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Insulin glargine derivative and application thereof

A technology for insulin glargine and arginine, which is applied in the field of insulin glargine derivatives, and can solve problems such as low yield, high production cost, and complicated insulin glargine process

Active Publication Date: 2021-12-10
NINGBO KUNPENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The procedure for preparing insulin glargine by this method is complicated, and the yield is low, resulting in extremely high production costs.

Method used

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  • Insulin glargine derivative and application thereof
  • Insulin glargine derivative and application thereof
  • Insulin glargine derivative and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Construction and expression of embodiment 1 insulin glargine expression strain

[0189] To construct the insulin glargine expression plasmid, the construction method refers to the existing technology in the field, specifically, refer to the description in the examples in the patent application number 201910210102.9. The DNA fragment of the fusion protein FP-TEV-R-G was cloned into the NcoI-XhoI site downstream of the araBAD promoter of the expression vector plasmid pBAD / His A (purchased from NTCC, kanamycin resistance) to obtain the plasmid pBAD-FP -TEV-R-G. Plasmid map such as figure 1 shown.

[0190] Then the DNA sequence of pylRs was cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (purchased from NTCC Company, chloramphenicol resistance), and at the same time, the pylRs was inserted downstream of the proK promoter by PCR. DNA sequence of tRNA (pylTcua) of aminoacyl-tRNA synthetase. This plasmid was nam...

Embodiment 2

[0203]Example 2 Dissolution and renaturation of inclusion bodies

[0204] Add 8mol / L urea solution to the obtained inclusion body, adjust the pH to 8.0-9.0 with sodium hydroxide, stir at room temperature for 1-3h, control the protein concentration to 10-20mg / mL, and add β-mercaptoethanol to a final concentration of 10-20mmol / L, continue stirring for 0.5-1.0h.

[0205] Add the inclusion body solution dropwise to the renaturation buffer, dilute 5-10 times for renaturation, maintain the pH of the renaturation solution at 9.0-10.5, temperature 2-8°C, and stir for 10-20 hours for renaturation.

Embodiment 3

[0206] Embodiment 3 Fusion proteolysis

[0207] Use 10KD ultrafiltration membrane to concentrate the refolding solution 8-10 times. Add dilute hydrochloric acid to the refolding solution to adjust the pH to 7.5-9.0. The protein concentration of the refolding concentrate was determined by the Bradford method, and the total protein amount was calculated. Add recombinant trypsin under stirring, the ratio of recombinant trypsin to the total protein of refolding solution is 1:3000~1:10000, add 30mmol / L succinic acid or 30mmol / L L-lysine, and the enzymatic digestion temperature is 15- At 25°C, the digestion time is 14-20 hours.

[0208] After 10 hours of enzyme digestion, the content of Boc-insulin glargine in the enzyme digestion solution was detected by HPLC. When the difference between the concentrations of Boc-insulin glargine detected for two consecutive hours was less than 3%, the enzyme digestion was completed. Finally, the concentration of Boc-insulin glargine in the dige...

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Abstract

The invention provides an insulin glargine derivative and a preparation method thereof. Specifically, the invention provides a fusion protein comprising a green fluorescent protein folding unit and insulin glargine or an active fragment thereof. Expression quantity of the fusion protein disclosed by the invention is remarkably improved, and the insulin glargine protein in the fusion protein is correctly folded and has biological activity. Moreover, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by protease, and compared with the target protein, molecular weight difference is large, and separation is easy. The invention also provides a method for preparing insulin glargine by using the fusion protein and a preparation intermediate.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to an insulin glargine derivative and application thereof. Background technique [0002] Diabetes is a major disease that threatens human health worldwide. In China, with the change of people's lifestyle and the acceleration of the aging process, the prevalence of diabetes is rising rapidly. Acute and chronic complications of diabetes, especially chronic complications that accumulate in multiple organs, cause disability and high mortality, seriously affect the physical and mental health of patients, and bring heavy burdens to individuals, families and society. [0003] Insulin glargine achieves long-lasting effect by changing the amino acid of recombinant human insulin glargine and slightly adjusting the formula. Insulin glargine replaces asparagine at position 21 of the human insulin A chain with a charge-neutral glycine to make the hexamer more stable. Adding two ar...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/20C07K1/18C07K1/06C12N15/62C12N15/70C12N1/21A61K38/28A61K47/64A61P3/10C12R1/19
CPCC07K14/62C12N15/70A61K47/64A61P3/10C07K2319/02C07K2319/50C07K2319/60A61K38/00A61K38/28C07K1/06C07K1/18C07K1/20C07K19/00C12N15/62C12N15/74C12N15/63Y02P20/55
Inventor 陈卫刘慧玲骆莉
Owner NINGBO KUNPENG BIOTECH CO LTD
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