A semi-recombinant preparation method of glp-1 analogs

Abstract

The invention discloses a semi-recombinant preparation method of GLP-1 analogs. The present invention ferments the Escherichia coli recombinant engineering strain with the fusion gene and the cis-solubilizing protein factor DsbA, induces expression to obtain the fusion protein, extracts and purifies the fusion protein, and conducts fixed-point chemical modification under specific in vitro conditions to obtain the fatty acid side chain The modified fusion protein molecules are finally digested with specific enzymes to obtain liraglutide and semaglutide molecules. The fusion gene has the following general structural formula: f-l-a; wherein, f is the gene encoding the chaperone protein, l is the gene encoding the connecting peptide, and a is the gene encoding the GLP-1 analogue. The invention combines the technical advantages of recombinant expression and chemical synthesis, has the characteristics of high fermentation yield, high proportion of soluble expressed protein, high modification accuracy rate, simple purification and preparation, etc., and is suitable for industrial scale-up production of liraglutide and semaglutide.

Description

technical field [0001] The invention relates to the technical field of DNA recombination and preparation of fusion proteins by chemical methods, in particular to a semi-recombinant preparation method of GLP-1 analogues. Background technique [0002] Human glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone with 37 amino acid residues, which is involved in the regulation of blood glucose metabolism, gastrointestinal secretion and metabolism, and food intake. GLP-1 stimulates insulin secretion in a glucose-dependent manner, stimulates insulin biosynthesis, promotes β-cell rescue, reduces glucagon secretion, gastric emptying, and food intake, thereby achieving a stable hypoglycemic effect and less likely to induce hypoglycemia. [0003] Due to the excellent effect of GLP-1 and its analogues in the treatment of type 2 diabetes, it has gradually occupied an important position in the diabetes treatment drug market in recent years. The biologically active GLP-1 in the hu...

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Problems solved by technology

[0010] There are two problems in the Escherichia coli production process disclosed above: one, the above-mentioned process has adopted enterokinase as a tool enzyme, and this is because Lys26, Arg34 and Arg36 etc. are contained in the Arg34-GLP-1 (7-37) fragment and can be The cleavage sites recognized by common digestive enzymes (trypsin, chymotrypsin, lysyl endonuclease, etc.) are prone to miscut GLP-1 fragments; although enterokinase can specifically recognize DDDDK linker peptides, it will not miscut GLP -1 fragment, but its enzyme activity is low. Generally, commercialized enterokinase can only digest 50μg-500μg fusion protein per unit of enzyme activity, and the digestion time is as long as 12-24h. For the digestion temperature (generally requires 4℃ -25°C) and many buffer restrictions (cannot contain more than 200mM salt, or more than 2M urea, 200mM imidazole), and often need to dialyze or replace the enzyme digestion buffer system during use; and its price is high and the amount of use is large , there is a risk of enzyme residue, which is not conducive to industrial production and cost reduction; 2. The above-mentioned processes all need to obtain the fusion protein precursor through various purification methods, and then perform enzyme digestion to obtain Arg34-GLP-1(7-37) Fragment, and then modify the Arg34-GLP-1 (7-37) fragment to obtain liraglutide molecule, the steps are cumbersome

This virtually increases downstream purification steps and costs, resulting in complex product preparation steps

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