Methods for improving expression levels of foreign proteins by means of phospholipase fusion expression

Patent metadata

Field	Value
Publication number	US-10323247-B2
Application number	US-201815995134-A
Country	US
Kind code	B2
Filing date	Jun 1, 2018
Priority date	Jan 15, 2016
Publication date	Jun 18, 2019
Grant date	Jun 18, 2019

Abstract

The invention discloses a method for improving the extracellular expression level of a foreign protein by means of phospholipase fusion expression. Four proteins, PLA 2 , MBP, CBD and SUMO, are used as a fusion tag to construct a fusion gene. Compared with an original protein MOH without any fusion tag, the extracellular expression level and enzymatic activity of all the four fusion proteins are increased to some degree. Among them, the fusion protein using PLA 2 as the fusion tag has the highest expression level, which is 7.4 times higher than that of the original protein. Compared with other fusion tags, PLA 2 has a low molecular weight and the fusion protein having PLA2 as the fusion tag has the highest expression level (up to 12 g·L −1 in a 7 L fermentation tank for high-density fermentation). It is shown that the secretory expression of a foreign protein can be effectively increased by using PLA 2 as a fusion tag.

First claim

What is claimed is:

1. A fusion gene, comprising a gene fragment encoding a fusion tag, a gene fragment encoding a linker peptide, and a gene fragment encoding a foreign protein, wherein the linker peptide has the amino acid sequence of SEQ ID NO:1, wherein the fusion tag is selected from a group of a phospholipase A2 and mutants thereof consisting of
(a) a phospholipase A2 having the amino acid sequence of SEQ ID NO: 3;
(b) a mutant of the sequence (a), wherein said mutant of the sequence (a) has the first four amino acids deleted from the sequence (a);
(c) a mutant of the sequence (a), wherein said mutant of the sequence (a) has the amino acid at position 69, 70, 73, 74, 77 or 90 mutated to alanine in the sequence (a); and
(d) a phospholipase A2 mutant having 90% or more homology to the sequence (a).

2. The fusion gene of claim 1 , wherein the foreign protein is a prolyl endopeptidase.

3. The fusion gene of claim 2 , wherein the prolyl endopeptidase has the amino acid sequence of SEQ ID NO: 4.

4. The fusion gene of claim 1 , wherein the fusion gene is inserted into an expression vector.

5. The fusion gene of claim 4 , wherein the expression vector with the fusion gene is transformed in a host microorganism to obtain an genetically engineered microorganism expressing the fusion gene.

6. The fusion gene of claim 5 , wherein the genetically engineered microorganism is yeast Pichia pastoris comprising the fusion gene of claim 1 inserted into vector pPICZαA.

7. A method for expressing a foreign protein, comprising constructing a fusion gene of claim 1 and expressing the fusion gene.

8. The method of claim 7 , wherein the foreign protein is a prolyl endopeptidase.

9. The method of claim 8 , wherein the fusion tag comprises a phospholipase A2 having the amino acid sequence of SEQ ID NO: 3.

10. The method of claim 8 , wherein the fusion tag comprises a mutant of SEQ ID NO:3, wherein said mutant of SEQ ID NO:3 has a point mutation to alanine at position 69, 70, 73, 74, 77 or 90.

Assignees

• Yu Xiaowei
• Xu Yan
• Univ Jiangnan

Inventors

• Yu Xiaowei
• Xu Yan

Classifications

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• C12Y301/01004

  Phospholipase A2 (3.1.1.4) · CPC title

• C12N9/20

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12N15/81

  for yeasts · CPC title

• C07K14/38

  from Aspergillus · CPC title