Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

FGF21 Fusion Proteins and Method of Inhibiting Degradation Thereof

A technology of FGF21 and fusion protein, which is applied in the direction of fusion polypeptide, chemical instruments and methods, animal/human protein, etc., can solve the problems of time-consuming and other problems, and achieve the effect of simple operation, inhibition effect and reduction of degradation

Active Publication Date: 2020-05-01
DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, design of molecular variants and subsequent validation are time consuming

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • FGF21 Fusion Proteins and Method of Inhibiting Degradation Thereof
  • FGF21 Fusion Proteins and Method of Inhibiting Degradation Thereof
  • FGF21 Fusion Proteins and Method of Inhibiting Degradation Thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Construction of expression vector

[0047] A genetically engineered cell line that expresses GLP-1 analogues and FGF21 analogues is constructed.

[0048] According to CHO's preferred codon optimization fusion protein sequence A-B-C-D-E-F, where A is the signal peptide, B is the glucagon-like peptide-1 variant (GLP-1), C is the linker peptide, D is the IgG4 variant, and E is the linker Peptide, F is FGF21 variant.

[0049] A: signal peptide

[0050] Nucleotide sequence (SEQ ID NO: 1)

[0051] atgcggtttttcttcgtgttcctggccatcgtgctgtttcagggcatccacgga

[0052] Amino acid sequence (SEQ ID NO:2)

[0053] MRFFFVFLAIVLFQGIHG

[0054] B: GLP-1 variant

[0055] Nucleotide sequence (SEQ ID NO:3)

[0056] cacggagaaggaacctttacctccgacgtgtcttcttacctggaggaacaggcagctaaggagtttatcgcttggctggtgaaaggaggagga

[0057] Amino acid sequence (SEQ ID NO:4)

[0058] HGEGFTTSDVSSYLEEQAAKEFIAWLVKGGG

[0059] C: connecting peptide

[0060] Nucleotide sequence (SEQ ID NO:5)

[00...

Embodiment 2

[0076] Example 2: Construction of CHO stably transfected cell pool

[0077] A CHO stably transfected cell pool expressing fusion protein A-B-C-D-E-F was constructed.

[0078] The expression vector constructed in Example 1 was linearized with restriction endonuclease, precipitated with ethanol, extracted with phenol and chloroform, and dissolved for use.

[0079] After the CHOK1 host cells were revived and cultured with Cellvento CHO 200 medium, when the cell density was about 8×10 5 Cells / mL were harvested for transfection. Transfected cells about 1×10 7 Cell, about 40 μg of linearized plasmid, was transfected by electroporation (Bio-Rad, Gene pulser Xcell). Cells were cultured in 20 mL Cellvento CHO 200 medium after electric shock. On the second day of culture, the cells were collected by centrifugation, and resuspended in 20 mL Cellvento CHO 200 medium for pressurized culture. When the cell density is about 0.6×10 6 cell / mL, the obtained mixed clones were subcultured w...

Embodiment 3

[0080] Embodiment 3: mass spectrometry detection

[0081] The obtained CHOK1 stably transfected cell pool was inoculated into Cellvento 200 medium, and C1 inhibitor was added to the medium, respectively high concentration addition group (2 μg / mL), medium concentration addition group (1 μg / mL), low concentration addition group ( 0.5μg / mL), 37°C, 8% CO 2 After culturing for 3 days under the same conditions, the temperature was lowered to 30° C. to continue culturing for 4 days. After protein A purification and DTT reduction, mass spectrometry detection was performed. The molecular weight of the intact undegraded protein is about 51.5Kda, and the molecular weight of the main degraded protein is about 34.2Kda.

[0082] The results showed that the high-concentration addition group could inhibit the degradation of FGF21 fusion protein, and the inhibitory effect was dose-dependent. The higher the addition concentration, the less the proportion of degraded protein (see figure 2 )....

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses an FGF21 fusion protein. The fusion protein comprises (1) a glucagon-like polypeptide 1 (GLP-1) variant of the amino acid sequence shown in SEQ ID NO: 4, a connecting peptide of the amino acid sequence shown in SEQ ID NO: 6, a Fc portion of human IgG4 variant antibody having amino acid sequence shown in SEQ ID NO: 8 and a fibroblast growth factor 21 (FGF21) variant of the amino acid sequence set forth in SEQ ID NO: 10; or (2): a fusion protein which is derived from (1) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (1) and hasthe activity of the FGF21 fusion protein. The invention further discloses a method for inhibiting degradation of the FGF21 fusion protein. The method comprises the step of adding the C1 protease inhibitor into a culture medium. The method provided by the invention can effectively inhibit the degradation of the FGF21 fusion protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a FGF21 fusion protein and a method for inhibiting the degradation of the FGF21 fusion protein. Background technique [0002] Fibroblast growth factor 21 (fibroblast growth factor 21, FGF21) is a hormone-like protein with pleiotropic effects. Important metabolic regulators of homeostasis play a role. [0003] It has been reported in the literature that in CHO cells stably expressing recombinant FGF21, FGF21 is prone to degradation, and the degradation is at the position of the peptide bond between the 19th Arg and the 20th Tyr from the N-terminus, and the degradation comes from the protease of CHO cells itself. [0004] Takeshi Shimomura et al. disclosed that recombinant human apolipoprotein E (Apo-E) expressed by CHO-322 cells was degraded into 24KDa and 23KDa fragments in serum-free medium, and the degradation was inhibited in medium containing fetal bovine serum. The inhibitor m...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10
CPCC07K14/50C07K14/605C12N15/85C12N5/0682C07K2319/30C07K2319/00C12N2800/107C12N2800/22C12N2510/02C12N2501/734
Inventor 李宇晟李利佳陈超欧晓涛尚龙万志滔杨佩向婷陈小锋李文佳
Owner DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com