Expression vectors with promoter and nucleic acid

Patent metadata

Field	Value
Publication number	US-10494622-B2
Application number	US-201715712652-A
Country	US
Kind code	B2
Filing date	Sep 22, 2017
Priority date	May 31, 2011
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

The aim of the invention is to improve the secretion of a protein from a host cell in order to increase the product yield of protein in a fermentation process. This is achieved by an expression vector comprising a) a promoter sequence and b) a nucleic acid sequence that codes for a protein. The protein comprises a signal peptide and an additional amino acid sequence, and the signal peptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence specified in SEQ ID NO: 2, at least 80% identical to the amino acid sequence specified in SEQ ID NO: 4, at least 80% identical to the amino acid sequence specified in SEQ ID NO: 6, or the signal peptide comprises an amino acid sequence that is structurally homologous to at least one of said sequences.

First claim

The invention claimed is:

1. An expression vector comprising:
a) a promoter sequence; and
b) a nucleic acid that encodes a protein comprising a signal peptide and a subtilisin, wherein the signal peptide comprises an amino acid sequence which is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4, wherein the signal peptide is heterologous to the subtilisin, and
wherein the expression vector achieves improved secretion of the protein from a host cell containing the expression vector as compared to secretion achieved by the expression vector having a signal peptide other than the signal peptide of b).

2. The expression vector of claim 1 , wherein the signal peptide has the amino acid sequence of SEQ ID NO: 4.

3. The expression vector of claim 1 , wherein the signal peptide is arranged N-terminal to the subtilisin in the protein encoded by the nucleic acid sequence b).

4. The expression vector of claim 1 , wherein the protein encoded by the nucleic acid sequence b) further comprises a connecting sequence arranged between the signal peptide and the subtilisin sequence of the protein, the length of the connecting sequence consisting of between 1 and 50 amino acids.

5. The expression vector of claim 1 , wherein the subtilisin comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 10 and has the amino acid glutamic acid (E) or aspartic acid (D) at position 99 in the numbering according to SEQ ID NO: 10.

6. A nonhuman host cell comprising the expression vector of claim 1 .

7. The host cell of claim 6 , wherein it is a bacterium.

8. A method for preparing a protein, comprising the steps of:
(a) culturing the host cell of claim 6 ; and
(b) isolating the protein from the culture medium or from the host cell.

9. The host cell of claim 7 , wherein the bacterium is selected from the group consisting of the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas.

10. The host cell of claim 7 , wherein the bacterium is selected from the group consisting of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor , and Stenotrophomonas maltophilia.

11. The expression vector of claim 1 , wherein the expression vector achieves improved secretion of the protein from a host cell containing the expression vector as compared to secretion achieved by the expression vector having signal peptide SubC ( Bacillus licheniformis ).

Assignees

• Basf Se

Inventors

• Degering Christian
• Eggert Thorsten
• Evers Stefan
• Maurer Karl-Heinz
• Bongaerts Johannes

Classifications

• C12N15/625 Primary

  containing a sequence coding for a signal sequence · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/75

  for Bacillus · CPC title

• C12N9/50 Primary

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title