Vector including a translation-impaired dihydrofolate reductase gene cassette and ubiquitously acting chromatin opening element

Patent metadata

Field	Value
Publication number	US-11781146-B2
Application number	US-201917054935-A
Country	US
Kind code	B2
Filing date	May 13, 2019
Priority date	May 24, 2018
Publication date	Oct 10, 2023
Grant date	Oct 10, 2023

Abstract

The present invention provides mammalian cell expression vectors that impart to mammalian host cells an ability to produce high levels of foreign gene-derived proteins. A ubiquitously acting chromatin opening element (UCOE) is introduced into an expression vector that has a plasmid DNA integrated into a transcriptional hot spot on the chromosome of a dihydrofolate reductase gene-deficient host cell so that it allows for selection of strains that grow in hypoxanthine-thymidine (hereinafter denoted as HT)-free medium, whereby transformants will produce a protein of interest in increased amounts.

First claim

The invention claimed is:

1. An expression vector comprising the following (a), (b) and (c):
(a) a translation-impaired dihydrofolate reductase gene cassette (translation-impaired DHFR gene cassette) comprising a region with altered codons, wherein the altered codons comprise GCA for alanine, CGA for arginine, AAU for asparagine, GAU for aspartic acid, UGU for cysteine, CAA for glutamine, GAA for glutamic acid, GGU for glycine, CAU for histidine, UUA for leucine, AAA for lysine, CCA for proline, UUU for phenylalanine, UCA for serine, ACU for threonine, UAU for tyrosine, and/or GUA for valine, and wherein the region with altered codons accounts for 30% or more of the full length of the DHFR gene from the 5′ end of the DHFR gene;
(b) a gene cassette comprising a cloning site for integration of a foreign gene between a transcriptionally active promoter and a stable polyadenylation signal; and
(c) a ubiquitously acting chromatin opening element (UCOE),
wherein the UCOE comprises the nucleotide sequence as shown in SEQ ID NO: 1.

2. The expression vector of claim 1 , wherein the translation-impaired DHFR gene cassette of (a) uses a promoter derived from a gene of a non-mammalian cell or a promoter whose enhancer portion has been removed.

3. A method for producing a transformant that produces a foreign gene-derived protein, which comprises integrating a foreign gene into the expression vector of claim 1 , and transforming a dihydrofolate reductase gene-deficient host cell with the expression vector.

4. A method for producing a foreign gene-derived protein, which comprises the following (a) to (d):
(a) integrating a foreign gene into the expression vector of claim 1 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector;
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium; and
(d) collecting the foreign gene-derived protein from the cultured transformant.

5. The method of claim 4 , wherein a chemically defined medium (CD medium) or a CD medium supplemented with non-animal-based additives is used for culturing in (c).

6. A method of screening for a transformant that produces a foreign gene-derived protein, which comprises the following (a), (b) and (c):
(a) integrating a foreign gene into the expression vector of claim 1 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector; and
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium.

7. A foreign gene expression vector which has a foreign gene integrated into the expression vector of claim 1 .

8. A host cell which has been transformed with the foreign gene expression vector of claim 7 .

9. A method for producing a transformant that produces a foreign gene-derived protein, which comprises integrating a foreign gene into the expression vector of claim 2 , and transforming a dihydrofolate reductase gene-deficient host cell with the expression vector.

10. A method for producing a foreign gene-derived protein, which comprises the following (a) to (d):
(a) integrating a foreign gene into the expression vector of claim 2 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector;
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium; and
(d) collecting the foreign gene-derived protein from the cultured transformant.

11. A method of screening for a transformant that produces a foreign gene-derived protein, which comprises the following (a), (b) and (c):
(a) integrating a foreign gene into the expression vector of claim 2 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector; and
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium.

12. A foreign gene expression vector which has a foreign gene integrated into the expression vector of claim 2 .

13. The expression vector according to claim 1 , wherein the expression vector comprises more than one UCOE.

14. The expression vector according to claim 13 , wherein the UCOEs are introduced in such positions that the gene cassette of (b) is sandwiched therebetween.

15. The expression vector according to claim 2 , wherein the expression vector comprises more than one UCOE.

16. The expression vector according to claim 15 , wherein the UCOEs are introduced in such positions that the gene cassette of (b) is sandwiched therebetween.

17. A method for producing a transformant that produces a foreign gene-derived protein, which comprises integrating a foreign gene into the expression vector of claim 13 , and transforming a dihydrofolate reductase gene-deficient host cell with the expression vector.

18. A method for producing a foreign gene-derived protein, which comprises the following (a) to (d):
(a) integrating a foreign gene into the expression vector of claim 13 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector;
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium; and
(d) collecting the foreign gene-derived protein from the cultured transformant.

19. A method of screening for a transformant that produces a foreign gene-derived protein, which comprises the following (a), (b) and (c):
(a) integrating a foreign gene into the expression vector of claim 13 ;
(b) transforming a dihydrofolate reductase gene-deficient host cell with the expression vector; and
(c) culturing the resultant transformant in a hypoxanthine-thymidine-free medium.

20. A foreign gene expression vector which has a foreign gene integrated into the expression vector of claim 13 .

Assignees

• Univ Hokkaido Nat Univ Corp
• Fuso Pharmaceutical Ind

Inventors

• Suzuki Yasuhiko
• Nakagawa Miki
• Kameda Yayoi
• KONNAI Satoru
• Okagawa Tomohiro
• Maekawa Naoya
• Goto Shinya
• Sajiki Yamato
• Ohashi Kazuhiko
• Murata Shiro
• Kitahara Yuzuru
• Yamamoto Keiichi

Classifications

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12N15/85 Primary

  for animal cells · CPC title

• C12Y105/01003 Primary

  Dihydrofolate reductase (1.5.1.3) · CPC title

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title