Disulfide-bond formation between SAV muteins and cysteine-containing tags.

<p>(A) Schematic of reducing and non-reducing SDS-PAGE analyses of disulfide-bonded complexes. Two BLA-L-SBP(A18C) tags (orange for BLA and grey for SBP) are covalently linked to a tetrameric SAVSBPM32 (blue) by disulfide bonds (shown as dashed lines). All samples were boiled prior to gel loading. One set of samples was analysed under reducing conditions resulting in separation of streptavidin subunits (16.5 kDa) from BLA-L-SBP(A18C) (36 kDa) as shown in panel B. The second set of samples was not reduced resulting in disulfide- bonded heterodimeric BLA-L-SBP(A18C)-SAVSBPM32 (51.5 kDa) complexes and streptavidin subunits (16.5 kDa) as shown in panel C. (B) Protein analysis by SDS-PAGE under reducing conditions. The ratio of SAV to BLA fusion proteins was 1 μM tetrameric SAV to 4 μM fusion protein in lanes 5–7. +ME indicates the presence of β-mercaptoethanol in the sample loading buffer. (C) Protein analysis by SDS-PAGE under non-reducing conditions. The ratio of SAV to BLA fusion proteins was 1 μM tetrameric SAV to 4 μM fusion protein in lanes 5–7.-ME indicates the absence of β-mercaptoethanol in the sample loading buffer. (D) Both the expected and apparent molecular masses for each species are shown. M96 and M32 represent SAVSBPM96 and SAVSBPM32, respectively. A18C and CPFB(-2) [or -2] represent BLA-L-SBP(A18C) and the non-biotinylated BLA-L-CPFB(-2), respectively.