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Structure of a novel proinsulin aspart and method for preparing insulin aspart

A technology of insulin aspart and sequence, which is applied in the direction of insulin, chemical instruments and methods, botany equipment and methods, etc., can solve the problems of inefficiency, time-consuming, expensive, etc., achieve simple purification process, avoid the problem of impurities, and remove and purify The effect of steps

Active Publication Date: 2021-12-10
AMPHASTAR NANJING PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process utilizes cyanogen bromide to cleave the N-terminal peptide tag followed by refolding, which is inefficient, expensive and time-consuming
Furthermore, this patent is limited to use in the preparation of human insulin

Method used

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  • Structure of a novel proinsulin aspart and method for preparing insulin aspart
  • Structure of a novel proinsulin aspart and method for preparing insulin aspart
  • Structure of a novel proinsulin aspart and method for preparing insulin aspart

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0099] Example 1: Construction of an Escherichia coli clone expressing formula I novel proinsulin.

[0100] One is designed for expressing the proinsulin protein sequence as shown in formula I in escherichia coli. The N-terminal leading amino acid sequence can enhance expression, protect proinsulin aspart, and prevent degradation by Escherichia coli. The preferred leader amino acid sequence is MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP (SEQ ID NO: 1);

[0101] The C-terminal of the leader amino acid sequence is connected to the B chain of insulin aspart through arginine or lysine residues, and the leader peptide is removed by trypsin cleavage. The C-peptide of proinsulin is shortened to an arginine or lysine residue, and the precursor sequence of the novel proinsulin is:

[0102]

[0103] Full sequence of proinsulin proaspart band leader peptide MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICS...

example 2

[0105] Example 2: Expression of proinsulin proaspart fusion protein

[0106] The WCB obtained in Example 1 was cultured at 37° C. for 8 hours in LB medium (containing 1.5% w / v yeast powder and 0.5% w / v sodium chloride) to obtain a cell seed solution. The seed solution was inoculated into BFM medium (containing 0.6% w / v diammonium hydrogen phosphate, 0.4% w / v ammonium chloride, 1.35% w / w potassium dihydrogen phosphate, 0.139% w / w magnesium sulfate 7 water, 0.28 %w / w citric acid monohydrate, 0.8%w / w glucose, 0.3%w / w yeast powder and 1%mL / L trace element solution) were further cultivated for 8h to obtain the secondary seed liquid, and then inoculated according to the volume ratio of 1:10 to the fermenter. Cultivate for about 16 hours until the OD600 of the fermentation broth is about 150, and then add IPTG at a final concentration of 1 mM to the fermenter to induce the expression of proinsulin proaspart. Induced for 12 hours, the fermentation process was completed. Bacteria we...

example 3

[0109] Example 3: Refolding of proinsulin aspart

[0110] The inclusion body solution was filtered with a 1 μm PP filter element, the temperature was controlled at 20° C., the pH was adjusted to 11.0, and then refolded for 24-48 hours to obtain refolded proinsulin aspart.

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Abstract

The invention provides a structural design of novel proinsulin aspart and a preparation method of insulin aspart. The main steps include designing the sequence of proinsulin aspart, constructing recombinant insulin aspart engineering bacteria, inducing the expression of insulin aspart fusion protein in the form of inclusion bodies through engineering bacteria, and then obtaining mature insulin aspart through denaturation, renaturation, enzyme digestion and separation and purification. Insulin aspart API. The invention avoids the dangerous and cumbersome step of cutting with hydrogen bromide by changing the sequence of the recombinant leader peptide and C-peptide; shortens the C-peptide to 1 amino acid to reduce the quality loss of enzyme digestion conversion.

Description

technical field [0001] The invention relates to the technical field of polypeptide preparation methods, in particular to the structural design of a novel short proinsulin aspart and the method for preparing insulin aspart therefrom. Background technique [0002] Insulin is a hormone that regulates glucose metabolism in animals. This hormone is composed of two peptide chains, the A chain and the B chain. The A chain has 21 amino acids and the B chain has 30 amino acids, a total of 51 amino acids. where A 7 (Cys)-B 7 (Cys), A 20 (Cys)-B 19 (Cys) 4 cysteines form 2 disulfide bonds connecting the A chain and the B chain. In chain A there is A 6 (Cys) and A 11 (Cys) form intrachain disulfide bonds. Diabetes mellitus is characterized by elevated blood glucose levels due to deficient insulin secretion and / or increased hepatic glucose production. Insulin is a medicine for people with diabetes. [0003] The general goal of insulin analog development is to mimic physiologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/28C07K14/62C12N15/17C12N15/63
CPCC07K14/62C12N15/70C12Y115/01001C07K2319/50C07K2319/35A61K38/00
Inventor 汤传根潘尚书刘晓锐李宬崔怀言陈松张昊宁丁捷飞
Owner AMPHASTAR NANJING PHARMA
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