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Purification and enzyme digestion transformation method of recombinant human insulin precursor

A recombinant human insulin and conversion method technology, which is applied in the field of preparation of recombinant human insulin, can solve the problems of cumbersome operation, low sample yield in process steps, complicated sample processing procedures, etc., and achieve the effect of improving purity yield and simplifying purification steps

Active Publication Date: 2015-12-02
SHANDONG EHUA BIOLOGICAL PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant insulin precursors are divided into inclusion bodies and secreted proteins. Secreted proteins often use cationic chromatography columns, but preliminary purification is required before samples are purified by ions. As mentioned in Chinese patent CN1854299A, insulin precursors are separated by ion chromatography Preliminary purification needs to be carried out by salting out or microfiltration steps before purification, the sample processing procedure is complicated, the operation is cumbersome, and the increase of process steps will inevitably lead to the decrease of sample yield

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  • Purification and enzyme digestion transformation method of recombinant human insulin precursor
  • Purification and enzyme digestion transformation method of recombinant human insulin precursor
  • Purification and enzyme digestion transformation method of recombinant human insulin precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preliminary Example 1 Preparation of Fermentation Supernatant Containing Recombinant Human Insulin Precursor

[0037] Refer to "Process Research on Transformation of Recombinant Insulin Precursor into Human Insulin and Insulin Detemir", Liu Haifeng, East China University of Science and Technology, PhD thesis (2013).

[0038] The artificially synthesized ILP gene and plasmid pPIC9K were digested with XhoI and EcoRI respectively, and the corresponding target fragment and large plasmid fragment were recovered. The two fragments were connected with T4DNA ligase to construct the recombinant plasmid pPIC9K::ILP, and then the constructed recombinant Plasmid pPIC9K::ILP was prepared in large quantities, linearized, transformed into host cell P.pastorisGS115 by electroporation, coated with MGY (His-) plates, and selected high-copy positive clones (among them, the amino acid sequence of the constructed insulin precursor protein (ILP) (SEQ ID NO.1) is: eeaeaeaepk (spacer peptide) ...

Embodiment 2

[0052] Example 2 Purification and enzymatic conversion method of recombinant human insulin precursor

[0053] The loading sample is the fermentation supernatant of Preliminary Example 1. The fermentation supernatant is diluted to 10 times the original volume with deionized water, and an appropriate amount of acetic acid is dissolved in the diluted sample to a final concentration of 20 mmol / L.

[0054] Ion chromatography purification method adopts SSepharoseFF (XK26 / 200, 50ml) on the chromatography workstation; the balance liquid is 20mmol / L acetic acid+0.01mol / LNaCl, the washing liquid A is 20mmol / L acetic acid+0.1mol / LNaCl, the washing liquid B is 5mmol / L hydrochloric acid, and the eluent is 0.15mol / L boric acid buffer;

[0055] All solutions except the eluent (including loading sample, balance solution, washing solution A, and washing solution B) were adjusted to pH 3.5 with hydrochloric acid or sodium hydroxide, and the pH of the eluent was adjusted to 8.5, and the column t...

Embodiment 3

[0065] Example 3 Purification and enzymatic conversion method of recombinant human insulin precursor

[0066]The loading sample is the fermentation supernatant of Preliminary Example 1, and the fermentation supernatant is diluted to 10 times the original volume with deionized water. Measure an appropriate amount of acetic acid and add it to the diluted sample to a final concentration of 20mmol / L.

[0067] The ion chromatography purification method adopts SPSepharoseFF (XK26 / 200, 50ml) on the chromatography workstation; the balance liquid is 20mmol / L acetic acid+0.01mol / LNaCl, the washing liquid A is 20mmol / L acetic acid+0.1mol / LNaCl, the washing liquid B is 5mmol / L hydrochloric acid, and eluent is 0.1mol / L tris (Tris);

[0068] All solutions except the eluent (including loading sample, balance solution, washing solution A, and washing solution B) were adjusted to pH 4.0 with hydrochloric acid or sodium hydroxide, and the pH of the eluent was adjusted to 8.0 with hydrochloric ...

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Abstract

The invention discloses a purification and enzyme digestion transformation method of a recombinant human insulin precursor and belongs to the field of preparation of recombinant human insulin and analogues thereof. In the invention, fermentation supernatant of a secretorily expressed recombinant human insulin precursor is purified by using a cation exchange chromatography column, two different washing solutions are used during washing of chromatography, the elution of target proteins is performed more quickly, the elution time is shortened, the precipitation of the target proteins is reduced and the volume of eluted samples is reduced; an eluting solution with pH 7.0 to 9.0 is used during elution, phase change operation does not need to be performed to eluted products, the eluted products are directly added into trypsin for enzyme digestion transformation and the transformation efficiency is high. The method disclosed by the invention is simple to operate, the primary purification and phase change operation of the fermentation supernatant are completed through one step of chromatography purification, the purity of the samples can be improved from 14 percent to 95 percent, the purified samples are directly subjected to enzyme digestion, the enzyme digestion transformation efficiency is above 95 percent, the production process is simplified and the cost is saved.

Description

technical field [0001] The invention relates to a method for purification and enzymatic conversion of recombinant human insulin precursor, which belongs to the field of preparation of recombinant human insulin. Background technique [0002] Diabetes has become the third chronic non-communicable disease that threatens human health after cardiovascular and cerebrovascular diseases and malignant tumors. At present, the number of people with diabetes in China has replaced India as the number one in the world. The World Health Organization (WHO) predicts that the number of people with diabetes in the world will exceed 300 million by 2025. [0003] Insulin products are one of the most effective diabetes treatment drugs. With the development of science and technology, from the extraction of animal insulin to organic synthetic insulin, semi-synthetic human insulin, to the recombinant human insulin that now occupies the main market, the industrial production process of insulin has u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K1/18C12P21/06
CPCC07K14/62
Inventor 刘海峰周祥山应欢张元兴田守生解福生方喆黄菁庞甲佩史兆松
Owner SHANDONG EHUA BIOLOGICAL PHARMA
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