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GLP-1(7-37) polypeptide analogue

1. GLP-1M, peptide technology, applied in the direction of fusion peptide, peptide, hybrid peptide, etc., can solve the problems of increasing the risk of drug side effects, reducing biological activity, and half-life of only 13 hours

Inactive Publication Date: 2018-04-06
北京亦庄国际蛋白药物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its shortcoming is that the half-life in vivo is only 13 hours, and it needs to be administered every day
And according to the latest structural information analysis, although GLP-1 fatty acid modification has greatly improved its half-life in vivo, but because fatty acids hinder its binding to receptors, resulting in a decrease in its biological activity, the measurement is tens to hundreds of times that of other products
This greatly increases the cost of its medication and increases the risk of drug side effects

Method used

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  • GLP-1(7-37) polypeptide analogue
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Experimental program
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Embodiment l

[0043] Embodiment 1: the design of the GLP-1M gene of codon optimization, synthesis and expression plasmid construction

[0044] According to the designed protein sequence, use software to optimize the codon design of its polynucleotide sequence. After optimization, add the necessary sequence between promoter and rbs sequence, rbs sequence, rbs and start expression codon at the 5 end of its polynucleotide sequence ATG spacer sequence, ATG and six histidine codons and other sequences. The above sequence was entrusted to Shanghai Jierui Company to synthesize, and the synthetic gene was inserted into the expression vector pET24(+) between BamH1 / SalI to complete the construction of the expression plasmid. The sequence involved is as follows

[0045] Protein sequence GLP-1M(A8S / V33R)

[0046] H SEGTFTSDVS SYLEGQAAKE FIAWLRRGRG

[0047] Synthetic polynucleotide sequence for expressing SUMO-GLP-1M(A8S / V33R) fusion protein

[0048] ATTTTGTTTAACTTTAATAAGGAGATATACCATGCATCACCATCATCAC...

Embodiment 2

[0053] Example 2: SUMO-GLP-1M (A8S / V33R) and SUMO-GLP-1M (A8S / V33R / G16E / A24E) protein expression

[0054] The recombinant vector with correct sequencing results was transformed into Escherichia coli BL21 (DE3) (E. coli) host cells, and used as an engineered bacterium expressing recombinant proteins to express SUMO-GLP-1M protein. The engineering culture medium is 2×YT medium (10g / L tryptone; 5g / L yeast powder; 10g / L NaCl). Pick a single spot of bacteria containing the recombinant plasmid and place it in 10 ml of 2×YT medium, shake at 230 revolutions per minute (rpm), and culture overnight at 37°C. Transfer 5ml of overnight bacteria into 500ml of 2×YT liquid medium, shake and culture at 37°C until the recombinant engineered bacteria grow to OD600nm≈0.4~1, add IPTG at a final concentration of 0.2mM to induce, and carry out 16h recombinant protein at 30°C expression. see results figure 1

Embodiment 3

[0055] Example 3: Polypeptide purification, fatty acid modification and purification after modification

[0056] Cell collection and crushing: Centrifuge the fermentation culture, discard the supernatant, harvest the bacterial pellet, and weigh it; wash the pellet with buffer A (pH 8.0, 50mM PB, 500mM NaCl), and then resuspend it in buffer A for ultrasonication Crush, and then centrifuge the bacteriostasis solution with a high-speed centrifuge (16000 rpm, 30 min, 4° C.), and collect the supernatant.

[0057] Affinity chromatography and enzyme digestion: put 10ml of Ni affinity chromatography medium into the affinity column, equilibrate the chromatography column with buffer A, then load the sample, wash with Buffer L until no protein flows out, and the affinity is completed. The SUMO-GLP-1M fusion protein was collected by elution with Buffer A containing 300mM imidazole. Add SUMO protease at a mass ratio of 1:100 to digest the fusion protein overnight for 4 nights.

[0058] H...

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Abstract

The invention relates to a new GLP-1(7-37) analogue. The GLP-1(7-37) analogue contains mutant A8S and V33R. The polypeptide analogue has higher bioactivity and longer biological half life period afterbeing improved. The invention also relates to a fatty acid modified compound of the new GLP-1(7-37) analogue and application of the fatty acid modified compound in treatment of diabetes mellitus.

Description

technical field [0001] The present invention relates to the treatment of type two diabetes. More specifically, the present invention relates to GLP-1(7-37) polypeptide analogs and fatty acid modifications of the polypeptide analogs, the preparation methods of these modifications, and the use of these modifications in hypoglycemic drugs. Background technique [0002] Diabetes is a disorder of glucose metabolism caused by genetic and environmental factors, and has become the third major disease that threatens human health and life safety after tumors and cardiovascular and cerebrovascular diseases. Diabetes itself does not necessarily cause harm, but long-term blood sugar increases, large blood vessels and microvessels are damaged and endangers the heart, brain, kidney, peripheral nerves, eyes, feet, etc. According to the statistics of the World Health Organization, there are more than 100 complications of diabetes, which is currently the A disease with the most known complic...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K19/00C12N15/62A61K38/26A61K47/42A61P3/10
CPCC07K14/605A61K38/00C07K2319/30C07K2319/31
Inventor 许峥李峰张华吴玲
Owner 北京亦庄国际蛋白药物技术有限公司
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