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Preparation method of insulin aspart

A technology for insulin aspart and preparations, which is applied in the biological field and can solve problems such as weak expression ability, complicated process, and increased drug production costs

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

This method is difficult and complex in reverse technology such as host bacterial transformation.
Due to the weak expression ability of Saccharomyces cerevisiae, the production of insulin aspart is not high, and the highest level of shaker culture of engineering bacteria is 21.5mg / L, which increases the production cost of drugs to a certain extent

Method used

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  • Preparation method of insulin aspart
  • Preparation method of insulin aspart
  • Preparation method of insulin aspart

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Construction and expression of embodiment 1 insulin aspart expression strain

[0179] The construction of the insulin aspart expression plasmid refers to the description in the examples in the patent application number 201910210102.9. The DNA fragment containing 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.

[0180] 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 named pEvol-pylRs-pylT. Plasmid map such as figure 2 shown.

[0181] The c...

Embodiment 2

[0193] Example 2 Dissolution and renaturation of inclusion bodies

[0194]Add 8 mol / L urea solution to the obtained inclusion bodies, adjust the pH to 9.0-10.0 with sodium hydroxide, stir at room temperature for 1-3 hours, control the protein concentration to 10-20 mg / mL, and add β-mercaptoethanol to a final concentration of 15-20mmol / L, continue stirring for 0.5-1.0h.

[0195] Add the inclusion body solution dropwise to the renaturation buffer, dilute 5-10 times for renaturation, maintain the pH of the renaturation solution at 9.0-10.0, and stir for 10-20 hours for renaturation.

[0196] After 20 hours of renaturation, the fusion content of insulin aspart with correct renaturation and folding was detected by HPLC, and the renaturation rate was over 75%.

[0197] image 3 The SDS-PAGE electrophoresis of the insulin aspart fusion protein after the inclusion body denaturation is shown.

Embodiment 3

[0198] Embodiment 3 Enzymatic cleavage of fusion protein

[0199] Add dilute hydrochloric acid to the refolding solution to adjust the pH to 8.0-9.5, add recombinant trypsin at 1:3000, add carboxypeptidase B at 1:15000, enzymatic digestion temperature is 36-38°C, enzyme digestion time is 14-20h, Boc-insulin aspart obtained after enzyme digestion.

[0200] After 16 hours of enzyme digestion, the content of Boc-insulin aspart in the digestion solution was detected by HPLC. When the difference between the concentrations of Boc-insulin aspart detected for two consecutive hours was less than 3%, the enzyme digestion was completed. Finally, the concentration of Boc-insulin aspart in the digestion solution is 0.4-0.6 g / L, and the digestion rate is above 80%.

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Abstract

The invention provides an insulin aspart derivative and a preparation method thereof. Specifically, according to the method, insulin aspart fusion protein containing a green fluorescent protein folding unit is expressed in escherichia coli in a high-density mode, and enzyme digestion and purification are conducted on the fusion protein to prepare the insulin aspart. The method disclosed by the invention does not need to be carried out in a solid-phase organic system, process steps are reduced, environmental pollution is small, cost is lower, and the method is suitable for popularization.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to a method for preparing insulin aspart. 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 aspart, as a fast-acting insulin analogue, belongs to the third generation of insulin. It is composed of two chains A and B bridged by two half-amino disulfide bonds. Proline (Pro) at position 28 is replaced (mutated) with negatively charged aspartic acid (Asp), which uses charge repulsion to prevent insulin monomers or dime...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/20C07K1/18C07K1/06C12N15/62C12N15/70C12N1/21C12P21/06A61K38/28A61P3/10C12R1/19
CPCC07K14/62C12N15/70C12P21/06A61P3/10C07K2319/02C07K2319/50C07K2319/60A61K38/00Y02P20/55
Inventor 唐亚连陈卫李克朗
Owner NINGBO KUNPENG BIOTECH CO LTD
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