Expression of PACE in host cells and methods of use thereof

A recombinant expression vector suitable for expression in a selected eukaryotic host cell comprising a polynucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs;

• and a polynucleotide sequence encoding a precursor polypeptide which requires gamma-carboxylation for biological activity and which is a substrate for PACE, operably linked to an expression control sequence permitting expression of the polynucleotide sequence in the eukaryotic host cell.

A recombinant expression vector suitable for expression in a selected eukaryotic host cell comprising a polynucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs;

• and a polynucleotide sequence encoding a precursor polypeptide which is a precursor of a blood coagulation protein and which is a substrate for PACE, operably liked to an expression control sequence permitting expression of the polynucleotide sequence in the eukaryotic host cell.

A method for producing a desired mature polypeptide and allowing the cleavage of a heterologous precursor polypeptide by the expressed recombinant PACE comprising culturing a transformed eukaryotic host cell comprising a heterologous polynucleotide encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs, and a polynucleotide encoding a precursor polypeptide, wherein the precursor polypeptide is a substrate for the encoded PACE and requires gamma-carboxylation for biological activity, under conditions that allow expression of both recombinant PACE and the precursor polypeptide.

A method of increasing the yield of a selected biologically active protein having a precursor which is a substrate for PACE comprising culturing a transformed mammalian or insect host cell comprising a first nucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs, operably linked to a heterologous expression control sequence permitting expression of the nucleotide sequence and a second nucleotide sequence encoding the precursor of said selected biologically active protein, said protein requiring gamma-carboxylation for biological activity, operably linked to a heterologous expression control sequence permitting expression of the second nucleotide sequence, wherein the action of said PACE on the expression product of the second nucleotide sequence permits the production of said biologically active protein.

A method of increasing the yield of a selected biologically active protein having a precursor which is a substrate for PACE comprising culturing a transformed mammalian or insect host cell comprising a first nucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs, operably linked to a heterologous expression control sequence permitting expression of the nucleotide sequence and a second nucleotide sequence encoding the precursor of said selected biologically active protein, operably linked to a heterologous expression control sequence permitting expression of the second nucleotide sequence, wherein the action of said PACE on the expression product of the second nucleotide sequence permits the production of said biologically active protein, wherein the biologically active protein is a blood coagulation protein selected from the group consisting of Factor IX, Protein C, Protein S, Prothrombin Factor X, and Factor VII.

A method of increasing the yield of a biologically active protein having a precursor which is a substrate for PACE comprising culturing a transformed mammalian or insect host cell comprising a first nucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs, operably linked to a heterologous expression control sequence permitting expression of the nucleotide sequence, and a second nucleotide sequence encoding the precursor of von Willebrand factor, operably linked to a heterologous expression control sequence permitting expression of the second nucleotide sequence, wherein the action of said PACE on the expression product of the second nucleotide sequence permits the production of biologically active von Willebrand Factor.

A method of increasing the yield of a biologically active protein having a precursor which is a substrate for PACE comprising culturing a transformed mammalian or insect host cell comprising a first nucleotide sequence encoding mammalian endopeptidase protein precursor processing enzyme, PACE, capable of cleaving precursor polypeptides at basic amino acid pairs, operably linked to a heterologous expression control sequence permitting expression of the nucleotide sequence and a second nucleotide sequence encoding the precursor of bone-gamma carboxyglutamate protein, operably linked to a heterologous expression control sequence permitting expression of the second nucleotide sequence, wherein the action of said PACE on the expression product of the second nucleotide sequence permits the production of biologically active bone-gamma carboxyglutamate protein.