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Formic acid cleavage site peptide, correlative biomaterial and applications of formic acid cleavage site peptide and correlative biomaterial in production of calcitonin

A technology of formic acid cutting and biomaterials, applied in calcitonin, applications, plant products, etc., can solve the problems of low biological activity, difficulty in large-scale production, and many by-products

Active Publication Date: 2016-01-06
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The high price is the main reason that affects the wide application of calcitonin. At present, the calcitonin in domestic injections in China is 5,200 yuan / mg, and the price of the original drug is as high as 0.8-10,000 yuan / gram
The reason for the high price of calcitonin is that currently clinically used calcitonin is mainly produced by chemical synthesis, which has the problems of many by-products and difficulty in large-scale production
Calcitonin expressed in Escherichia coli or yeast by genetic engineering method has low biological activity, only 100-200 international units per mg, because the 32 amino acids at the C-terminal cannot be amidated, so it cannot be used clinically. The above two reasons Leading to the current high market price of calcitonin

Method used

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  • Formic acid cleavage site peptide, correlative biomaterial and applications of formic acid cleavage site peptide and correlative biomaterial in production of calcitonin
  • Formic acid cleavage site peptide, correlative biomaterial and applications of formic acid cleavage site peptide and correlative biomaterial in production of calcitonin
  • Formic acid cleavage site peptide, correlative biomaterial and applications of formic acid cleavage site peptide and correlative biomaterial in production of calcitonin

Examples

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Effect test

Embodiment 1

[0089] Example 1. Screening of Asp-Pro formic acid cleavage site peptides suitable for Asp-Pro formic acid cleavage method (formic acid cleavage method of peptide bond D-P (Asp-Pro))

[0090] In this example, four kinds of Asp-Pro formic acid cleavage site peptides in Table 1 are designed, and the names are CA1, CA2, CA3 and CA4 respectively. Asp-Pro formic acid cleavage site peptide CA3 with high cleavage efficiency was screened among the formic acid cleavage site peptides.

[0091] The four fusion proteins FP1, FP2, FP3 and FP4 were obtained by fusing salmon calcitonin with the extramembrane region of the glycoprotein through the four Asp-Pro formic acid cleavage site peptides, and the Asp-Pro formic acid cleavage site peptide in FP1 It is CA1, the Asp-Pro formic acid cleavage site peptide in FP2 is CA2, the Asp-Pro formic acid cleavage site peptide in FP3 is CA3 (amino acid sequence such as SEQIDNo.2), the Asp-Pro formic acid cleavage site peptide in FP4 It is CA4. The am...

Embodiment 2

[0128] Example 2. Using Asp-Pro Formic Acid Cleavage Site Peptide CA3 to Cultivate Salmon Calcitonin Transgenic Rapeseed

[0129] 2.1 Construction of fusion protein G-sCT-SO gene expression vector

[0130] The fusion protein G-sCT was obtained by linking salmon calcitonin with the extramembrane region of the glycoprotein through the Asp-Pro formic acid cleavage site peptide CA3. In order to express the fusion protein G-sCT in the oil body of rapeseed, the fusion protein G- One end of sCT was connected with sesame oleosin to obtain fusion protein G-sCT-SO. The amino acid sequence of the fusion protein G-sCT-SO is SEQIDNo.4, in SEQIDNo.4, the 8th-150th is the sequence of the sesame oil body protein, the 153-208th is the sequence of the extramembrane region of the glycoprotein, and the 209th-214th Position 215-247 is the sequence of salmon calcitonin.

[0131] Artificially synthesized fusion protein G-sCT-SO gene expression cassette shown in SEQIDNo.5, SEQIDNo.5 is salmon calci...

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Abstract

The invention discloses a formic acid cleavage site peptide, a correlative biomaterial and applications of the formic acid cleavage site peptide and the correlative biomaterial in production of calcitonin. The provided formic acid cleavage site peptide is named after CA3, and is a peptide with an amino acid sequence shown in SEQ ID No.2. The formic acid cleavage site peptide CA3 is employed as a fusion protein for formic acid cleavage site construction, the cleavage rate is 85.3% in the following formic acid cleavage conditions: the formic acid (a cleavage agent) concentration is 40% (volume percent), the cleavage temperature is 43 DEG C and the cleavage time is 2.5h. The fusion protein containing the formic acid cleavage site peptide CA3 carries out formic acid cleavage to generate aim protein, the formic acid cleavage time is short, the cleavage efficiency is high, the cleavage efficiency of the fusion protein is raised effectively, the purification cost of the aim protein is saved, a new idea is provided for industrial production of protein recombination, and technical support is provided for production of protein products.

Description

technical field [0001] The invention relates to formic acid cleavage site peptides and related biological materials in the field of biotechnology and their application in the production of calcitonin. Background technique [0002] Recombinant expression has the advantages of fast expression speed, low cost, and high production efficiency, making it a convenient method for polypeptide synthesis. The key technology of protein recombinant expression is the purification of the target protein. Recombinant expression generally adopts the expression method of fusion protein. Therefore, the cleavage and separation of the target protein from the fusion protein is the key to recombinant expression. For protein products, the cleavage and separation of fusion protein Efficiency is extremely important. Currently, fusion protein cleavage reactions mainly include chemical cleavage and enzymatic cleavage. [0003] The method of enzymatic hydrolysis has relatively mild reaction conditions,...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K19/00C12N15/11C12N15/62C07K1/12C12N15/84A01H5/00C07K14/585
Inventor 刘昱辉张华光李梅
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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