Recombinant inactive core streptavidin mutants

Die vorliegende Erfindung betrifft Muteine von Avidin und Streptavidin mit einer verringerten Bindungsaffinität für Biotin sowie deren Verwendung als Entstörungsreagenzien in Verfahren zur Bestimmung eines Analyten, z.B. in diagnostischen Tests wie etwa in Immuno- und Nukleinsäurehybridisierungsassays. Weiterhin betrifft die Erfindung die Verwendung von Muteinen von Avidin und Streptavidin als regenerierbare Systeme zur Bindung von Biotin, z.B. zur Analyse biotinylierter Moleküle, zur Untersuchung von Rezeptor-Ligand-Wechselwirkungen sowie zur Affinitätsreinigung biotinylierter Moleküle. A new biotin-bindable polypeptide is selected from avidin or streptavidin muteins that differ from the native polypeptide by at least one amino acid and have a binding affinity for biotin of less than 10 l/mole; the biotin-bindable polypeptide may be present as a polymeric conjugate, e.g. with another polypeptide or protein, especially bovine serum albumin. Also claimed are: (1) a nucleic acid coding for a polypeptide as above; (2) a vector containing at least one copy of a nucleic acid as in (1); and (3) a cell transformed with a vector as in (2). Also new are: (A) the use of a biotin-bindable polypeptide selected from avidin or streptavidin muteins as a regenerable system for binding biotin, where at least one amino acid of the native polypeptide is substituted and the mutein has a binding affinity for biotin of 10 -10 l/mole; and (B) a biotin-bindable regenerable solid phase coated with a streptavidin or avidin mutein having a binding affinity for biotin of 10 -10 l/mole.