Protein production method
View Patent ↗This invention relates to a method for producing a protein of interest, comprising introducing a protein expression vector which comprises a gene fragment a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell; integrating the gene fragment inserted between a pair of the transposon sequences, into a chromosome of the mammalian cell to obtain a mammalian cell capable of expressing the protein of interest; and suspension-culturing the mammalian cell; and a suspension mammalian cell capable of expressing the protein of interest.
1. A method selected from the group consisting of (I), (II) and (III):
(I) a method for producing a protein of interest, comprising the steps of:
(1) introducing an expression vector into a mammalian cell adapted to suspension culture, wherein the expression vector comprises a DNA sequence encoding a protein of interest and a selectable marker, and wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals;
(2) integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell to obtain a mammalian cell capable of expressing the protein of interest; and
(3) culturing the mammalian cell to produce the protein of interest,
(II) a method for producing a protein of interest, comprising the steps of:
(1) simultaneously introducing expression vector (a) and expression vector (b) into a mammalian cell adapted to suspension culture, wherein expression vector (a) comprises a DNA sequence encoding a protein of interest and a selectable marker, wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals, and wherein expression vector (b) comprises a DNA sequence encoding a transposase which recognizes the transposon sequences, and wherein the transposase has an activity of integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell;
(2) transiently expressing the transposase encoded by expression vector (b) to integrate the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell; and
(3) culturing the mammalian cell to produce the protein of interest, and
(III) a method for obtaining a mammalian cell capable of expressing a protein of interest, comprising the steps of:
(1) introducing an expression vector into a mammalian cell adapted to suspension culture, wherein the expression vector comprises a DNA sequence encoding a protein of interest and a selectable marker, and wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals; and
(2) integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell,
wherein in the methods of (I)-(III), the transposon sequences are nucleotide sequences derived from a pair of Tol1 transposons or derived from a pair of Tol2 transposons, and the mammalian cell adapted to suspension culture is selected from the group consisting of: a CHO cell adapted to suspension-culture; a PER.C6 cell; a rat myeloma YB2/0 cell; and a mouse myeloma NS0 cell adapted to suspension-culture.
2. The method according to claim 1 ,
(a) wherein the mammalian cell adapted to suspension culture is a cell capable of surviving and proliferating in a serum-free medium; and/or
(b) wherein the DNA sequence encoding the selectable marker is a cycloheximide resistance gene.
3. The method according to claim 1 , wherein the mammalian cell adapted to suspension culture is a CHO cell selected from the group consisting of: CHO-K1; CHO-K1SV; DUKXB11; CHO/DG44; Pro-3; and CHO-S.
4. The method according to claim 2 , wherein the cycloheximide resistance gene is a gene encoding a mutant of human ribosomal protein L36a.
5. The method according to claim 4 , wherein the mutant is a mutant in which the residue corresponding to the proline at position 54 of the wild-type human ribosomal protein L36a is substituted with a different amino acid.
6. The method according to claim 5 , wherein the different amino acid is glutamine.
7. The method according to claim 1 ,
(I) wherein the nucleotide sequences derived from a pair of Tol2 transposons are the nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO:2 and the nucleotide sequence shown in SEQ ID NO:3; or
(II) wherein the nucleotide sequences derived from a pair of Tol1 transposons are the nucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequence shown in SEQ ID NO:15.
8. A mammalian cell capable of producing a protein of interest, selected from the group consisting of (I) and (II):
(I) a mammalian cell adapted to suspension culture comprising an expression vector which comprises a DNA sequence encoding a protein of interest and a selectable marker, and wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals to allow integration of the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell; and
(II) a mammalian cell adapted to suspension culture comprising expression vectors (a) and (b), wherein expression vector (a) comprises a DNA sequence encoding a protein of interest and a selectable marker, wherein the DNA sequence encoding the protein of interest and the selectable marker is flanked by transposon sequences at both terminals, and wherein expression vector (b) comprises a DNA sequence encoding a transposase which recognizes the transposon sequences, and wherein the transposase has an activity of integrating the DNA sequence encoding the protein of interest and the selectable marker into a chromosome of the mammalian cell,
wherein in the cells of (I)-(II), the transposon sequences are nucleotide sequences derived from a pair of Tol1 transposons or derived from a pair of Tol2 transposons, and the mammalian cell adapted to suspension culture is selected from the group consisting of: a CHO cell adapted to suspension-culture; a PER.C6 cell; a rat myeloma YB2/0 cell; and a mouse myeloma NS0 cell adapted to suspension-culture.
9. The cell according to claim 8 ,
(a) wherein the mammalian cell adapted to suspension culture is a cell capable of surviving and proliferating in a serum-free medium; and/or
(b) wherein the DNA sequence encoding the selectable marker is a cycloheximide resistance gene.
10. The cell according to claim 8 , wherein the mammalian cell adapted to suspension culture is a CHO cell selected from the group consisting of: CHO-K1; CHO-K1SV; DUKXB11; CHO/DG44; Pro-3; and CHO-S.
11. The cell according to claim 9 , wherein the cycloheximide resistance gene is a gene encoding a mutant of human ribosomal protein L36a.
12. The cell according to claim 11 , wherein the mutant is a mutant in which the residue corresponding to the proline at position 54 of the wild-type human ribosomal protein L36a is substituted with a different amino acid.
13. The cell according to claim 12 , wherein the different amino acid is glutamine.
14. The cell according to claim 8 ,
(I) wherein the nucleotide sequences derived from a pair of Tol2 transposons are the nucleotide sequence shown in SEQ ID NO:2 and the nucleotide sequence shown in SEQ ID NO:3; or
(II) wherein the nucleotide sequences derived from a pair of Tol1 transposons are the nucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequence shown in SEQ ID NO:15.