Streptavidin reductive amination

Conjugation of HRP by reductive amination can be done by oxidizing the carbohydrate on the enzyme and subsequently coupling to the amines on avidin or streptavidin (Fig. 364). 

Protocol for the Preparation of Avidin—HRP or Streptavidin—HRP by Reductive Amination... 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

The streptavidin variant is generated as a part of a screen of modifications. In this work, no asymmetric induction was obtained without protein, and the wild-type streptavidin (Sav) gave a reduction product of up to 57 % e.e. (/ ). However, the S112A mutant of Sav gave an amine in up to 96 % e.e. (R), and in other cases (e.g. S112K mutant) the enantioselectivity could even be reversed. In addition, up to 4000 turnovers of the synthetic enzyme were able to be achieved [133-137]. Computational and molecular modelling has been applied to this process to aid optimization [138]. 

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