding the upstream peptide MHHHHHHRSK (SEQ ID 1 ⁇ 0: 4) upstream of the multi-copy short peptide
  • the fusion protein can be efficiently expressed in Pichia or E. coli. Based on this, the present invention has been completed.
  • the fusion protein of the present invention is in a non-natural form.
  • the elements of the entire fusion protein are linearly connected and there is generally no disulfide bond in the structure. Therefore, the fusion protein has a linear structure, no space folding, and is easily digested by various proteins.
  • the element that plays a key role in stable and efficient expression is the upstream peptide A, namely MHHHHHHRSK (SEQ ID NO: 4). It enables the fusion protein containing multiple copy elements Cl-K- (B-C2-K) n not only to be expressed, but also to be stably present in the host cell. In addition, due to the short upstream elements, the proportion of the protein of interest in the fusion protein is high. (ii) Short peptides
  • C1 and C2 are each independently a short peptide having a length of 10 to 40 amino acids. Since the fusion protein is cleaved into a short peptide, an enzyme (such as trypsin, carboxypeptidase B, etc.) that targets basic amino acids such as lysine (Lys) is used. Therefore, the sequences of C1 and C2 must not contain basic amino acids such as Lys and Arg. Suitable examples include proinsulin C peptide (31aa), ⁇ -human atrial natriuretic peptide (28aa), hepatitis B pre-SI antigen peptide (28aa), and the like.
  • C1 and C2 may be the same or different. When the two are the same, the fusion protein is cleaved to form the desired short peptide of 20 to 40 amino acids in length, as well as a linker, upstream peptide, and downstream peptide of 4 to 15 amino acids in length. Due to the different lengths, short peptides can be easily purified.
  • a preferred case is that both C1 and C2 are proinsulin C peptides.
  • the length of the two should differ by 8-10 amino acids in order to separate C1 and C2 after cutting.
  • C1 and C2 are two kinds of short peptides that can be used in combination, then it is not necessary to separate the two. At this time, C1 and C2 may have no length difference.
  • n is an integer of 3-30, preferably, n is an integer of 5-25, and more preferably an integer of 8-15.
  • the molecular weight of the fusion protein is about 6-60KD, which is suitable for Pichia and E. coli expression systems.
  • the upper limit of n can exceed 20, such as 25, 30, 35, 40 or even higher.
  • B is a linker.
  • the length of the linker is usually 4-10 amino acids. The shorter the length, the higher the proportion of the short peptide in the fusion protein, and the larger the difference between the length of the short peptide and the linker is to facilitate separation, so the short linker is more suitable for the present invention.
  • the carboxy terminus of the linker should be Lys or Arg.
  • a preferred linker is AGSK (SEQ ID NO: 5).
  • D is a downstream peptide, and its role is to provide the cleavage site of carboxypeptidase with Lys before the downstream peptide.
  • the coding sequence of a downstream peptide can also provide restriction sites for cloning operations.
  • Downstream peptides are optional.
  • D should be 3-15 amino acids in length, and the first 3 amino acids are downstream peptides of AGS.
  • Suitable examples include (but are not limited to): AGSLNSLGRPRINS (SEQ ID NO: 6), AGSLNSP (SEQ ID NO: 7) Construction of fusion genes, vectors and host cells
  • the construction of the fusion gene, the vector, and the host cell can be performed according to a conventional method.
  • Vectors and host cells suitable for use in the present invention are not particularly limited.
  • the coding DNA is first designed based on the amino acid sequence of the fusion protein of the present invention.
  • Codon optimization is performed based on the host cell selected, such as Pichia or E. coli.
  • the designed fusion protein coding sequence is fully synthesized, digested and cloned into an expression vector.
  • the expression vector is transfected into Pichia pastoris or Escherichia coli using conventional methods, and after high-resistance screening, the host cells with high expression can be obtained. Production and purification of fusion proteins
  • the transformed host cells expressing the fusion protein can be cultured with conventional methods and conditions, thereby expressing the fusion protein, and used conventional methods (such as salting out method, centrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, HPLC, etc. ) For separation and purification.
  • conventional methods such as salting out method, centrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, HPLC, etc.
  • suitable media include (but are not limited to) the following media:
  • a preferred shake flask culture condition is: picking a single clone on a YPD plate, using BMGY as a shake flask culture medium, and culturing to OD 6 . . 2-20, the expression was induced by adding 1% methanol. After 24 hours of induction, the concentration of the target protein in the culture supernatant could reach 50-200 mg / L.
  • pilot-scale and large-scale production the pilot-scale expression and expression conditions of P. Pastoris engineered cells need to be optimized.
  • Pilot fermentation refers to fermenter fermentation.
  • the preferred production conditions are as follows:
  • a low-salt medium can be selected during fermentation in the fermentor, and certain changes can be made based on the low-salt medium, but the ionic composition is similar to the low-salt medium.
  • the fermentation and induction temperature is maintained at 25-31 ° C.
  • the pH during the induction period is controlled at 4-8, preferably pH 5-7.
  • DO dissolved oxygen
  • the type of feeding should include glycogen such as glycerin, methanol, and glucose, which can be fed separately or mixed.
  • the amount of PTMi in the initial culture stage is 1-4ml / L culture solution, and the amount of 2-20ml / L feed is added in the feed stage.
  • the methanol concentration in the induction period is concerned, conventional induction concentrations can be used in the present invention, and the methanol concentration is usually controlled at 0.5-2%.
  • the fusion protein of the present invention is an excellent raw material for preparing short peptide monomers. After enzymatic digestion and separation, To short peptide monomers.
  • the enzymes used are enzymes that target alkaline amino acids, such as trypsin, carboxypeptidase B, and the like.
  • the method of enzymatic digestion can be a two-step method, that is, first digesting to form a short peptide with K (such as C1K or C2K), and then cutting off K to obtain a short peptide monomer. It is also possible to use a single-step method, that is, double-enzyme digestion, to directly obtain short peptide monomers.
  • the conditions for the enzyme digestion are not particularly limited, and conventional conditions can be used. E.g.
  • the fusion protein concentration is 0.1-100m g / ml, preferably l-50mg / ml;
  • the ratio of enzyme to protein is 1: 10-1: 5000, preferably 1: 50-1: 500;
  • the digestion temperature is 15-35 ° C, preferably 20-30 ° C;
  • Digestion ⁇ is 5.0-10.0, preferably 6.0-9.0;
  • the buffer system is phosphate buffer, acetate buffer, Tris, or carbonate buffer, and the buffer concentration is 10-200mM;
  • the digestion time is 1-50 hours, preferably 1-10 hours.
  • conventional techniques such as cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, reversed-phase chromatography, affinity chromatography, etc.
  • the preferred method is to subject the digested sample to ion exchange chromatography, hydrophobic chromatography, and reversed phase chromatography.
  • the prepared short peptide monomers can be made into corresponding preparations by conventional methods.
  • an appropriate auxiliary material such as 1-10% mannitol (or sucrose, lactose, etc.) stabilizer can be added to the purified stock solution, and at the same time, surfactants, antioxidants and other protection can be added.
  • the main advantages of the present invention are-i.
  • the expression level is high and stable;
  • the preparation method is simple and easy to purify, so the cost is low;
  • Example 1 Construction method of Pichia pastoris expressing human proinsulin C peptide fusion protein-engineered cells
  • the full-length amino acid sequence of the fusion protein is:
  • the gene sequence of the fusion protein (EcoRI sites are added at both ends) is shown in SEQ ID NO: 1, where 0RF is the 25th -Position 1056, and immediately downstream of the sequence is a stop codon.
  • This gene was cloned into PUC19 for sequencing and verification, and then digested with EcoRI, and cloned into a plasmid pPIC9 (Invitrogen) to be also digested with EcoRI. Then, the chromosomes of P. Pastoris GS1 15 (Invitrogen) were transformed and integrated, and multi-copy integrated transformed cells were selected by dot hybridization, and then engineered cells were selected ( Figure 1).
  • the engineered cells can be either the methanol-type 0Vhit + ;) or the methanol-type (Muf;).
  • the second fusion protein was constructed in the same manner as in Example 1, except that the downstream peptide D was different. .
  • Example 3 Construction method of Pichia pastoris expressing human proinsulin C peptide fusion protein-In this example, several schemes for comparing Pichia pastoris expressing human proinsulin C peptide fusion protein were compared.
  • Scheme 1 is Example 1.
  • the structure of the fusion protein in Scheme 2 is: (His) 6 -Lys- C peptide- Lys- C peptide- Lys.
  • the recipient bacteria is E. coli BL21 (DE3).
  • Example 1 The fusion protein gene sequence in Example 1 was verified to be free of codons that are particularly rare in E. coli.
  • the gene was cloned into PUC19 for sequencing and verification, and then cloned into the expression vector pET-30a (+). After sequencing confirmed that there was no change in the expression frame, it was transformed into E. coli BL21 (DE3) to obtain engineered bacteria.
  • the confirmed engineering strain was inoculated in 10 mL of LB (kanamycin concentration of 100 g / mL) culture medium.
  • the Pichia engineered cells obtained in Example 1 were fermented by the following method:
  • Fermentation medium Low-salt fermentation medium
  • methanol is added for induction.
  • the methanol concentration is controlled at 0.5-1% during the entire induction period, and the induction time is 20-50 hours.
  • Example 4 The engineered E. coli cells obtained in Example 4 were fermented by the following method:
  • Seed solution LB medium
  • Fermentation medium M9 medium (3.5L)
  • IPTG IPTG was added at one time for induction.
  • the final IPTG concentration was 1 mM.
  • the whole induction process was controlled by feeding glucose in a pH range of 6.9 to 7.2, and the induction time was 3 hours.
  • Enzymes used TPCK-trypsin (Sigma), carboxypeptidase B (Sigma)
  • the digested product obtained in Example 7 is purified, and the specific conditions are as follows:
  • Buffer A 10mM PB Buffer B: 10mMPB + 1M NaCl
  • the enzyme digested product obtained in Example 7 was subjected to ultrafiltration or dialysis to desalinate and remove a small amount of impurities. Then, load this sample. After loading, rinse with buffer A until the UV baseline is stable, and then use a linear gradient to reduce 8% from 0-100 using 20CV. Samples appeared at around 20% B concentration and collected in separate tubes. The sample recovery rate is over 60% and the purity is over 95%.
  • Example 8 To the purified stock solution obtained in Example 8, 10% mannitol was added, divided, lyophilized, and dried to prepare a C-peptide powder injection. The stability test showed that the powder injection was stable. The activity measurement shows that the activity of the human proinsulin C peptide prepared by the method of the present invention is the same as that of the standard. Industrial applicability
  • the expression of the fusion protein of the present invention by an expression system such as Pichia pastoris has the advantages of high expression and high stability, and the prepared fusion protein can be short peptides such as C peptide after simple digestion and separation monomer.
  • the method of the invention greatly reduces the complexity of the process and greatly improves the yield, and is particularly suitable for large-scale production of short peptides such as C peptide.

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Description

一种适合于高效表达的融合蛋白及其生产方法 技术领域
本发明涉及了基因工程领域。 更具体地， 本发明涉及一种适合于高效表达 的融合蛋白及其生产方法。 该融合蛋白特别适合高效生产不含赖氨酸的短肽。 背景技术
毕赤酵母和大肠杆菌这两种表达系统的适合表达的产物的分子量均在 5到 200KD左右。 对于分子量在 1到 5KD的短多肽， 或者不能表达， 或者虽能表达， 但通常情况下都存在表达量偏低的问题。
此外， 低分子量的短肽表达产物还存在不易进行检测的问题。 对于常规的
SDS-PAGE电泳来说， 常用分子量标准的分子量就在 14到 65KD。 短肽的分子量 在检测范围之外 (SDS-PAGE电泳凝胶的浓度最高为 20%左右， 此浓度下检测的 分子量线性范围在 10到 40KD), 因此容易跑出凝胶而流失， 无法得到准确的检 验结果。
许多常见病可用短肽进行治疗。例如， 糖尿病肾病 (DN)是糖尿病患者常见 的慢性并发症之一， 并已成为终末期肾病 (ESRD)的主要原因。 约 35%的 1型糖 尿病患者和 15%的 2型糖尿病患者最终将发展为糖尿病肾病。 胰岛素原 C肽是胰 岛 P细胞分泌的由 31个氨基酸组成的多肽。 研究已表明， 胰岛素原 C肽具有改 善糖尿病远期并发症 (包括糖尿病肾病)的作用。此外， 研究还表明， C肽单独应 用或与胰岛素联合应用， 可以减少肾小球细胞外基质积聚导致系膜扩张。 C肽 的这一作用是其治疗糖尿病微血管病变包括糖尿病肾病的主要机制。 因此在胰 岛素中等控制糖代谢情况下， 联合 c肽治疗可以更明显改善糖尿病肾病的发生 和发展。 鉴于糖尿病患者人数众多， 因此 C肽等短肽药物的需求是很大。
目前国际市场上短肽类药物多为化学合成， 价格昂贵。 因此， 迫切需要工 艺简单、 成本低廉的大规模生产短肽的基因工程方法。
多拷贝是一种提高短肽表达水平的有效手段。 构建多拷贝基因可以表达较 大分子量的融合蛋白， 达到高效表达和易检测的目的。 ，
对于高效表达的多拷贝融合蛋白或其编码基因， 必须处理好以下问题： （a> 目的基因 (或目的蛋白）的选择； （b).长度 (即拷贝数)； （c).连接接头， 所述自勺连接 包括融合蛋白与表达质粒的连接、目的蛋白单体之间的连接、信号肽选择等；（d). 表达后的酶切等。不恰当的选择都会影响表达水平，甚至使融合蛋白不能表达。
一种常见的方法是将多拷贝的短肽与分子量较大的肽 (约 10-20KD)融合， 从而使表达的融合蛋白能稳定地在宿主细胞中存在。 这种方法的缺点是， 所需 的短肽在融合蛋白中所占比例较小， 因此效率不够高。 此外， 与 GST等形成的 融合蛋白在纯化时多一步酶切和纯化， 工艺较复杂。
另一种理想的方法是将在多拷贝的短肽的上游和下游添加长度较短的上 游序列和下游序列， 这样可以显著提高短肽在融合蛋白中所占比例。 但是目前 用这种方法的所构建的融合蛋白大多不稳定， 难以获得大量的表达产物。
因此， 本领域迫切需要开发一种高效、 稳定表达多拷贝短肽的方法。 发明内容
本发明的目的就是提供一种高效、 稳定表达多拷贝短肽的方法。
本发明的另一目的是提供有关的融合蛋白、 载体和宿主细胞。 在本发明的第一方面， 提供了一种融合蛋白， 其结构式是：
A-Cl-K- (B-C2-K) n-D 其中 A为丽匪 HHRSK (SEQ ID NO： 4)，
C1和 C2各自独立地是长度为 20-40氨基酸且序列中不含 Lys的短肽；
K是 Lys;
B是 AGSK (SEQ ID NO: 5)；
D是长度为 3- 15个氨基酸且前 3个氨基酸为 AGS的下游肽； n为 3- 30的整数。 在另一优选例中， 所述的 C1和 C2是相同的， 更佳地 C1和 C2都是胰岛素原 C肽。 在另一优选例中， 所述的 C1和 C2是不同的， 且两者的长度相差 8-10个氨基酸。 在另一优选例中， 所述的 C1和 C2选自： 胰岛素原 C肽、 α-人心房钠尿肽、 乙肝 前 S1抗原肽。
在另一优选例中， 所述融合蛋白具有 SEQ ID N0: 2或 3所示的氨基酸序列。 在本发明第二方面， 提供了一种 DNA分子， 它编码本发明上述的融合蛋白。 在本发明第三方面， 提供了一种表达载体， 它含有本发明上述的 DNA分子。 在本发明的第四方面， 提供了一种宿主细胞， 它含有本发明所述的表达载 体， 或者在其基因组中整合有本发明所述的 DNA分子。 较佳地， 所述的宿主细 胞是毕赤酵母或大肠杆菌。
在本发明的第五方面， 提供了一种融合蛋白的生产方法， 包括步骤- (a)培养本发明所述的宿主细胞， 从而表达所述的融合蛋白；
(b)分离融合蛋白。
在本发明的第六方面， 提供了一种生产短肽的方法， 包括步骤-
(c)用胰蛋白酶和羧肽酶 B酶切本发明所述的融合蛋白， 产生短肽 C1和 C2;
(d)分离短肽 C1和 C2。
较佳地， 在所述的方法， 所述的 C1和 C2是相同的， 都是胰岛素原 C肽。 附图说明
图 1.毕赤酵母表达人胰岛素原 c肽融合蛋白质粒构建图。
图 2.毕赤酵母表达人胰岛素原 C肽融合蛋白 SDS-PAG.E电泳图。 泳道 a: 分子 量标准（从上到下分子量为 96KD、 66KD、 43KD、 31KD、 20. 1KD、 14. 4KD)； b : 空白对照； c : 培液上清（C肽融合蛋白）。
图 3.大肠杆菌表达人胰岛素原 C肽融合蛋白 SDS-PAGE电泳图（摇瓶鉴定）。 泳道 a、 b、 c、 d分别表示诱导时间为 0小时、 1小时、 2 小时和 3小时（其中目的 蛋白含量分别是： 0%， 11. 47%, 11. 88%, 12. 36%)； e: 分子量标准（从上到下分子 量为 96KD、 66KD、 43KD、 31KD、 20. 1KD、 14. 4KD)；
图 4. 毕赤酵母发酵表达人胰岛素原 C肽融合蛋白 SDS-PAGE电泳图。 泳道 a: 分子量标准（从上到下分子量为 96KD、 66KD、 43KD、 31KD、 20.亂 14. 4KD)； b.诱导前； c-g: 诱导 8、 16、 24、 32、 48hr。
图 5.大肠杆菌发酵表达人胰岛素原 C肽融合蛋白 SDS-PAGE电泳图。 泳道 a、 b、 c、 d分别表示诱导时间为 0小时、 1小时、 2 小时和 3小时（其中目的蛋白含 量分别是： 0%， 24. 87%, 27. 89%, 30. 33%)； e : 分子量标准（从上到下分子量为 96KD、 66KD、 43KD、 31KD、 20. 1KD、 14. KD) 0
图 6显示了酶切后 C肽的 HPLC分析结果。
图 7显示了纯化后 C肽的 HPLC分析结果。 具体实施方式
本发明人经过广泛而深入的研究发现， 在多拷贝短肽上游添加上游肽 MHHHHHHRSK (SEQ ID 1^0 : 4)所形成的 - 1-1^(8-€2-1¾11-0的结构结合了多种优 势， 可以使融合蛋白在毕赤酵母或大肠杆菌中高效表达。 在此基础上完成了本 发明。 融合蛋白的结构
本发明融合蛋白为非天然的形式， 整个融合蛋白中各元件线性连接且结构 上一般不存在二硫键， 因此融合蛋白呈线形结构， 没有空间折叠， 易被各种蛋 白酶酶切。
(i)上游肽
在本发明通式为 A-Cl-K-(B-C2-K)n-D的融合蛋白中， 对稳定高效表达起关 键作用的元件是上游肽 A， 即 MHHHHHHRSK (SEQ ID NO : 4)。 它使得含多拷贝元件 Cl-K-(B-C2-K)n的融合蛋白不仅能够表达， 而且能够稳定存在于宿主细胞中。 此外， 由于上游元件的很短， 因此融合蛋白中目的蛋白的比例很高。 (ii)短肽
在本发明融合蛋白中， C1和 C2各自独立地是长度为 10-40氨基酸的短肽。 由 于在将融合蛋白切割为短肽时，使用针对碱性氨基酸如赖氨酸 (Lys)的酶 (如胰蛋白 酶、羧肽酶 B等）， 因此 C1和 C2的序列中不可含有碱性氨基酸如 Lys和 Arg。合适的例 子包括胰岛素原 C肽 (31aa)、 α-人心房钠尿肽 (28aa)、 乙肝前 SI抗原肽 (28aa)等。
C1和 C2可相同， 也可不同。 当两者相同时， 融合蛋白被切割后就形成长度为 20 - 40氨基酸的所需的短肽， 以及长度为 4- 15氨基酸的接头、 上游肽和下游肽。 由 于长度不同， 因此可以方便地纯化出短肽。 一种优选情况是 C1和 C2都是胰岛素原 C 肽。
当 C1和 C2不同时， 两者的长度宜相差 8- 10个氨基酸， 以便在切割后将 C1和 C2 分离开。当然，如果 C1和 C2是可以联用的两种短肽，那么就不一定需要将两者分开， 此时 C1和 C2可以没有长度差异。
在本发明融合蛋白中， n为 3-30的整数， 较佳地， n为 5-25的整数， 更佳地 为 8-15的整数。 此时， 融合蛋白的分子量约为 6-60KD, 适合毕赤酵母和大肠杆 菌表达系统。 当然， 对于其他适合表达系统， n的上限可以超过 20， 例如 25， 30, 35， 40甚至更高。
(iii)接头 '
在本发明融合蛋白中， B为接头。 接头的长度通常为 4-10个氨基酸。 长度 越短， 则短肽在融合蛋白中的比例越高， 而且短肽与接头的长度差越大从而更 有利于分离， 因此短接头更适用于本发明。此外，接头的羧基端应为 Lys或 Arg。 一种优选的接头是 AGSK (SEQ ID N0: 5)。 (iv)下游肽
在本发明融合蛋白中， D是下游肽， 其作用是与下游肽之前的 Lys—起提供羧 肽酶的酶切位点。 有时， 下游肽的编码序列还可提供用于克隆操作的限制性位点。
下游肽是可有可无的。 当8为4631( (3£0 ]1^0 : 5)时， D宜为长度 3- 15个氨基酸， 且前 3个氨基酸为 AGS的下游肽。 合适的例子包括（但并不限于） ： AGSLNSLGRPRINS (SEQ ID NO： 6)、 AGSLNSP (SEQ ID NO： 7)。 融合基因、 载体和宿主细胞的构建
在本发明中， 一旦确定了融合蛋白的序列， 则融合基因、 载体和宿主细胞 的构建都可按常规方法进行。 适用于本发明的载体和宿主细胞没有特别限制。
通常， 先根据本发明融合蛋白的氨基酸序列设计出编码 DNA。 较佳地， 可 根据选用的宿主细胞如毕赤酵母或大肠杆菌， 进行密码子优化。
然后， 选择表达载体， 如毕赤酵母系统的 pPIC9或 pPIC9K， 或大肠杆菌系 统的 pET30(a)等， 并且根据选定的载体， 设计酶切位点。
接着， 全合成所设计的融合蛋白编码序列， 酶切后克隆入表达载体。
最后， 用常规方法将表达载体转染毕赤酵母或转化大肠杆菌， 经过抗性筛 选等方法， 就可获得高表达的宿主细胞。 融合蛋白的生产和纯化
对于转化的表达融合蛋白的宿主细胞， 可用常规方法和条件进行培养， 从 而表达出融合蛋白，并用常规方法 (如盐析方法、离心、分子筛层析、吸附层析、 离子交换层析、 HPLC等)进行分离纯化。
以毕赤酵母培养与表达为例，合适的培养基包括 (但并不限于)以下培养基：
Figure imgf000006_0001
一种优选的摇瓶培养条件是： 挑取 YPD平板上的单克隆， 以 BMGY为摇瓶 培养液， 培养至 OD6。。2-20， 加入 1%浓度甲醇诱导表达， 诱导 24小时后， 培液 上清的目的蛋白浓度可达到 50-200mg/L。
为了中试和大规模生产， 需要对 P.Pastoris工程细胞的中试表达与表达条件 进行优化。 中试发酵指的是发酵罐发酵。 优选的生产条件如下：
1.对于培养基的选择而言， 发酵罐发酵时可选择低盐培养基， 也可在低盐 培养基基础上进行一定更改， 但所含离子成份与低盐培养基相似。
Figure imgf000007_0001
2. 就温度的控制而言， 发酵及诱导温度保持在 25-31 °C。
3. 就诱导期的 pH值而言， 诱导期 pH控制在 4-8， 较佳地为 pH5-7。
4. 就溶氧 (DO)的控制而言 DO控制在 20-90%。 溶氧的维持可以用氧气 /空 气混合气体的通入来解决。
5. 就补料的流加而言， 补料种类宜包括甘油、 甲醇、 葡萄糖等糖原， 可 单独补料或混合补料。
6. 就 ΡΤΜ (混合微量元素)的量： 起始培养阶段 PTMi的量为 l-4ml/L培液， 在补料阶段加入量为 2-20ml/L补料。
7.就诱导期甲醇浓度而言， 常规诱导浓度都可用于本发明， 通常甲醇浓度 控制在 0.5-2%。
8.就诱导时间而言， 通常为 10-100小时， 较佳地为 20-60小时。 短肽单体的制备
本发明的融合蛋白是制备短肽单体的优良原料。 经过酶切和分离， 就可得 到短肽单体。
通常， 所用的酶是针对碱性氨基酸的酶， 如胰蛋白酶、 羧肽酶 B等。 酶切 的方法可用二步法， 即先酶切形成带 K的短肽 (如 C1K或 C2K), 再将 K切除， 得 到短肽单体。 也可使用单步法， 即用双酶混合酶切， 直接获得短肽单体。
酶切的条件没有特别限制， 可采用常规条件。 例如
融合蛋白浓度是 0.1-100mg/ml， 较佳地 l-50mg/ml;
酶与蛋白比例是 1 : 10-1： 5000, 较佳地， 1： 50-1： 500；
酶切温度是 15-35°C， 较佳地 20-30°C ;
酶切 ρΗ是 5.0-10.0，较佳地 6.0-9.0;
缓冲体系是磷酸盐缓冲液、 醋酸盐缓冲液、 Tris、 或碳酸盐缓冲液， 缓冲 液浓度 10-200mM;
酶切时间是 1-50小时， 较佳地 1-10小时。 对于酶切混合物， 可用常规技术 (如阳离子交换层析、 阴离子交换层析、凝 胶过滤层析、 疏水层析、 反相层析、 亲和层析等)纯化短肽单体。优选方法是将 酶切后的样品进行离子交换层析、 疏水层析和反相层析。
对于制备的短肽单体， 可用常规方法将其制成相应的制剂。 例如当短肽是 胰岛素原 C肽时， 可在纯化后的原液加入适当辅料， 如 1-10%甘露醇 (或蔗糖、 乳糖等)稳定剂，同时还可添加表面活性剂、抗氧化剂等保护剂，及适当缓冲液， 分装， 制成 C肽水溶液剂， 或者冷冻抽干， 制成 C肽的注射用粉针剂。 本发明的主要优点在于- i. 表达量高且稳定；
ii. 短肽 (如人胰岛素原 C肽）的产量高；
iii. 制备方法简单， 易纯化所以成本低；
iv. 目的蛋白若用真核细胞表达系统-毕赤酵母进行表达， 与哺乳动物有 较强同源性， 产生的毒副作用较少。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法，通常按照常规条件，例如 Sambrook等人， 分子克隆：实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂 商所建议的条件。
实施例 1毕赤酵母表达人胰岛素原 C肽融合蛋白-工程细胞的构建方法 在本实施例中， 选择 C1 = C2 =胰岛素原 C肽， n= 8。 1 . 融合蛋白全长氨基酸序列为：
顧 HHHHHRSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPG AGSLQPLALEGSLQKAGSKEAEDLQVGQVEL6GGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVEL GGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQV GQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQV6QVEL6GGPGAGSLQPLALEGSLQKAGSKEA EDLQVGQVEL6GGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKA GSLNSLGRPRINS (SEQ ID NO : 2)
2. 构建
根据 SEQ ID N0 : 2 , 并根据毕赤酵母密码子偏爱性， 全合成该融合蛋白的 基因序列 (两端加上 EcoRI位点）， 如 SEQ ID N0 : 1所示， 其中， 0RF为第 25- 1056 位， 并且在该序列下游立刻是终止密码子。
将该基因克隆到 PUC19中测序验证后，用 EcoRI酶切，克隆入待同样用 EcoRI 酶切的质粒 pPIC9(Invitrogen公司）。 然后， 转化、 整合入毕赤酵母 (P.Pastoris)GS1 15(Invitrogen公司）的染色体，通过点杂交筛选出多拷贝整合的转 化细胞， 进而筛选出工程细胞 (图 1)。 工程细胞可以是快速利用甲醇型 0Vhit+;)或 慢速利用甲醇型 (Muf；)。
3. 表达
经过筛选， 最后得到高效表达株 (经检验， 其基因拷贝整合数 20个）。
BMGY培养液摇瓶试验表明， 诱导 24小时后， 培养上清目的蛋白占总蛋白 50% 以上， 表达水平 lOOug/ml以上 (图 2)。 根据其消耗甲醇的速度， 判断此工程细胞 株为 Mut+。 实施例 2毕赤酵母表达人胰岛素原 C肽融合蛋白-工程细胞的构建方法 在本实施例中， 选择 C1 = C2 =胰岛素原 C肽， n= l l。
该与实施例 1相同的方法构建第二种融合蛋白， 不同点仅在于下游肽 D不 同。 .
MHHHHHHRSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPG AGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVEL GGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQV GQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEA EDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKA GSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEG SLQKAGSKEAEDLQVGQVELGGGPGAGSLQPLALEGSLQKAGSLNSP (SEQ ID NO : 3) 经过筛选，也得到高效表达株 (经检验，其基因拷贝整合数 20个)。 BMGY 培养液摇瓶试验表明， 诱导 24小时后， 培养上清目的蛋白占总蛋白 50%以上， 表达水平 100ug/ml以上。 根据其消耗甲醇的速度， 判断此工程细胞株为 Mut+。 实施例 3毕赤酵母表达人胰岛素原 C肽融合蛋白-工程细胞的构建方法 在本实施例中， 比较了毕赤酵母表达人胰岛素原 C肽融合蛋白的几种方案。 方案 1是实施例 1。
方案 2中融合蛋白的结构是： （His) 6-Lys- C肽- Lys- C肽- Lys。
方案 3中融合蛋白的结构与实施例 1基本相同， 不同点仅在于 n=4。
结果表明， 方案 2表达目的产物得到的量很低， 难以检测出来。 这表明，
6His的上游肽以及连接肽的设计形成一个有利于高表达和分泌的构象。 方案 3 - 得到了所需的表达蛋白。 · 实施例 4.大肠杆菌表达人胰岛素原 C肽融合蛋白-工程细胞的构建方法 选择合适的表达载体和宿主菌系统： 表达载体质粒 pET-30a(+)来自
Novagen公司， 受体菌为大肠杆菌 BL21 (DE3)。
实施例 1中的融合蛋白基因序列经查证没有大肠杆菌特别稀有的密码子， 将该基因克隆到 PUC19中测序验证后， 克隆入表达载体 pET-30a(+)中。测序确认 没有表达框改变后， 转化入大肠杆菌 BL21 (DE3)， 获得工程菌。
接种确证的工程菌株于 10mL LB (卡那霉素浓度为 lOO g/mL)培养液中， 在
37Ό , 250rpm条件下培养至 0D麵 =0. 5- 1. 0， 此时细胞处于对数生长期； 无菌条 件下加入 IPTG至终浓度为 lmM， 继续在 37°C， 250rpm条件下诱导表达， 3小时后 取样， SDS - PAGE检测， 在 41KD处有目的蛋白表达（图 3) 。 实施例 5.毕赤酵母表达人胰岛素原 C肽融合蛋白的中试发酵
实施例 1所得的毕赤酵母工程细胞用以下方法发酵：
NBS BioFlo 3000发酵罐（5L) :
1. 种子液： BMGY培养基
2. 发酵培养基： 低盐发酵培养基
3. 发酵培养阶段：
温度： 30 °C
H: 5. 0
DO : ^30%
甲醇诱导前湿细胞重： 88g/L
4. 发酵诱导阶段- 温度： 30 °C
pH: 6. 5
DO : ^20%
甲醇补料中？ ！^量 1 Oml/L
甲醇诱导结束湿细胞重： 257g/L
在培养阶段后期， 甘油被耗尽后流加甲醇进行诱导， 整个诱导过程甲醇浓 度控制在 0. 5- 1%，诱导时间 20- 50 小时。
诱导 48小时后， 人胰岛素原 C肽融合蛋白表达量占发酵上清液总蛋白 47%， 表达量达 lmg/ml (用改良 Lowry法测）（图 4) 。 实施例 6大肠杆菌表达人胰岛素原 C肽融合蛋白的中试发酵
实施例 4所得的大肠杆菌工程细胞用以下方法发酵：
NBS BioFlo 3000发酵罐（5L) :
1.种子液： LB培养基
2. 发酵培养基： M9培养基（3. 5L)
3. 发酵培养阶段- 温度： 37 V
pH: 7. 0
DO : 80%
诱导前 0D6Q。： 1. 52
4. 发酵诱导阶段：
温度： 35 V
pH: 6. 5
DO : 50%
葡萄糖补料中微量元素母液（PTM)的量： 10ml/L
IPTG诱导结束 0D謹： 5. 72
在培养阶段后期， 0D漏上升到 1. 5左右， 一次性加入 IPTG进行诱导。 IPTG 终浓度为 lmM。 整个诱导过程通过流加葡萄糖补料来控制在 pH在 6. 9到 7. 2范围 内，诱导时间 3 小时。
· 诱导 3小时后， 离心， 得到 25. 7克菌体。 人胰岛素原 C肽融合蛋白表达量占 发酵菌体总蛋白 35%， 蛋白含量为 1. 02mg/ml (用改良 Lowry法测）。 目的蛋白总 量为 1. 25克， 表达量达 0. 357g/L发酵液（图 5)。 实施例 7.毕赤酵母表达人胰岛素原 C肽融合蛋白一酶切
实施例 5所得的毕赤酵母工程细胞表达产物上清经初步纯化， 进行单步法 酶切， 酶切条件如下：
所用的酶： TPCK-胰蛋白酶 (Sigma)、 羧肽酶 B(Sigma公司）
缓冲体系 10mM PB(pH7.4)
C肽融合蛋白与 TPCK-胰蛋白酶比例 200: 1(W/W)
C肽融合蛋白与羧肽酶 B 比例 200: 1(W/W)
酶切温度： 25 Ό
酶切时间： 6小时。 酶切得到的 C肽经 HPLC分析， HPLC鉴定条件如下：
C18分析柱 Φ 4. 6*250mm
λ ： 230nm
缓冲液 A: 0. 1%TFA 缓冲液 B: 0. 08%TFA 70%乙腈
0-5min 0% 缓冲液 B
5-10min 30% 缓冲液 B
10-30min 60% 缓冲液 B
30-3 lmin 100% 缓冲液 B 结果如图 6所示， 酶切的主要产物的保留时间为 27± lmin， 与 C肽单体的保 留时间相符， 这表明酶切后产生了 C肽单体。 实施例 8.酶切产物纯化
实施例 7所得的酶切产物经过纯化， 具体条件如下：
层析介质： Q-Sepharose FF (Pharmacia)
缓冲液 A: 10mM PB 缓冲液 B: lOmMPB + 1M NaCl
梯度： 0-100% 缓冲液 B 20柱体积
流速： 60cm/h
将实施例 7所得的酶切产物超滤或是透析以脱盐和去少部分杂质。 然后， 将此样品上样，上样完毕后用缓冲液 A冲洗至 UV基线平稳，而后使用线形梯度， 用 20CV使 8%从0- 100。 在 20%B浓度左右出现样品， 分管收集。 样品回收率达到 60%以上， 纯度达到 95%以上。
由于此样品盐浓度较高， 将收集的样品使用 G- 50 Sephadex脱盐， 使用缓 冲液 A为缓冲液， 样品回收率在 90%以上， HPLC纯度在 98%以上（图 7)。
样品经鉴定， 其序列与天然人胰岛素原 C肽完全相同， 证实获得的是人胰 岛素原 C肽。 实施例 9 纯化产物的制剂
对实施例 8中获得的纯化后原液加入 10%甘露醇， 分装， 冷冻抽干， 制成 C 肽的注射用粉针剂。 经稳定性试验表明， 该粉针剂稳定。 经活性测定表明， 本 发明方法制得的人胰岛素原 C肽的活性与标准品相同。 工业实用性
综上所述， 通过毕赤酵母等表达系统来表达本发明的融合蛋白， 其优点在 于： 高表达、 高稳定， 而且制得的融合蛋白经简单酶切和分离后即可 C肽等短 肽单体。 本发明方法使工艺复杂程度大大降低， 产率得到较大提高， 特别适合 大规模生产 C肽等短肽。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种融合蛋白， 其特征在于， 其结构式是：
A-Cl-K- (B-C2-K) n-D
其中 A为匪 HHHHHRSK (SEQ ID NO : 4)，
CI和 C2各自独立地是长度为 20- 40氨基酸且序列中不含 Lys的短肽；
K是 Lys;
B是 AGSK (SEQ ID NO : 5)；
D是长度为 3-15个氨基酸且前 3个氨基酸为 AGS的下游肽；
n为 3- 30的整数。
2. 如权利要求 1所述的蛋白， 其特征在于， 所述的 C1和 C2是相同的。
3. 如权利要求 1所述的蛋白， 其特征在于， 所述的 C1和 C2是不同的， 且两者 的长度相差 8- 10个氨基酸。
4.如权利要求 1所述的蛋白，其特征在于，所述的 C1和 C2选自：胰岛素原 C肽、 α-人心房钠尿肽、 乙肝前 S1抗原肽。
5. 如权利要求 1所述的蛋白， 其特征在于， 具有 SEQ ID N0 : 2或 3所示的氨基 酸序列。
6. —种 DNA分子， 其特征在于， 它编码权利要求 1所述的融合蛋白。
7. 一种表达载体， 其特征在于， 它含有权利要求 6所述的 DNA分子。
8. —种宿主细胞， 其特征在于， 它含有权利要求 7所述的表达载体， 或者 在其基因组中整合有权利要求 6所述的 DNA分子。
9. 如权利要求 8所述的宿主细胞， 其特征在于， 所述的宿主细胞是毕赤酵 母或大肠杆菌。
10. 一种融合蛋白的生产方法， 其特征在于， 包括步骤：
(a)培养权利要求 6所述的宿主细胞， 从而表达所述的融合蛋白；
(b)分离融合蛋白。
11. 一种生产短肽的方法， 其特征在于， 包括步骤：
(c)用胰蛋白酶和羧肽酶 B酶切权利要求 1所述的融合蛋白， 产生短肽 C1和
C2 ;
(d)分离短肽 C1和 C2。
PCT/CN2003/000426 2003-06-03 2003-06-03 Proteine de fusion pouvant etre exprimee tres efficacement et son procede de production WO2004106525A1 (fr)

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US10/559,059 US7795384B2 (en) 2003-06-03 2003-06-03 Fusion protein suitable for high efficiency expression and the production method thereof
AU2003244053A AU2003244053A1 (en) 2003-06-03 2003-06-03 A fusion protein suitable to be expressed high effectively and the production method thereof
PCT/CN2003/000426 WO2004106525A1 (fr) 2003-06-03 2003-06-03 Proteine de fusion pouvant etre exprimee tres efficacement et son procede de production

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WO1993010152A1 (en) * 1991-11-16 1993-05-27 Smithkline Beecham Biologicals S.A. HYBRID PROTEIN BETWEEN CS FROM PLASMODIUM AND HBsAG
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CN1298742C (zh) 2007-02-07
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AU2003244053A1 (en) 2005-01-21
US20080045695A1 (en) 2008-02-21

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