Method for producing proteins
AssigneeKyowa Hakko Kirin Co., Ltd.
InventorsMegumi Kurokawa, Yoko Hayashi, Masayoshi Tsukahara
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Abstract
This invention relates to a method for producing a protein of interest, comprising introducing an expression vector which comprises a gene fragment comprising a DNA encoding the protein of interest and a selectable marker gene and also comprises a pair of transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell; integrating the gene fragment inserted between the pair of transposon sequences into a chromosome of the mammalian cell; obtaining a suspension mammalian cell producing the protein of interest; and suspension-culturing the suspension mammalian cell, and a suspension mammalian cell which expresses the protein of interest by the method.
Claims42
A method for producing a protein of interest, comprising introducing an expression vector which comprises a gene fragment comprising a DNA encoding the protein of interest and an attenuated selectable marker gene and also comprises a pair of transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell;
- integrating the gene fragment comprising the DNA encoding the protein of interest inserted between the pair of transposon sequences into a chromosome of the mammalian cell;
- obtaining a mammalian cell which expresses the protein of interest;
- and suspension-culturing the mammalian cell.
A method for producing a protein of interest, comprising the following steps (A) and (B):
- (A) a step of simultaneously introducing the following expression vectors (a) and (b) into a suspension mammalian cell;
- integrating a gene fragment inserted between a pair of transposon sequences into a chromosome of the mammalian cell by a transiently expressed transposase;
- and obtaining a suspension mammalian cell which expresses the protein of interest:
- (a) an expression vector which comprises the gene fragment comprising a DNA encoding the protein of interest and an attenuated selectable marker gene and also comprises the pair of transposon sequences at both terminals of the gene fragment,
- (b) an expression vector which comprises a DNA encoding the transposase which recognizes the transposon sequences and has activity of transferring the gene fragment inserted between the pair of transposon sequences into the chromosome,
- (B) a step of suspension-culturing the suspension mammalian cell which expresses the protein of interest to produce the protein of interest.
A suspension mammalian cell, in which an expression vector which comprises a gene fragment comprising a DNA encoding a protein of interest and an attenuated selectable marker gene and also comprises a pair of transposon sequences at both terminals of the gene fragment is introduced, and, wherein the gene fragment inserted between the pair of transposon sequences is integrated into a chromosome of the suspension mammalian cell, and the suspension mammalian cell produces the protein of interest.
A suspension mammalian cell, which has a chromosome into which a gene fragment inserted between a pair of transposons is integrated and which produces a protein of interest obtainable by simultaneously introducing the following vectors (a) and (b):
- (a) a protein expression vector which comprises a gene fragment comprising a DNA encoding the protein of interest and an attenuated selectable marker gene and also comprises the pair of transposon sequences at both terminals of the gene fragment,
- (b) an expression vector which comprises a DNA encoding a transposase which recognizes the transposon sequences and has activity of transferring the gene fragment inserted between the pair of transposon sequences into the chromosome.
An expression vector, which comprises a gene fragment comprising a DNA encoding a protein of interest and an attenuated selectable marker, and also comprises a pair of transposon sequences at both terminals of the gene fragment.
Dependencies are inferred from the claim text.