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Preparation method for yeast recombinant expression of insulin aspart

A technology of insulin aspart and yeast is applied in the field of recombinant protein preparation under biotechnology, which can solve the problems of high enzyme dosage and low yield, and achieve the effects of high enzyme cleavage efficiency, high coupling rate and low miscleavage rate.

Active Publication Date: 2019-12-03
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of this method is lower than 70%, and the enzyme consumption is on the high side

Method used

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  • Preparation method for yeast recombinant expression of insulin aspart
  • Preparation method for yeast recombinant expression of insulin aspart
  • Preparation method for yeast recombinant expression of insulin aspart

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0038] Example 1. Upstream construction and screening of recombinant insulin aspart

[0039](1) Design its cDNA sequence based on the amino acid sequence of recombinant human proinsulin proaspart, and design two stop codon sequences TGA and TAA behind the sequence, and design XhoI (CTCGAG) and NtoI ( GCGGCCGC) restriction enzyme site. Entrust Dalian TAKARA Engineering Co., Ltd. to carry out the whole gene synthesis.

[0040] (2) Insert the Pro-Asp cDNA synthesized by the above-mentioned whole gene into the PMD18-T-Pro-Asp plasmid. PMD18-T-Pro-Asp and pPICZα A were digested with restriction enzymes respectively, and two target fragments (Pro-Asp and pPICZα A large fragment) were recovered, linked by T4 and transformed into P. Pastoris X-33 host bacteria In this method, engineering bacteria (pPICZαA-Pro-Asp / X-33) were obtained.

[0041] (3) By screening highly expressed engineering bacteria and optimizing the fermentation process, recombinant proinsulin aspart was obtained. ...

example 2

[0051] Example 2. Preparation of insulin aspart

[0052] (1) Purification to obtain proinsulin aspart and enzymatic digestion to obtain missing B30-insulin aspart: 5L of Pichia pastoris fermentation broth, centrifuged, and the supernatant was subjected to copper ion chelation chromatography and SP cation exchange chromatography to obtain aspart Proinsulin; then add 20mM Zn ion to precipitate proinsulin aspart, centrifuge, dissolve the precipitate with 100ml 50mM Tris, the protein content is 1g, adjust the pH to 8.8, and add lysyl in the ratio of enzyme to protein at 1:5000 Peptidase (Lys-C) 0.2 mg, digestion at 30°C with stirring. After 16 hours, samples were taken for RP-HPLC analysis, and the enzyme digestion efficiency reached 90%, and the pH was adjusted to 5.5 for isoelectric point precipitation.

[0053] (2) Coupling of desB30-insulin aspart to obtain insulin aspart ester: take isoelectric point precipitate desB30-insulin aspart, centrifuge, dissolve with 5ml 10% glacia...

example 3

[0057] Example 3. Optimization of the preparation process of insulin aspart

[0058] (1) Purification to obtain proinsulin aspart and enzymatic digestion to obtain DesB30-insulin aspart: Pichia pastoris fermentation broth, centrifugation, supernatant per copper ion chelation chromatography and SP cation exchange chromatography for proaspart; Then add Zn to precipitate proinsulin aspart, centrifuge, dissolve the precipitate with 300ml 50mM Tris, the protein content is 2.5g, adjust the pH to 9.0, add lysyl endopeptidase (Lys -C) 0.25mg, digested with stirring at 37°C. After 16 hours, samples were taken for RP-HPLC analysis, and the enzyme digestion efficiency reached 95%, and the pH was adjusted to 5.0 for isoelectric point precipitation.

[0059] (2) Deletion of B30-insulin aspart coupling to obtain insulin aspart ester: take the isoelectric point precipitate DesB30-insulin aspart and centrifuge, dissolve with 10ml 10% glacial acetic acid, the protein concentration is 100mg / ml...

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Abstract

The invention discloses a preparation method for insulin aspart through recombinant expression by using yeast, and concretely relates to a preparation method for insulin aspart through recombinant expression by using pichia yeast. Concretely, the method comprises the following technological process: effectively secreting and expressing human aspart proinsulin, performing lysyl endopeptidase single enzyme digestion to obtain insulin aspart deleting B30, coupling with a threoninate, and performing deprotection, anti-phase purification and crystallization. The method is relatively suitable for industrialized preparation of recombinant insulin aspart.

Description

technical field [0001] The invention belongs to the field of preparation of recombinant protein under biotechnology. The invention discloses a preparation method for recombinantly expressing insulin aspart by yeast. Background technique [0002] Diabetes mellitus is a metabolic disorder characterized by hyperglycemia caused by defective insulin secretion or insulin resistance. At present, China has become the country with the largest number of diabetic patients in the world. Diabetes has become the third most harmful disease after cardiovascular and cerebrovascular diseases and tumors. Diabetic patients are often accompanied by a variety of complications, which can easily lead to kidney, eye, heart and blood vessel damage, dysfunction and failure of many organs, which is extremely harmful. [0003] The deviation between the peak of postprandial glucose and the peak arrival time of injected insulin brings difficulties to the application of insulin injection, thus promoting...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C07K14/62C12P21/06C12R1/84C12R1/865
Inventor 冯一建范开何勇
Owner CHONGQING PEG BIO BIOTECH CO LTD
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