Production of activated protein C
AssigneeZymoGenetics, Inc.
InventorsDonald C. Foster, Mark J. Murray, Kathleen L. Berkner
Life of this patent
Estimating the term…
Abstract
Methods are disclosed for producing a protein which has substantially the same biological activity as human protein C or human activated protein C. The protein is produced by mammalian host cells transfected with a plasmid capable of integration in mammalian host cell DNA. The plasmid includes a promoter followed downstream by a nucleotide sequence which encodes a protein having substantially the same structure and/or activity as human protein C or human activated protein C the nucleotide sequence being followed downstream by a polyadenylation signal.
Claims52
An isolated DNA sequence which codes for human protein C or human activated protein C, said sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is Lys or Arg and n=1, 2 or 3, between the light and heavy chains.
A DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of factor VII, factor IX, factor X, prothrombin and protein S, positioned upstream of and operably linked to a DNA sequence which codes for human protein C or human activated protein C.
A DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 158 as shown in FIG. 2 with a non-acidic amino acid residue selected from the group consisting of Ala, Ser, Thr, and Gly.
A DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 154 as shown in FIG. 2 with an amino acid residue selected from the group consisting of Lys, Arg and Leu.
A DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of Lys-Arg at residues 156-157 as shown in FIG. 2 with Lys-Lys or Arg-Arg.
A DNA sequence which codes for human activated protein C, said sequence further coding for the amino acid sequence R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 -X-R.sub.5 -R.sub.6 -R.sub.7 -R.sub.8, wherein each of R.sub.1 -R.sub.8 is Lys or Arg and X is a peptide bond or a spacer peptide of 1-12 amino acids, between the light and heavy chains.
A DNA sequence which codes for human activated protein C, said sequence coding for the amino acid sequence L-R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 -X-R.sub.5 -R.sub.6 -R.sub.7 -R.sub.8 -H, wherein L is the light chain of protein C, each of R.sub.1 -R.sub.8 is Lys or Arg, X is a peptide bond or a spacer peptide of 1-12 amino acids, and H is the heavy chain of activated protein C.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R1, R2, R3 and R4 is Lys or Arg and n=1, 2 or 3, between the light and heavy chains.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C.
Cultured mammalian cells stably transfected with an expression vector, said expression vector including a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 158 as shown in FIG. 2 with a non-acidic amino acid residue selected from the group consisting of Ala, Ser, Thr and Gly.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 154 as shown in FIG. 2 with an amino acid residue selected from the group consisting of Lys, Arg and Leu.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of Lys-Arg at residues 156-157 as shown in FIG. 2 with Lys-Lys or Arg-Arg.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said cells further transfected with the KEX1 or KEX2 gene of Saccharomyces cerevisiae.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human activated protein C, said sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is Lys or Arg and n=0, 1, 2 or 3, between the light chain and the heavy chain, said cells further transfected with the KEX1 or KEX2 gene of Saccharomyces cerevisiae.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human activated protein C, said sequence further coding for the amino acid sequence R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 -X-R.sub.5 -R.sub.6 -R.sub.7 -R.sub.8, wherein each of R.sub.1 -R.sub.8 is Lys or Arg and X is a peptide bond or a spacer peptide of 1-12 amino acids, between the light and heavy chains.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 158 as shown in FIG. 2 with a non-acidic amino acid residue selected from the group consisting of Ala, Ser, Thr and Gly;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said mammalian host cell.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of residue 155 as shown in FIG. 2 with an amino acid residue selected from the group consisting of Lys, Arg and Leu;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said mammalian host cell.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said DNA sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is Lys or Arg and n=1, 2 or 3, between the light and heavy chains;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of factor VII, factor IX, factor X, prothrombin and protein S, positioned upstream from a promoter, the promoter being operably linked to a DNA sequence which codes for human protein C or human activated protein C;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said protein further including the substitution of Lys-Arg at residues 156-157 as shown in FIG. 2 with Lys-Lys or Arg-Arg;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
A method for producing human protein C or human activated protein C, comprising:
- transfecting a cultured mammalian host cell with an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said cell further transfected with the KEX1 or KEX2 gene of Saccharomyces cerevisiae;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
A method for producing activated protein C, comprising:
- transfecting a cultured mammalian host cell with an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of Protein C, Factor VII, Factor IX, Factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human activated protein C, said sequence further coding for the amino acid sequence R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 -X-R.sub.5 -R.sub.6 -R.sub.7 -R.sub.8, wherein each of R.sub.1 -R.sub.8 is Lys or Arg and X is a peptide bond or a spacer peptide of 1-12 amino acids, between the light and heavy chains;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
A DNA sequence which codes for human protein C or human activated protein C, said sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3, wherein each of R.sub.1, R.sub.2 and R.sub.3 is Lys or Arg and n=1, 2 or 3, between the light chain of protein C, as shown in FIG. 2 from amino acid residue 1 to residue 155, and the heavy chain of protein C.
Cultured mammalian cells transfected with an expression vector capable of integration into mammalian cell DNA, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3, wherein each of R.sub.1, R.sub.2 and R.sub.3 is Lys or Arg and n=1, 2 or 3, between the light chain of protein C, as shown in FIG. 2 from amino acid residue 1 to residue 155, and the heavy chain of protein C.
A method for producing human protein C or human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin, and protein S, operably linked to a DNA sequence which codes for human protein C or human activated protein C, said DNA sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3, wherein each of R.sub.1, R.sub.2 and R.sub.3 is Lys or Arg and n=1, 2 or 3, between the light chain of protein C, as shown in FIG. 2 from amino acid residue 1 to residue 155, and the heavy chain of protein C;
- growing said cultured mammalian host cell in an appropriate growth medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian host cell.
An isolated DNA sequence which codes for human activated protein C, said DNA sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 is Lys or Arg and n=0, 1, 2 or 3, between the light and heavy chains.
Cultured mammalian cells stably transfected with an expression vector, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin and protein S, operably linked to a DNA sequence which codes for human activated protein C, said DNA sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is Lys or Arg and n=0, 1, 2 or 3, between the light and heavy chains.
A method for producing human activated protein C, comprising:
- introducing into a cultured mammalian host cell an expression vector capable of directing the expression of a cloned gene in a mammalian cell, said expression vector comprising a promoter operably linked to a DNA sequence which codes for a pre-pro peptide of a human protein selected from the group consisting of protein C, factor VII, factor IX, factor X, prothrombin and protein S, operably linked to a DNA sequence which codes for human activated protein C, said DNA sequence further coding for the amino acid sequence (R.sub.1).sub.n -R.sub.2 -R.sub.3 -R.sub.4, wherein each of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is Lys or Arg and n=0, 1, 2 or 3, between the light and heavy chains;
- growing said cultured mammalian host cell in an appropriate medium;
- and
- isolating the protein encoded by said expression vector and produced by said cultured mammalian cell.
Dependencies are inferred from the claim text.