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Novel human fibroblast growth factor 18 and soluble recombinant expression method, preparation method, preparation and applications thereof

A technology for human fibroblasts and growth factors, which is applied in the preparation methods of fibroblast growth factors, peptides, growth factors/inducing factors, etc., can solve the problems of cumbersome process, harsh renaturation conditions, unstable peptide bonds, etc. To achieve the effect of simple process and beneficial to industrial production

Active Publication Date: 2019-06-04
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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  • Novel human fibroblast growth factor 18 and soluble recombinant expression method, preparation method, preparation and applications thereof
  • Novel human fibroblast growth factor 18 and soluble recombinant expression method, preparation method, preparation and applications thereof
  • Novel human fibroblast growth factor 18 and soluble recombinant expression method, preparation method, preparation and applications thereof

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preparation example Construction

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

[0043] (1) Use pressure homogenate to crush at low temperature, and collect the supernatant by centrifugation;

[0044] (2) Anion chromatography was used to capture FGF18△N in the supernatant of broken cells;

[0045] (3) Copper ions are used as the correct pairing accelerator of disulfide bonds;

[0046] (4) The target product was captured by heparin affinity chromatography after the correct disulfide bond pairing.

[0047] The preparation method of novel human fibroblast growth factor 18 disclosed by 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 bacteria;

[0049] S2 Select two suitable endonucleases to double-enzyme-cut the plasmid vector and the N-terminal deletion of the human fibroblast growth factor 18 ...

example 1

[0071] Example 1. N-terminal deletion of human fibroblast growth factor 18 genetically engineered strain construction and induced expression

[0072] The cDNA sequence (SEQ ID NO: 2) designed based on the amino acid sequence (SEQ ID NO: 1) of human fibroblast growth factor 18 with N-terminal deletion. The whole gene was synthesized and inserted into the vector plasmid pUC-57 to obtain the pUC-57-FGF18△N plasmid. By using NdeI and BamHI restriction sites, the FGF18△N gene was connected to the carrier plasmid pET-30a to construct the pET-30a-FGF△N recombinant plasmid, and the above plasmid was transformed into E. coli expression host strain BL21(DE3)PlysS , after expression screening, construct pET-30a-FGF18△N / BL21(DE3)PlysS recombinant engineering bacteria. Streak inoculation of the engineering strains, select the bacterial lawn and inoculate it into the test tube containing the medium, transfer it to the triangle agent after activation, and cultivate it overnight to become th...

example 2

[0073] Example 2. Disulfide bond formation of N-terminally deleted human fibroblast growth factor 18

[0074] After the bacteria were resuspended, the bacteria were broken by high-pressure homogenization, and FGF18△N was captured by anion chromatography, diluted with diluent (20mM Tris, pH7.5) at an equal volume ratio of 2:1, added with a final concentration of 5uM copper ions, and stirred at a low speed at 4°C for 4h. As detected by HPLC, the disulfide bond pairing rate was not lower than 90%, and FGF18△N with high activity was obtained.

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Abstract

The invention discloses a novel human fibroblast growth factor 18. The amino acid sequence of the growth factor is as 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) selecting a plasmid containing a T7 promoter as an expression vector and corresponding escherichia coli as a host bacterium; (2) acquiring recombinants of cDNA corresponding to the SEQ ID NO:1 and expressed strains; and (3) performing induction, wherein the induction expression conditions of the expressed strains are as follows: a temperature of 15-30 DEG C, lactose induction or IPTG induction, and induction culture time of 4-16 hours. According to the preparation method of the N terminal-deleted human fibroblast growth factor 18 and a preparation of the N terminal-deleted human fibroblast growth factor 18, the N terminal-deleted human fibroblast growth factor 18 is used for treating osteoarthritis or articular cartilage injury.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a novel human fibroblast growth factor 18 and its soluble recombinant expression method, preparation method, preparation and application. Background technique [0002] Fibroblast Growth Factor (FGF) is a structurally related protein encoded by members of the FGF gene family. Promote DNA synthesis and cell division, mainly secreted by fibroblasts, endothelial cells, bone cells, inert cells, the relative molecular weight is generally 17 ~ 34kD. Fibroblast growth factor 18 (fibroblast growth factor 18, hereinafter referred to as FGF18) was first isolated from mouse embryos by Japanese scientist Ohbayashi in 1998, and plays an important role in the process of bone growth and development. [0003] According to reports in the 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 o...

Claims

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Application Information

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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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