Fusion protein and its preparation method for intermediate polypeptide of Semaglutide

Abstract

The invention discloses a fusion protein and its preparation method for intermediate polypeptide of Semaglutide. The invention belongs to the technical field of genetic engineering and polypeptide preparation. The fusion protein comprises a fusion peptide, a protease cleavage site and a target main molecular sequence. By optimizing the fusion peptide sequence, changing the isoelectric point and hydrophilicity of the protein, the highest expression of the fusion protein is effectively increased to 13.1 g/L. Meanwhile, the properties of fusion protein are also improved, which is conducive to the development of subsequent extraction, enzyme digestion and purification processes. The yield of intermediate polypeptide after enzyme digestion is 3.62 g/L. The production cost of Semaglutide intermediate polypeptide Arg34GLP-1(9-37) is reduced from the source, which is conducive to industrial scale-up and suitable for industrial production.

Claims

1. A fusion peptide which is characterized in that the fusion peptide sequence is set forth as SEQ ID NO: 5.

2. A fusion protein which is characterized in that: the fusion protein comprising the fusion peptide described in claim 1, and the fusion protein consists of fusion peptide-DDDDK-Arg34GLP-1(9-37) or fusion peptide-DDDDK-Arg34GLP-1(11-37); the amino acid sequence of Arg34GLP-1(9-37) is set forth as SEQ ID NO: 1; the amino acid sequence of Arg34GLP-1(11-37) is set forth as SEQ ID NO: 2.

3. A gene encoding the fusion protein of claim 2.

4. A recombinant expression vector which is characterized in that it contains the gene of claim 3.

5. The recombinant expression vector of claim 4, characterized in that: the recombinant expression vector comprises pET family, Duet family, pGEX family, pHY300, pHY300PLK, pPIC3K, pPIC9K, or pTrc family vectors; the pET family comprises pET-24a(+), pET-28a(+), pET-29a(+) and pET-30a(+); the Duet family comprises pRSFDuet-1 and pCDFDuet-1; the pTrc family comprises pTrc99a.

6. Recombinant microbial cells which are characterized in that they express the fusion protein described in claim 2.

7. The recombinant microbial cells of claim 6, which are characterized in that: the host of the microbial cells is Escherichia coli, Bacillus subtilis, or Pichia pastoris; the E. coli includes E. coli JM109 (DE3), E. coli HMS174(DE3), E. coli BL21(DE3), E. coli Rostta2(DE3), E. coli Rostta gami(DE3), E. coli Rostta2(DE3), E. coli DH5?, E. coli W3110 and/or E. coli K12.

8. A preparation method for Semaglutide intermediate polypeptide Arg34GLP-1(9-37), which is characterized in that: the Semaglutide intermediate polypeptide Arg34GLP-1(9-37) is produced by fermentation of recombinant microbial cell described in claim 6.

9. The preparation method of claim 8, which is characterized in that: the recombinant microbial cells are cultured at 35.sup.?40? C. for 10.sup.?12 hours to obtain seeds solution, and then cultured in TB medium with the inoculum amount of 0.1%.sup.?2% (v/v) until the OD.sub.600 value of the fermentation solution reached 6.sup.?8, and IPTG with a final concentration of 0.05.sup.?1 mM is added for induction; the fermentation is ended after induction for 14.sup.?18 hours at 25.sup.?40? C.; the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide is obtained by homogenization of fermented cells, extraction, and enzyme digestion.

10. The preparation method of claim 8, which is characterized in that: the recombinant microbial cells are cultured in LB medium at 35.sup.?40? C. for 8.sup.?12 hours to obtain pre-culture seeds solution, which is then transferred to the base fermentation medium for further 8?12 hours to obtain seeds solution; the seed solution is transferred to the base fermentation medium for fermentation; when the value of OD.sub.600 reached 100-200, IPTG with a final concentration of 0.05?1 mM is added for induction, the fermentation is ended after induction for 8.sup.?18 hours at 25.sup.?40? C., and the cells were collected; the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide is obtained by homogenization of fermented cells, extraction, and enzyme digestion.

11. The preparation method of claim 9, which is characterized in that: the inclusion bodies (IBs) precipitation is obtained by centrifuging and collecting after lysing the fermented cells, and the IBs are washed with a washing buffer; the washed IBs are dissolved with dissolved buffer at a pH of 6.0.sup.?10.0 and a protein concentration of 5?55 g/l; the dissolved fusion protein is enzyme digested by enterokinase (EK) at 30.sup.?35? C. for 20.sup.?24 hours to obtain the mixed solution containing intermediate polypeptide, fusion peptide and binding peptide; the mixed solution can be separated to obtain the intermediate polypeptide sample with the required purity.

Description

BRIEF DESCRIPTION OF FIGURES

[0040] FIG. 1 is the schematic diagram of the construction of recombinant plasmid in EXAMPLE 1.

[0041] FIG. 2 is the HPLC chromatogram of main peak of ion exchange purification in EXAMPLE 19.

DETAILED DESCRIPTION

[0042] In order to facilitate those skilled in the art to understand the present invention, the technical scheme of the invention will be further described in combination with specific embodiments below. However, the following contents shall not limit the scope of protection requested by the claims of the invention in any way.

[0043] Materials, reagents, etc. used in the following embodiments are all commercially available unless otherwise specified.

[0044] BFM-Medium: Diammonium hydrogen phosphate 6 g/L, ammonium chloride 4 g/L, potassium dihydrogen phosphate 13.5 g/L, magnesium sulfate 7 hydrate 1.39 g/L, citric acid 1 hydrate 2.8 g/L, yeast powder (Angie 802) 3 g/L, trace elements (large intestine) 10 mL/L, pH was adjusted to 7.0 with 10 M NaOH.

Example 1: Construction of Recombinant Strain Expressing Semaglutide Intermediate Polypeptide Fusion Protein

[0045] Designed a fusion protein sequence for expression in E. coli: fusion peptide-DDDDK-Arg34GLP-1(9-37).

[0046] The fusion peptide not only can improve protein expression in the host, but also can protect the intermediate polypeptide Arg34GLP-1(9-37) from internal degradation during synthesis in the E. coli. The amino acid sequence of fusion peptide was MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLV (SEQ ID NO: 3). The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. Therefore, the complete amino acid sequence of fusion protein was MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLVDDDD KEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO:12), the isoelectric point (pI) of the fusion protein was 6.2, and the mean hydrophilicity was 0.

[0047] The nucleotide sequence encoding the fusion protein was synthesized by PCR technology, and the obtained nucleic acid sequence was inserted into the corresponding enzyme cleavage site of plasmid pET-28a(+) through the Nco I/Hind III site. The recombinant plasmid map was shown in FIG. 1. The recombinant plasmid was transferred to host E. coli BL21(DE3), and recombinant strain S1 expressing the intermediate polypeptide fusion protein of Semaglutide was obtained.

Example 2: Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0048] Culture the recombinant strain S1 of Example 1 in LB media at 37? C. for 12 h to recover the seeds solution. The recovered seeds solution were inoculated into TB media with 0.2% (v/v) inoculation amount. When the cell concentration reaches O.D. (optical density) 600 nm of about 6-8, IPTG with a final concentration of 0.1 mM was added for induction. Induce for another 16 h at 37? C. to finish the fermentation. The cells were collected by centrifugation.

Example 3: Yield Detection of Semaglutide Intermediate Polypeptide Fusion Protein Expression in Shaker System

[0049] The fermentation cells of Example 2 were washed and disrupted by ultrasonic cell disruptor. The IBs were collected by centrifugating the disrupted suspension. The whole cells and IBs were detected by SDS-PAGE, and the purity of target protein was detected by optical densitometer. At the same time, BCA kit was used to detect the total protein content of whole cells and IBs. The expression of intermediate polypeptide fusion protein was obtained by multiplying the total protein amount and electrophoretic purity. The yield of fusion protein from recombinant strain S1 after fermentation and induction was 1.56 g/L, and 0.95 g/L IBs were obtained after disruption and washing.

Example 4: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0050] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 4. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 4, the isoelectric point (pI) of the fusion protein was 6.4, and the mean hydrophilicity was 0. The recombinant strain S2 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S2 after fermentation and induction was 1.44 g/L, and 1.32 g/L IBs were obtained after disruption and washing.

Example 5: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0051] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 5. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 5, the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was 0. The recombinant strain S3 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S3 after fermentation and induction was 1.73 g/L, and 1.43 g/L IBs were obtained after disruption and washing.

Example 6: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0052] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 6. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 6, the isoelectric point (pI) of the fusion protein was 6.4, and the mean hydrophilicity was 0. The recombinant strain S4 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S4 after fermentation and induction was 1.36 g/L, and 1.17 g/L IBs were obtained after disruption and washing.

Example 7: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0053] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 7. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 7, and the isoelectric point (pI) of the fusion protein was 6.4, and the mean hydrophilicity was-0.1. The recombinant strain S5 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S5 after fermentation and induction was 0.93 g/L, and 0.87 g/L IBs were obtained after disruption and washing.

Example 8: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0054] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 8. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 8, and the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was ?0.1. The recombinant strain S6 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S6 after fermentation and induction was 1.16 g/L, and 1.08 g/L IBs were obtained after disruption and washing.

Example 9: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0055] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 9. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 9, and the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was ?0.1. The recombinant strain S7 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S7 after fermentation and induction was 1.24 g/L, and 1.14 g/L IBs were obtained after disruption and washing.

Example 10: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0056] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 10. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 10, and the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was ?0.1. The recombinant strain S8 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S8 after fermentation and induction was 1.15 g/L, and 1.04 g/L IBs were obtained after disruption and washing.

Example 11: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0057] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 11. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(9-37) of Semaglutide through a DDDDK residue. The complete fusion protein contains the fusion peptide sequence of SEQ ID NO: 11, and the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was 0. The recombinant strain S9 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S9 after fermentation and induction was 1.45 g/L, and 1.25 g/L IBs were obtained after disruption and washing.

Example 12: Construction of Recombinant Strain Expressing Semaglutide Intermediate Polypeptide Fusion Protein

[0058] Designed a fusion protein sequence for expression in E. coli: fusion peptide-DDDDK-Arg34GLP-1(11-37).

[0059] The fusion peptide not only can improve protein expression in the host, but also can protect the intermediate polypeptide Arg34GLP-1(11-37) from internal degradation during synthesis in the E. coli. The amino acid sequence of fusion peptide was MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLVHA (SEQ ID NO:4). The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(11-37) of Semaglutide through a DDDDK residue. Therefore, the complete amino acid sequence of fusion protein was MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHKFVNQHLCGSHLVALYLVHADD DDKTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO:13) the isoelectric point (pI) of the fusion protein was 6.7, and the mean hydrophilicity was 0.

[0060] The recombinant strain S10 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S10 after fermentation and induction was 1.69 g/L, and 1.40 g/L IBs were obtained after disruption and washing.

Example 13: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0061] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 5. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(11-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 5, and the isoelectric point (pI) of the fusion protein was 7.6, and the mean hydrophilicity was 0.1. The recombinant strain S11 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S11 after fermentation and induction was 1.17 g/L, and 1.11 g/L IBs were obtained after disruption and washing.

Example 14: Construction of Recombinant Strain and Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Shaker System

[0062] The amino acid sequence of fusion peptide was shown as SEQ ID NO: 11. The C-terminal of the fusion peptide sequence was connected to the intermediate polypeptide Arg34GLP-1(11-37) of Semaglutide through a DDDDK residue. The complete fusion protein contained the fusion peptide sequence of SEQ ID NO: 11, and the isoelectric point (pI) of the fusion protein was 7.6, and the mean hydrophilicity was 0.1. The recombinant strain S12 was constructed according to the operation of Example 1, and the fusion protein was expressed according to the operation of Example 2 and Example 3. The yield of fusion protein from recombinant strain S12 after fermentation and induction was 1.21 g/L, and 1.13 g/L IBs were obtained after disruption and washing. The results of the above examples were summarized in Table 1 below.

TABLE-US-00003 TABLE 1 Mean Yield of Recombinant Sequence of Hydro- fusion Yield of strain fusion petide pI philicity protein(g/L) IBs(g/L) S1 SEQ ID NO: 3 6.2 0 1.56 0.95 S2 SEQ ID NO: 4 6.4 0 1.44 1.32 S3 SEQ ID NO: 5 6.7 0 1.73 1.43 S4 SEQ ID NO: 6 6.4 0 1.36 1.17 S5 SEQ ID NO: 7 6.4 ?0.1 0.93 0.87 S6 SEQ ID NO: 8 6.7 ?0.1 1.16 1.08 S7 SEQ ID NO: 9 6.7 ?0.1 1.24 1.14 S8 SEQ ID NO: 10 6.7 ?0.1 1.15 1.04 S9 SEQ ID NO: 11 6.7 0 1.45 1.25 S10 SEQ ID NO: 4 6.7 0 1.69 1.4 S11 SEQ ID NO: 5 7.6 ?0.1 1.17 1.11 S12 SEQ ID NO: 11 7.6 ?0.1 1.21 1.13

Example 15: Fermentation of Recombinant Strain in Fermentor System

[0063] Cultured the recombinant strain S3 of Example 1 in LB media at 37? C. for 8 h to recover the pre-culture seeds solution. The recovered seeds were inoculated into BFM media to further culture for another 8 hours to obtain seeds solution, which was then inoculated into fermentor containing BFM media with a volume of 6 L. When the O.D. (optical density, 600 nm) value of the fermentation solution reached 150, IPTG with a final concentration of 0.1 mM was added for induction. Induce for another 12 h at 37? C. to finish the fermentation. The cells were collected by centrifugation.

Example 16: Extraction and Enzyme Digestion of Semaglutide Intermediate Polypeptide Fusion Protein

[0064] The harvested cells in Example 15 were diluted with buffer at a volume ratio of 1:2 (that is, the cells from 1 L fermentation were diluted by 2 L buffer). The buffer contains 25 mM Tris, 10 mM EDTA-2Na, and pH was 7.5?8.0. The IBs precipitation was obtained by centrifugation after homogenization of cells, and washed with washing buffer at a volume ratio of 1:2 (that was, the IBs precipitation from 1 L fermentation were washed by 2 L washing buffer). The washing buffer contained 25 mM Tris, 0.25 M urea, 1% Tween 20, and pH was 7.5. The washing operation of IBs precipitation was repeated once. The washed IBs were dissolved by dissolved buffer at a protein concentration of 25 g/L. The dissolved buffer contained 25 mM Tris, 0.1 mM EDTA-2Na, and pH was adjusted to 7.5.sup.?8.0 for 0.5 h dissolution. The dissolved fusion protein was enzyme digested by enterokinase (EK) at 35? C. for 24 h to obtain the mixed solution containing intermediate polypeptide, fusion peptide, and binding peptide. The yield of fusion protein was 13.1 g/L and the yield of digested intermediate polypeptide was 3.62 g/L by HPLC.

Example 17: Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Fermentor System

[0065] The recombinant strain S2 of Example 4 was fermented, induced, extracted and enzyme digested according to the operation of Example 15 and Example 16. The yield of fusion protein was 11.4 g/L and the yield of digested intermediate polypeptide was 3.08 g/L by HPLC.

Example 18: Expression of Semaglutide Intermediate Polypeptide Fusion Protein in Fermentor System

[0066] The recombinant strain S9 of Example 11 was fermented, induced, extracted and enzyme digested according to the operation of Example 15 and Example 16. The yield of fusion protein was 10.3 g/L and the yield of digested intermediate polypeptide was 2.77 g/L by HPLC.

Example 19: Purification of Semaglutide Intermediate Polypeptide

[0067] The mixture solution containing 20.3 g target protein obtained in Example 16 was purified by ion exchange, and the washing peak 1, main peak and regeneration peak were obtained in turn. The main peak sample was taken for HPLC, and the elution time of HPLC was 15.833 min. As shown in FIG. 2, the purity of target protein was 92.5%. The sample of Semaglutide intermediate polypeptide collected was 17.5 g and the yield was 86.2%.

Comparative Example 1

[0068] Using the fusion peptide sequence published at present, which was similar to the fusion peptide sequence from this invention. The fusion peptide was used as leading peptide, and the sequence was MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGL

[0069] TEGLHGFHVHKFVNQHLCGSHLVALYLV (SEQ ID NO:14). The recombinant strain S13 was constructed and the fusion protein was expressed according to the operation of Example 1.sup.?3. The yield of fusion protein from recombinant strain S13 after fermentation and induction was 0.85 g/L, and 0.61 g/L IBs were obtained after disruption and washing. The recombinant strain S13 was fermented, induced, extracted and enzyme digested according to the operation of Example 15 and Example 16. The yield of fusion protein was 6.5 g/L and the yield of digested intermediate polypeptide was 1.75 g/L by HPLC. Using the fusion peptide sequence in the invention, the yield of the intermediate polypeptide after enzyme digestion was 2.77.sup.?3.62 g/L, which was higher than the data in the comparative example.

[0070] The lead peptide sequences used in Liraglutide intermediate polypeptides preparation in this invention were not applicable to all GLP-1, such as the Semaglutide.

[0071] Although the present invention has been disclosed in terms of preferred embodiments, it is not intended to limit the present invention, and any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention, so the scope of protection of the present invention shall be determined by the claims.

[0072] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.