Synthesis and characterisation of a small protein domain for Fab fragment labelling
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Labelling of antibodies is traditionally done by conjugation to free amine or carboxyl groups, whish may result in unspecific labelling and the risk of influecning the antigen binding site of the antibody. In this project, the use of an immunoglobulin binding protein domain, C2, modified with a photoactivable amino acid analogue, benzoylphenylalanine (BPA), enables covalent coupling to the antibody Fab fragment. This can be used as a new strategy for site-specific labelling. Previous studies include an introduction of a double mutation to the C2 domain that removes the Fc-binding properties of the rotein, making sure that the binding occurs only at the antibody Fab fragment. Incorporation of a BPA gives the domain crosslinking abilities to human antibodies. In this project, an affinity analysis shows that this previous BPA mutation removes the affinity of C2 for the murine Fab fragment, which could explain the absence of crosslinking abilities towards mouse antibodies. This thesis includes solid phase peptide synthesis of novel C2.BPA variants where the most promising candidate, with an additional benzoylphenylalanine substitution, shows crosslinking towards both human and murine antibodies of various subclasses.
Place, publisher, year, edition, pages
antibody, synthesis, covalent linkage, BPA, fab
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-173603OAI: oai:DiVA.org:kth-173603DiVA: diva2:853815