The biotin-streptavidin interaction is among the strongest known in nature. Here, we report the site-directed incorporation of biotin and 2-iminobiotin bearing non-canonical amino acids (ncAA) into proteins. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. Using the high affinity binding of biotin lysine, we could specifically isolate the bacterial protein RecA and analyze its interaction partners. Furthermore, we successfully transferred our biotinylation approach to mammalian cells. The stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin by the different biotin-analogs makes this approach an attractive tool for protein interaction studies, protein immobilization and the generation of well-defined protein drug conjugates.