Preparation method of liraglutide intermediate polypeptide

Abstract

The invention belongs to the technical field of preparation methods of polypeptides and particularly relates to a preparation method of liraglutide intermediate polypeptide GLP-1 (glucagon-like peptide-1) (7-37). The method comprises the following main steps: constructing recombinant liraglutide engineering bacteria, expressing liraglutide intermediate fusion protein in a form of an inclusion bodyunder Echerichia coli induction, and performing denaturation, renaturation, enzyme digestion, separation and purification to obtain the liraglutide intermediate polypeptide GLP-1 (7-37). Expression is changed into intracellular insoluble inclusion body expression by changing recombinant sequence signal peptides, and expression quantity is increased greatly; the washed inclusion body is subjectedto alkali dissolution, a large quantity of denaturant is not needed, the inclusion body with high concentration of protein concentration being 20-30 g / L is added to an inclusion body dissolution buffer, denaturation and renaturation time does not exceed 1 h, and enzyme digestion can be performed after dissolution; procedures are reduced, operation volume is reduced, reagent cost is reduced, and industrialized enlargement is facilitated; UniSP-50XS cation exchange is adopted for separation and purification, and separation degree is high. The purity of the liraglutide intermediate polypeptide prepared with the method reaches 87% or higher, and the yield is higher than 85%.

Description

technical field [0001] The invention relates to the technical field of polypeptide preparation methods, in particular to a preparation method of a liraglutide intermediate polypeptide. Background technique [0002] Diabetes mellitus is due to the interaction of genetic and environmental factors, resulting in the absolute or relative insufficiency of insulin secretion and the decrease in the sensitivity of target tissue cells to insulin, causing a series of metabolic disorder syndromes such as protein, fat, water and electrolytes, among which hyperglycemia is the main symbol . In typical clinical cases, symptoms such as polyuria, polydipsia, polyphagia, and weight loss may appear, that is, symptoms of "three excesses and one deficiency". In recent years, with the improvement of living standards, changes in diet structure, most people move less and sit more, and other factors have led to a rapid increase in the incidence of diabetes worldwide. Among them, type 1 diabetes pat...

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Problems solved by technology

[0010] The object of the present invention is to provide a preparation method of the liraglutide intermediate polypeptide GLP-1 (7-37), through genetic recombination technology, using E. 37) After the fusion protein, a high-yield and high-purity polypeptide GLP-1 (7-37) is obtained through operations such as dissolving, renaturation, enzymatic digestion, and separation, which solves the problems of many impurities and high yield in the prior art. Low, the use of a large number of reagents is not friendly to the environment, soluble expression in cells leads to low expression, inclusion body dissolution and renaturation time is long, low protein concentration leads to too large renaturation volume, not suitable for large-scale production, and limits production capacity Ascension question

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