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A kind of human fibroblast growth factor 18 and its soluble recombinant expression method, preparation method, preparation and application

A technology of human fibroblasts and growth factors, which is applied to the preparation methods of fibroblast growth factors and peptides, growth factors/inducing factors, etc., can solve the problems of complicated process, unstable structure, harsh renaturation conditions, etc., and achieve The process is simple, the structure is stable, and it is beneficial to the effect of industrial production

Active Publication Date: 2022-06-17
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The patent (US7858341) discloses the use of genetic engineering technology, the prokaryotic expression system of Escherichia coli, and the expression of inactive FGF18 in the form of inclusion bodies, and then FGF18 is obtained after dissolving with denaturant, renaturation, and column chromatography, which has the disadvantage of cumbersome process
[0006] FGF18 is a heparin-binding protein with an isoelectric point of 9.86. The amino acid sequence contains a large number of basic amino acids (lysine and arginine), with a ratio as high as 20.6%. A large number of negatively charged amino acids aggregate and repel each other, resulting in unstable peptide bonds. , the N-terminus is easy to break, and it is very unstable when used in pharmaceutical preparations
The structure of FGF18 reported in the current literature or patents is a basic protein, the structure is unstable, and the N-terminus is easily broken; its preparation method is expressed in the form of inclusion bodies in E. coli, and disulfide bonds cannot be formed in the cell, and the pre-treatment is complicated. Harsh conditions make industrial production difficult

Method used

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  • A kind of human fibroblast growth factor 18 and its soluble recombinant expression method, preparation method, preparation and application
  • A kind of human fibroblast growth factor 18 and its soluble recombinant expression method, preparation method, preparation and application
  • A kind of human fibroblast growth factor 18 and its soluble recombinant expression method, preparation method, preparation and application

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preparation example Construction

[0042] A preparation method of human fibroblast growth factor 18, comprising the steps of:

[0043] (1) low-temperature crushing by pressure homogenization, and centrifugation to collect the supernatant;

[0044] (2) Anion chromatography was used to capture FGF18ΔN in the supernatant of cell disruption;

[0045] (3) Using copper ions as the correct pairing accelerator for disulfide bonds;

[0046] (4) After correct pairing of disulfide bonds, heparin affinity chromatography captures the target product.

[0047] The preparation method of human fibroblast growth factor 18 disclosed in the present invention comprises the following specific steps:

[0048] S1 selects the expression system containing the T7 promoter as the expression plasmid vector, and the corresponding Escherichia coli as the host strain;

[0049] S2 selects two suitable endonucleases, and then double-digests the plasmid vector and the N-terminal deletion of the gene of human fibroblast growth factor 18, then ...

example 1

[0071] Example 1. Construction and inducible expression of genetically engineered strains with N-terminal deletion of human fibroblast growth factor 18

[0072] The cDNA sequence (SEQ ID NO:2) was designed based on the amino acid sequence of human fibroblast growth factor 18 (SEQ ID NO:1) deleted at the N-terminus. The whole gene was synthesized and inserted into the vector plasmid pUC-57 to obtain the pUC-57-FGF18ΔN plasmid. By using NdeI and BamHI restriction sites, the FGF18△N gene was connected to the vector plasmid pET-30a to construct the pET-30a-FGF△N recombinant plasmid, which was transformed into E. coli expression host strain BL21(DE3)PlysS , After expression screening, pET-30a-FGF18ΔN / BL21(DE3)PlysS recombinant engineering bacteria were constructed. The engineering strain is streaked and inoculated, and the bacterial lawn is selected and inoculated in a test tube containing a medium. After activation, it is transferred to the triangular agent and cultivated overnig...

example 2

[0073] Example 2. Disulfide bond formation with N-terminal deletion of human fibroblast growth factor 18

[0074] After the bacteria were resuspended, high-pressure homogenization was used to break the bacteria. FGF18ΔN was captured by anion chromatography, diluted with diluent (20mM Tris, pH 7.5) in an equal volume ratio of 2:1, and the final concentration of 5uM copper ions was added. HPLC detection showed that the pairing rate of disulfide bonds was not less than 90%, and FGF18ΔN with high activity was obtained.

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Abstract

The present invention discloses a human fibroblast growth factor 18, the amino acid sequence of which is shown in SEQ ID NO: 1; the soluble recombinant expression method of the N-terminal deleted human fibroblast growth factor 18 comprises the following steps: ( 1) A plasmid containing a T7 promoter is selected as the expression vector, and the corresponding Escherichia coli is used as the host bacterium; (2) the recombinant of the cDNA corresponding to SEQ ID NO: 1 and the expression strain; (3) the induction expression condition of the expression strain is: temperature 15-30°C, lactose or IPTG induction, induction culture time 4-16 hours. The preparation method of the N-terminally deleted human fibroblast growth factor 18, the preparation of the N-terminally deleted human fibroblast growth factor 18, and the N-terminally deleted human fibroblast growth factor 18 for treating bone Arthritis or damage to the articular cartilage.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a human fibroblast growth factor 18 and a soluble recombinant expression method, preparation method, preparation and application thereof. Background technique [0002] Fibroblast Growth Factor (FGF) is a class of structurally related proteins encoded by members of the FGF gene family. Promote DNA synthesis and cell division, mainly secreted by fibroblasts, endothelial cells, osteocytes, inert cells, the relative molecular weight is generally 17 ~ 34kD. Fibroblast growth factor 18 (FGF18 for short) was first isolated from mouse embryos by Japanese scientist Ohbayashi in 1998 and plays an important role in bone growth and development. [0003] Literature (Ornitz and Marie, Genes & Development, 16:1446-1465) (discussing the role of all FGFs, including FGF18 in bone development), FGF18 has a wide range of physiological functions and has an impact on bone development, embryonic deve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/50C12N15/70C07K1/18C07K1/22A61K38/18A61P19/02A61P29/00
Inventor 曾鑫曹宇覃晓兰蔡春淞范玲玲范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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