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CN-121974995-A - Polypeptide for promoting soluble expression of GLP-1 analogue precursor polypeptide and application thereof

CN121974995ACN 121974995 ACN121974995 ACN 121974995ACN-121974995-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a series of polypeptides for promoting soluble expression of GLP-1 analogue precursor polypeptides and application thereof. The polypeptide comprises a GB 1-like polypeptide fragment, and particularly comprises an amino acid sequence shown as SEQ ID NO. 1-SEQ ID NO. 8. The series of polypeptides have small molecular weight, can effectively promote the soluble expression of the multi-copy GLP-1 analogue precursor polypeptide under high temperature induction, has a theoretical recovery rate of more than 60 percent, and can realize the low-cost, simple and convenient high-efficiency production of target proteins.

Inventors

  • XIE HAISONG
  • DING XU
  • Zhang gun
  • SHEN SHULI
  • LU WENYAO

Assignees

  • 杭州中美华东制药有限公司

Dates

Publication Date
20260505
Application Date
20251223

Claims (20)

  1. 1. A polypeptide that facilitates soluble expression of a GLP-1 analogue precursor polypeptide, comprising a GB 1-like polypeptide fragment, said GB 1-like polypeptide fragment being: （a）YKLILNGKTX 1 KGETTTX 2 AX 3 X 4 AX 5 TAEKX 6 FKQYAX 7 DNGVDGEWTYDDX 8 TK TFTVTE; Wherein X 1 is selected from L or I, wherein X 2 is selected from E or Y, wherein X 3 is selected from V or D, wherein X 4 is selected from D or N, wherein X 5 is selected from A or E, wherein X 6 is selected from V or I or F, wherein X 7 is selected from N or T or A, wherein X 8 is selected from A or D; or (b) an amino acid sequence which has more than 85% sequence identity to (a), or a polypeptide fragment which functions as the polypeptide fragment defined in (a).
  2. 2. The polypeptide according to claim 1, comprising an amino acid sequence selected from any one of the following groups SEQ ID NOs 1 to 8.
  3. 3. An isolated polynucleotide encoding the polypeptide of any one of claims 1 or 2.
  4. 4. A fusion protein comprising the polypeptide of any one of claims 1 or 2 and a GLP-1 analogue precursor polypeptide.
  5. 5. The fusion protein of claim 4, wherein the GLP-1 analog precursor polypeptide is liraglutide precursor P31 shown in SEQ ID NO. 9 or semraglutide precursor P29 shown in SEQ ID NO. 10.
  6. 6. The fusion protein of claim 5, wherein the liraglutide precursor P31 or the semraglutide precursor P29 is a multicopy tandem fusion protein.
  7. 7. A polynucleotide encoding the fusion protein of claim 6.
  8. 8. The polynucleotide according to claim 7, wherein the polynucleotide sequence is shown in SEQ ID NO. 11 or SEQ ID NO. 12.
  9. 9. A recombinant expression vector comprising the polynucleotide of any one of claims 3, 7 or 8.
  10. 10. The recombinant expression vector according to claim 9, wherein the recombinant expression vector is a prokaryotic expression vector.
  11. 11. The recombinant expression vector according to claim 10, wherein the recombinant expression vector is a plasmid.
  12. 12. The recombinant expression vector of claim 10, wherein the plasmid is pET-28a or pET-22a.
  13. 13. A host cell comprising the polynucleotide of any one of claims 3, 7 or 8 or the recombinant expression vector of any one of claims 9-12.
  14. 14. The host cell of claim 13, wherein the host cell is selected from the group consisting of eukaryotic cells and prokaryotic cells.
  15. 15. The host cell of claim 14, wherein the eukaryotic cell is a yeast.
  16. 16. The host cell of claim 14, wherein the prokaryotic cell is an E.coli cell.
  17. 17. The host cell of claim 16, wherein the E.coli cell is E.coli T7 express or BL21 (DE 3).
  18. 18. A method for preparing recombinant expression bacteria for soluble expression of GLP-1 analog precursor polypeptide, which is characterized in that the method comprises transforming the host cell of any one of claims 13-17 with the recombinant expression vector of any one of claims 9-12 to obtain recombinant expression cells.
  19. 19. A method for the soluble expression of a recombinant GLP-1 analog precursor polypeptide, which is characterized in that the method comprises the steps of expressing a foreign protein, culturing the host cell according to any one of claims 16-17 at 37 ℃, and adding IPTG with a final concentration of 0.1-1 mM to induce the expression of the foreign protein when the bacterial concentration OD 600 reaches 0.6-0.8.
  20. 20. Use of the polypeptide of any one of claims 1-2, the fusion protein of any one of claims 4-6, the polynucleotide of any one of claims 3, 7, 8, the recombinant expression vector of any one of claims 9-12, the host cell of any one of claims 13-17, to promote soluble expression of a GLP-1 analogue precursor polypeptide.

Description

Polypeptide for promoting soluble expression of GLP-1 analogue precursor polypeptide and application thereof Technical Field The invention relates to the technical field of biology, in particular to a series of polypeptides for promoting soluble expression of GLP-1 analogue precursor polypeptides and application thereof. Background In the 60 s of the 20 th century, mcIntyre and Elrick et al found that oral glucose was more able to promote insulin secretion than intravenous injection, i.e. "incretin effect". The incretin is an entero-derived hormone, and stimulates insulin secretion through a glucose concentration dependent mechanism after eating, and is an important target for treating type 2 diabetes. Incretins mainly include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), with GLP-1 acting as the core. Natural GLP-1 polypeptides consist of 37 amino acids and analogs developed based on natural GLP-1 have become first line therapeutic agents for type 2 diabetes and obesity, such as liraglutide and semraglutide. The liraglutide precursor polypeptide P31 is a fragment of a native GLP-1 polypeptide comprising the amino acid sequence from position 7 to position 37, and the semraglutide precursor polypeptide P29 comprises the amino acid sequence from position 9 to position 37 of the native GLP-1 polypeptide, the precursor polypeptide being an important intermediate for the preparation of GLP-1 analogues. Currently, GLP-1 analog precursor polypeptides are produced mainly by gene recombination techniques, and commonly used hosts include e.coli and yeast systems. The expression system of the escherichia coli has the advantages of rapid propagation, simple culture, convenient operation and genetic stability, but the excessive expression of the protein of the escherichia coli often forms insoluble inclusion bodies, and the relative molecular weight of the GLP-1 analogue precursor polypeptide is small, thus being very unfavorable for genetic engineering expression and separation and purification. Soluble expression facilitates proper folding of the protein, allows the protein to better remain active, and does not require denaturation and post-purification protein dialysis renaturation. One of methods for effectively improving the soluble expression of exogenous proteins in escherichia coli is fusion expression, specifically, splicing exogenous genes and specific genes together through gene operation, and expressing the exogenous genes and the specific genes under the same reading frame. The expression products of these specific genes often improve folding of the foreign protein, increase its solubility, increase its resistance to degradation by endogenous proteases, or express in secreted form, ultimately increasing the yield of soluble proteins. Commonly used fusion tag proteins are glutathione-S-transferase (GST), maltose Binding Protein (MBP), thioredoxin (Thioredoxin) and the like. The molecular weight of different tag proteins is different, and the influence on the correct folding and activity of the foreign proteins is different, so that the molecular weight and the characteristics of the tag proteins need to be considered when different foreign genes are fused and expressed. Disclosure of Invention Aiming at the problems of low solubility of GLP-1 analogue precursor polypeptide in prokaryotic cell recombinant expression, particularly low solubility in high temperature induction and/or low recovery rate of target protein caused by unbalanced molecular weight of fusion protein tags, the invention provides a GB1M series fusion protein tag which can promote high-solubility expression of GLP-1 analogue precursor polypeptide and improve the recovery rate of target polypeptide. In order to achieve the above object, the present invention provides a series of polypeptides for promoting the soluble expression of a GLP-1 analogue precursor polypeptide, wherein the polypeptides comprise GB 1-like polypeptide fragments, and the GB 1-like polypeptide fragments are as follows: （a）YKLILNGKTX1KGETTTX2AX3X4AX5TAEKX6FKQYAX7DNGVDGEWTYDDX8TK TFTVTE; Wherein X 1 is selected from L or I, wherein X 2 is selected from E or Y, wherein X 3 is selected from V or D, wherein X 4 is selected from D or N, wherein X 5 is selected from A or E, wherein X 6 is selected from V or I or F, wherein X 7 is selected from N or T or A, wherein X 8 is selected from A or D; or (b) an amino acid sequence which has more than 85% sequence identity to (a), or a polypeptide fragment which functions as the polypeptide fragment defined in (a). In a preferred technical scheme, the polypeptide comprises an amino acid sequence selected from any one of the following groups, wherein the amino acid sequence is shown as follows, and SEQ ID NO is 1-8: SEQ ID NO:1 YKLILNGKTIKGETTTYADNAETAEKIFKQYAADNGVDGEWTYDDDTKTFTVTE SEQ ID NO:2 YKLILNGKTIKGETTTYADNAETAEKIFKQYADDNGVDGEWTYDDDTKTFTVTE SEQ ID NO:3 YKLILNGKTLKGETTTYADNAETAEKIFKQYATDNGV