IgG antibodies are multi domain glycoproteins of immense therapeutic and biotechnological relevance. IgG is composed of multiple protein domains which share a common structural core. Recently, isolated IgG domains have been the subject of protein engineering efforts which have described methods for producing these antibody fragments independently of the full-length molecule across a range of hosts. Evidence suggests that these fragments can retain their post-translational modifications and express at yields comparable to that of full-length antibodies despite being a fraction of the size. In this proof-of-concept work, I determine the feasibility of directing the in vitro multi-part assembly of IgG1 from its constituent protein domains using split intein mediated protein trans-splicing (PTS) and identify considerations for efficient covalent coupling as a consequence of split inteins sensitivity to redox environment.

First, I identify 4 functionally orthogonal split inteins capable of directing simultaneous multi part assembly in vitro and show that the concentration of exogenous DTT can be significantly lowered without impacting PTS yield. I show that the presence of thioredoxin, a common split intein fusion partner, can influence PTS and identify 2-Mercaptoethylamine, an alternative to DTT that can facilitate PTS and avoid disrupting the inter-chain disulphide bond structure of IgG under certain conditions. I develop expression protocols for CH2 and CH3 split intein fusion proteins in bacterial hosts and mediate covalent assembly of the Fc domain with minimal scarring across a range of conditions. I go on to coordinate simultaneous multi-part assembly of an Fc fusion protein via PTS and probe the binding affinity of the spliced product to a biologically relevant receptor.

My results shed new light on split intein mediated PTS, provide novel approaches for coupling redox sensitive proteins without relying on the complexity of protein engineering and open new opportunities for the modular assembly of multi-domain proteins like IgG.