A kind of extraction method of insulin glargine precursor protein

A technology for insulin glargine and its extraction method, which is applied in the field of extraction of insulin glargine precursor protein, can solve the problems that the recovery rate of the precursor protein is not significantly improved, the protein content is not increased, and the content of the insulin glargine precursor protein is increased , Make up for the loss of the target protein and improve the production efficiency

Active Publication Date: 2017-01-11
YICHANG HEC CHANGJIANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking the adjustment of pH value as an example, existing reports such as US6218385 fermented broth add acid to inactivate the remaining microorganisms after fermentation, prevent broth autolysis and maintain a relatively low broth viscosity; CN200610011694. It is convenient to filter the capsule of bacteria; CN200710026682.3 is to add acid to the fermentation broth to prevent the target protein from being adsorbed by activated carbon; CN200810233835.6 is to add acid to the fermentation broth to prevent glutathione from being oxidized; Afterwards, the precipitate was diluted and acid was added to assist in dissolving natamycin, but none of the above reports involved the fermentation broth of insulin glargine precursor protein, and the starting point of adding acid was not to increase the protein content
At the same time, after the human insulin precursor fermentation broth was acidified before centrifugation, the recovery rate of its precursor protein was not significantly improved.
In addition, other reports of pretreatment of fermentation broth did not involve the increase of insulin glargine precursor protein content
[0007] As can be seen from this, there are almost no reports on how to improve the content of insulin glargine precursor protein in the extraction process in the prior art.

Method used

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  • A kind of extraction method of insulin glargine precursor protein
  • A kind of extraction method of insulin glargine precursor protein
  • A kind of extraction method of insulin glargine precursor protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Microbial strain: Pichia pastoris GS115 (commercially available)

[0029] C peptide sequence: amino acid sequence shown in SEQ ID NO:1

[0030] Fermentation broth: obtained by fermentation culture of Pichia pastoris GS115 introduced with the precursor gene of insulin glargine.

[0031] The pH of the fermentation broth was measured to be 5.0, then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control, and then 16 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and glacial acetic acid , 10% concentrated sulfuric acid and 95% phosphoric acid were adjusted to pH 1, 2, 3, 4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 1.

[0032] Table 1 HPLC detection results of insulin glargine precursors adjusted by different acids

[0033] pH1234 5 (control) 1.2mol / L hydrochloric acid173179171129100 ...

Embodiment 2

[0036] Microbial strain: Pichia pastoris GS115 (commercially available)

[0037] C peptide sequence: amino acid sequence shown in SEQ ID NO:1

[0038] Fermentation broth: same as Example 1

[0039] The pH value of the fermentation broth was measured to be 5.0, and then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control. Then 10 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and 95% Phosphoric acid was adjusted to pH 1.2, 1.5, 1.8, 2.1, 2.4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 2.

[0040] Table 2 HPLC detection results of insulin glargine precursors adjusted by different acids

[0041] pH1.21.51.82.12.4 5 (control) 1.2mol / L hydrochloric acid170172172179165100 95% phosphoric acid133137160185178100

[0042] It can be seen from Table 2 that adding acid treatment according to the met...

Embodiment 3

[0044] Microbial strain: Saccharomyces cerevisiae MT663 (commercially available)

[0045] C peptide sequence: amino acid sequence shown in SEQ ID NO:1

[0046] Fermentation broth: obtained by fermentation culture of Saccharomyces cerevisiae MT663 introduced with the precursor gene of insulin glargine.

[0047] The pH of the fermentation broth was measured to be 5.2, then the supernatant was centrifuged and the concentration of the precursor of insulin glargine was measured by HPLC as a standard value of 100 as a control, and then 10 parts of the fermentation broth of the same volume were taken, using 1.2mol / L hydrochloric acid and 95% Phosphoric acid was adjusted to pH 1.2, 1.5, 1.8, 2.1, 2.4, and then centrifuged to take the supernatant for comparison. The results are shown in Table 3.

[0048] Table 3 HPLC detection results of insulin glargine precursors adjusted by different acids

[0049] pH12345 5.2 (Control) 1.2mol / L hydrochloric acid161170160119107100 95% phosphoric acid13...

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Abstract

The invention relates to the field of biochemistry and discloses an extraction method of an insulin glargine precursor protein. The extraction method comprises the following steps of adding an acid into an insulin glargine precursor protein fermentation broth to adjust a pH value to 1-4, carrying out centrifugation and collecting a supernatant so that the insulin glargine precursor protein is obtained. Before bacterium centrifugation, the acid is used to adjust a pH value of the insulin glargine precursor protein fermentation broth to 1-4 so that after extraction, insulin glargine precursor protein content is improved, target protein loss caused by direct centrifugation removal of the bacteria is avoided and an insulin glargine yield is improved.

Description

[0001] This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on November 21, 2012, the application number is 201210475070.3, and the invention title is "a method for extracting insulin glargine precursor protein", the entire content of which is incorporated by reference In this application. Technical field [0002] The invention relates to the field of biochemistry, in particular to an extraction method of insulin glargine precursor protein. Background technique [0003] In the treatment of diabetes, insulin has an irreplaceable role in controlling blood sugar standards and preventing complications. It is currently one of the most effective treatments for diabetes. In recent years, with the development of insulin technology, insulin analogues have been developed. One of the long-acting insulin analogues, insulin glargine, is being accepted and used by more and more doctors and patients. [0004] Insulin glargine is a biosynthet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/14
Inventor 张鸿宁荣良张宏达封海生王超陈小锋李利佳徐军
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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