Enhancing the Expression Levels of Fabs in Escherichia coli

Enhancing the expression levels of Fabs (antigen-binding antibody fragments) in Escherichia coli is a difficult field that has a variety of potential exciting implications. The field has grown substantially in the past twenty years. The main area of difficulty is facilitating the entry of the antibody fragments into the periplasm of E. Coli, where the antibody fragments can be successfully expressed. Entry into the periplasm is difficult for antibody fragments due to their inability to fold in any other section besides the periplasm. Therefore it is necessary for the antibody to enter the periplasm in an unfolded state. Background research was done into inspecting the three primary methods of periplasmic entry: the Sec-dependent pathway, the SRP-dependent pathway (signal recognition particle) and the TAT-dependent pathway (twin arginine translocase). The Sec-dependent and SRP-dependent pathways were deemed more viable for expressing antibodies due to their ability to transfer an unfolded protein into the periplasm, which the TAT-dependent pathway cannot do. Academic research showed that the Sec-dependent and SRP-dependent pathways were equally viable methods, with more research being done into the Sec-dependent pathway, particularly of the OmpA signal sequence. Physical experiments were done using typical cloning procedures with slight modifications to the ligation step (Gibson Assembly was performed instead of normal ligation). These physical experiments showed that the Sec-dependent and SRP-dependent pathways were equally viable methods of periplasmic entry. The A4 and C6 antibodies were successfully expressed using these pathways. These antibodies were expressed on an SDS gel using 10% SDS. It was hypothesized that with further experimental modifications, using different signal sequences, Fabs can be expressed at higher and more consistent level.