Fig. 3.

Generation of cis-divalent streptavidin. (a) Amino acid sequence of core streptavidin (SA), along with Dead (Dd) containing a polyaspartate insertion in the 3/4 loop (red and underlined) and residues impairing biotin binding in blue. (b) Generation of the different valency forms of SA/Dd by mixed refold and ion exchange, with predicted pI of monomer and tetramer indicated. (c) Ion-exchange chromatogram of a mixture of tetramers containing different proportions of SA and Dd. (d) Analysis of peaks from ion exchange by SDS-PAGE with Coomassie staining, unboiled (left) to show tetramer mobility, or boiled (right) to show subunit composition.