Preparation method of liraglutide intermediate polypeptide by genetic recombination

Abstract

The patent relates to the field of biomedicine, and relates to a synthetic method of liraglutide intermediate polypeptide namely Arg34-GLP-1(7-37). The synthetic method includes the following steps: optimizing a liraglutide intermediate gene and constructing a recombinant expression vector; transforming escherichia coli BL21, fermenting and inducing expression of a fusion protein; and followed by,breaking cells by ultrasound, carrying out affinity purification, carrying out enzymatic digestion and passing through an affinity chromatography column to remove impurities, to obtain the liraglutide intermediate with high purity. With use of the genetic recombination method, cumbersome preparation and purification steps, excessive generation of impurities and pollutants and long production period in chemical synthesis method are avoided. Compared with previous methods, the method simplifies the production process, reduces production costs and generation of pollutants, and is more conduciveto large-scale production.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of a liraglutide intermediate polypeptide. [0002] technical background [0003] Diabetes is a common chronic disease whose symptoms include excessive urine production, constant hunger, weight loss, and vision loss. Sustained increase in blood sugar concentration can also easily cause diseases of various tissues and organs such as the heart, blood vessels, eyes, kidneys and nerves, and affect people's health. In 2017, there were 425 million diabetic patients in the world. Among all countries, China has the heaviest burden of diabetes, and the number of patients (114 million) accounts for nearly 27% of the world. It is estimated that by 2045, the number of diabetic patients in the world will further increase, reaching 700 million. [0004] Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue, which was launched in the EU in 2009, and was approved by my ...

AI Technical Summary

Problems solved by technology

For long-chain polypeptides such as liraglutide, although it can be synthesized automatically by solid phase, the production cycle is long and the yield is low

And through the chemical liquid phase synthesis, although the synthesis cycle can be effectively reduced, it will also generate a large amount of waste liquid that is not easy to be recycled.

[0010] Domestically reported patents for the synthesis of liraglutide intermediates by biological methods, such as CN108191981A, generally have two methods for obtaining liraglutide intermediates: one is to process inclusion bodies after ultrasonic crushing, and the main disadvantage is that a large amount of urea is used for denaturation. The renaturation efficiency of dilute renaturation is very low, which is not conducive to enzymatic digestion to obtain the target polypeptide; the second is to purify from the supernatant after ultrasonic breaking, because the protein in the supernatant has natural activity, which is conducive to subsequent enzyme digestion, and obtained by affinity purification. The purity of the target peptide will be relatively high

Images