Page 91 - Tyrosine-Based Bioconjugations - Jorick Bruins
P. 91

Preparation of Trifunctional Protein-antibody Conjugates
  Figure 6. SDS-PAGE analysis of SPOCQ product between 6 and Tras[LC]G4SG4Y (lane 1), and 6 and Tras[LC]G4SG4SG4Y (lane 7), and subsequent reactions with N3-lis (lanes 2 and 8), MeTz–TAMRA (lanes 3 and 9), MeTz–UCHT1 (lane 4 and 10), MeTz–IL-2 (lanes 5 and 11), and MeTz-ODN1826 (lanes 6 and 12).
each light chain (AT1002[HC]ST–[LC]G4SG4SG4Y).21 In theory, trifunctional antibodies would be accessible by either order of event, i.e. sortase A ligation before SPOCQ or vice versa. However, both UCHT1 and IL-2 were modified with TCO or MeTz functionalities by means of sortase ligation, thus generating constructs of a format similar to: protein–LPET–GGG–click probe. Thus, it was reasoned that SPOCQ modification of antibody prior to sortase ligation of a third functionality, was likely to cause havoc, due to sortase ligation being a reversible reaction,29 and
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