Fusion protein and method for preparing liraglutide intermediate polypeptide

Abstract

The invention belongs to the technical field of polypeptide preparation, and in particular, relates to a fusion protein and a method for preparing a liraglutide intermediate polypeptide GLP-1 (7-37).The method includes the main steps: constructing recombinant liraglutide intermediate engineering bacteria, expressing a GLP-1 (7-37) fusion protein by culture and induction of escherichia coli, and carrying out denaturation, renaturation, enzyme digestion and separated purification to obtain the intermediate polypeptide GLP-1 (7-37). Through changing a sequence of leading peptide, the expressionmode becomes an intracellular insoluble inclusion body expression, and the expression quantity is remarkably increased; inclusion bodies after washing are dissolved at high pH, and a large amount of denaturants are not needed to use, the inclusion bodies after washing are added to a inclusion body dissolved buffer solution at a high protein concentration of 5-40 g / L, the renaturation time does notexceed 1 h, and enterokinase digestion is carried out immediately after dissolution; the renaturation process can be reduced, the enzymatic digestion system is reduced, the cost of chemical reagentscan be reduced, and industrial amplification is facilitated; ion exchange separation and purification can be used, and the separation degree is high. The prepared liraglutide intermediate polypeptideGLP-1 (7-37) reaches 92% or more and the yield is more than 87%.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and polypeptide preparation methods, in particular to a fusion protein and a method for preparing 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...

AI Technical Summary

Problems solved by technology

The above-mentioned fusion protein obtains high-yield and high-purity polypeptide GLP-1 (7-37) through operations such as dissolution, renaturation, enzyme digestion, and separation, which solves the problems of many impurities, low yield, and use in the prior art. A large number of organic reagents are not friendly to the environment, soluble expression in cells leads to low expression, inclusion body dissolution and renaturation time are long, and low protein concentration leads to too large renaturation volume, which is not suitable for large-scale production and limits the possibility of increasing production capacity. question

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