Patent ID: 6156493
Filing Date: 2000-12-05
Classification: C07K

Abstract:
A method for separating a target substance from a heterogeneous suspension containing said target substance, comprising:a) reacting the suspension with a biotinylated binding component which binds to the target substance to form a biotinylated targeted substance complex;b) exposing the suspension containing the biotinylated target substance complex to a chimeric streptavidin tetramer comprising at least one modified monomer but less than four modified monomers, wherein the tetramer is formed by mixing wild-type monomers and a single type of modified monomer, and 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 asnaragine 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 strepavidin, 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; and(c) separating the biotinylated target substance complex from the suspension by means of the chimeric streptavidin tetramer to recover the target substance in enriched form.