Method for improving recombinant human albumin expression quality and reducing degradation and application

A technology for human albumin and mass reduction, applied in the field of genes, can solve problems such as affecting the quality of recombinant proteins in purification work, and achieve the effect of reducing wrong shearing and improving protein quality

Inactive Publication Date: 2020-07-03
TONGHUA ANRATE BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The yeast expression system has the advantages of high fermentation density, strong secretion ability, and low degree of glycosylation, but the expression of foreign protein is also accompanied by t

Method used

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  • Method for improving recombinant human albumin expression quality and reducing degradation and application
  • Method for improving recombinant human albumin expression quality and reducing degradation and application
  • Method for improving recombinant human albumin expression quality and reducing degradation and application

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0132] Example 1 Construction of engineering bacteria containing only human albumin gene

[0133] Recombinant engineering bacteria I

[0134] The transformed recombinant plasmid is pPic9-HSA, and its structural diagram is as follows figure 1 shown. The coding sequence of human albumin signal peptide is shown in SEQ ID NO.1, and the coding sequence of human albumin mature peptide is shown in SEQ ID NO.4.

Example Embodiment

[0135] Example 2 Construction of engineering bacteria co-expressing yeast protein disulfide isomerase

[0136] Recombinant engineering bacteria II

[0137] The pPicZA-PDI (structural schematic diagram shown in figure 2 shown) and pPic9-HSA plasmid were recombined into the KM71 cell genome twice, the coding sequence of human albumin signal peptide is shown in SEQ ID NO.1, and the coding sequence of human albumin mature peptide is shown in SEQ ID NO.3. The coding sequence of the yeast protein disulfide isomerase signal peptide is shown in SEQ ID NO.6, and the coding sequence of the mature peptide is shown in SEQ ID NO.7.

[0138] Recombinant engineering bacteria III

[0139] The pPic9-HSA-PDI (structural schematic diagram shown in image 3 shown) and pPicZA-HSA-PDI (schematic diagram shown in Figure 4 The plasmid was recombined into the KM71 cell genome twice, the coding sequence of human albumin signal peptide is shown in SEQ ID NO.1, and the coding sequence of human albu...

Example Embodiment

[0140] Example 3 Construction of engineering bacteria co-expressing human protein disulfide isomerase

[0141] Recombinant engineered bacteria IV

[0142] The pPicZA-PDI and pPic9-HSA plasmids were respectively recombined into the KM71 cell genome twice, the coding sequence of human albumin signal peptide is shown in SEQ ID NO.1, and the coding sequence of human albumin mature peptide is shown in SEQ ID NO.3. Show. The coding sequence of the human protein disulfide isomerase signal peptide is shown in SEQ ID NO.9, and the coding sequence of the mature peptide is shown in SEQ ID NO.11.

[0143] Recombinant engineering bacteria V

[0144] The transformed recombinant plasmid is pPic9-HSA-PDI, and the schematic diagram is as follows image 3 shown. The coding sequence of human albumin signal peptide is shown in SEQ ID NO.1, and the coding sequence of human albumin mature peptide is shown in SEQ ID NO.3. The coding sequence of the human protein disulfide isomerase signal pepti...

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Abstract

The present invention relates to the field of genes, and particularly relates to a method for improving the recombinant human albumin expression quality and reducing degradation and an application. The present invention relates to the method for improving the recombinant human albumin expression quality and reducing the degradation. By over-expressing protein disulfide isomerase (PDI) in recombinant host cells, wrong shear in a protein secretion pathway is reduced, protein degradation is reduced, and the quality of exogenous protein expression is improved.

Description

technical field [0001] The invention relates to the field of genes, in particular to a method and application for improving the expression quality of recombinant human albumin and reducing degradation. Background technique [0002] The yeast expression system has the advantages of high fermentation density, strong secretion ability, and low degree of glycosylation, but the expression of foreign protein is also accompanied by the expression of a certain amount of protease, so that the expressed foreign protein is degraded to varying degrees. It directly affects the subsequent purification work and the quality of the recombinant protein. [0003] Yeast cells contain secretory pathway proteases (kex2 protease, Yap3 protease) and vacuolar proteases (protease A, protease B, carboxypeptidase Y). The foreign protein polypeptide chain is synthesized in the ribosome, then enters the endoplasmic reticulum to be folded under the traction of the signal peptide, and after being processe...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/14C12N15/61C12R1/84C12R1/78C12R1/72
CPCC07K14/765C12N9/90C12N15/815C12Y503/04001
Inventor 项炜韩旭姜钧茹
Owner TONGHUA ANRATE BIOPHARMACEUTICAL CO LTD
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