Cysteine-depleted muteins of biologically active proteins
AssigneeCetus Corporation
InventorsDavid F. Mark, Leo S. Lin, Shi-da Yu Lu, Alice M. Wang
Life of this patent
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Abstract
Muteins of biologically active proteins such as IFN-.beta. and IL-2 in which cysteine residues that are not essential to biological activity have been deleted or replaced with other amino acids to eliminate sites for intermolecular crosslinking or incorrect intramolecular disulfide bridge formation. These muteins are made via bacterial expression of mutant genes that encode the muteins that have been synthesized from the genes for the parent proteins by oligonucleotide-directed mutagenesis.
Claims22
A synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biological activity, said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein.
A structural gene having a DNA sequence that encodes a synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is nonessential to said biologically activity, said native mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein.
A method for making a gene having a DNA sequence that encodes a synthetic mutein of a biologically active native protein which native protein has at least one cysteine residue that is free to form a disulfide link and is non-essential to said biological activity, said mutein having at least one of said cysteine residues substituted by another amino acid and said mutein exhibiting the biological activity of said native protein comprising:
- (a) hybridizing single-stranded DNA comprising a strand of a structural gene that encodes said protein with a mutant oligonucleotide primer that is complementary to a region of said strand that includes the codon for said cysteine residue or the anti-sense triplet paired with said codon, as the case may be, except for a mismatch with said codon or said antisense triplet which mismatch defines a triplet that codes for said other amino acid;
- (b) extending the primer with DNA polymerizes to form a mutational heteroduplex;
- and
- (c) replicating said mutational heteroduplex
Dependencies are inferred from the claim text.