Patent ID: 6165750
Filing Date: 2000-12-26
Classification: C07K

Abstract:
An isolated chimeric streptavidin tetramer comprising between one and three modified monomers,wherein the tetramer is formed by mixing wildtype monomers and a single type of modified monomer,wherein the chimeric tetramer has one or more of the following characteristics:(i) an overall altered binding affinity for biotin or a compound comprising biotin wherein a subunit modification consists of either a substitution of a hydrophobic amino acid for one or more amino acids present in the biotin binding domain or a deletion of one or more amino acids in the biotin binding domain,(ii) an altered subunit affinity conferring an enhanced sensitivity to hydrostatic pressure wherein a subunit modification consists of the substitution of an amino acid selected from the group consisting of a positively-charged amino acid, a negatively charged amino acid, or a cysteine for an amino acid present either in the region of a monomer-monomer interface or in the region of the dimer-dimer interface, and(iii) a molecular label permitting monitoring of subunit association into tetramers having specific stoichiometric ratios of modified and unmodified subunits wherein a subunit modification consists of the substitution of cysteine for asparagine at amino acid position 94 and covalent attachment of the label to the cysteine, and,(iv) a capacity to bind to a molecule other than streptavidin, biotin, or a compound comprising biotin, wherein a subunit modification consists of the substitution of cysteine for asparagine at amino acid position 94 and covalent attachment of the molecule to the cysteine.