DNAs encoding analog GM-CSF molecules displaying resistance to proteases which cleave at adjacent dibasic residues
AssigneeImmunex Corporation
InventorsMichael C. Deeley, Virginia L. Price
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Abstract
Amplified expression of recombinant DNA products is achieved in hosts expressing proteases that cleave at multi-basic amino acid residues. To this end, cDNAs encoding granulocyte-macrophage colony stimulating factor (GM-CSF) are mutated such that one or both of the arginine residues at positions 23 and 24 of the protein product are replaced by non-basic amino acid residues. The GM-CSF analogs thus obtained maintain the activity of the wild-type protein.
Claims27
An isolated DNA molecule encoding a polypeptide that is a variant of a naturally-occurring human granulocyte-macrophage colony stimulating factor, wherein (A) at least one amino acid of a pair of adjacent arginine amino acids occurring in said factor is replaced, in said polypeptide, with a non-basic amino acid and (B) said polypeptide displays biological activity in a human bone-marrow colony assay.
A recombinant DNA cloning vector comprising a DNA sequence encoding a polypeptide that is a variant of a naturally-occurring human granulocyte-macrophage colony stimulating factor, wherein (A) at least one amino acid of a pair of adjacent arginine amino acids occurring in said factor is replaced, in said polypeptide, with a non-basic amino acid and (B) said polypeptide displays biological activity in a human bone-marrow colony assay.
A recombinant DNA expression vector comprising a DNA sequence encoding a polypeptide that is a variant of a naturally-occurring human granulocyte-macrophage colony stimulating factor, wherein (A) at least one of a pair of adjacent arginine amino acids occurring in said factor is replaced, in said polypeptide, with a non-basic amino acid and (B) said polypeptide displays biological activity in a human bone-marrow colony assay.
A method of expressing a recombinant DNA in a host which expresses a protease that cleaves at a sequence of two adjacent basic amino acid residues, comprising the step of transfecting the host with a recombinant nucleic-acid expression vector comprises a DNA sequence encoding a polypeptide that is a variant of a naturally-occurring human granulocyte-macrophage colony stimulating factor, wherein (A) at least one amino acid of a pair of adjacent arginine amino acids occurring in said factor is replaced, in said polypeptide, with a non-basic amino acid and (B) said polypeptide displays biological activity in a human bone-marrow colony assay, whereby the amount of said polypeptide is increased.
Dependencies are inferred from the claim text.