Antigen-binding Fragments: Production for and Use in Crystallographic Studies

• Main Author: Liu, Feiyang (Victoria)
• Other Authors: Pai, Emil F.
• Language: en_ca
• Published: 2013
• Subjects: antigen-binding fragment; X-ray crystallography; HIV-1; broadly neutralizing monoclonal antibody; 0307
• Online Access: http://hdl.handle.net/1807/43090

An immunoglobulin (IgG) consists of two antigen-binding fragments (Fab) connected to a crystallisable fragment through hinge regions. This thesis mainly investigates the production methods of Fabs used in structural studies. A cost effective and time saving protocol has been established for the production of recombinant 2F5 Fab, a HIV-1 broadly neutralizing monoclonal antibody fragment, using an Escherichia coli expression system. The integrity of structure and antigen-binding capability of the produced 2F5 Fab was confirmed by determining the crystal structure of the Fab-antigen peptide complex. In parallel, 3H1 Fab, a fragment of an antibody which is involved in detecting misfolded superoxide dismutase, which is related to familial amyotrophic lateral sclerosis, was produced by papain proteolysis of its parent IgG molecule. Both Fab production methods resulted in high yields of pure Fab samples that are crystallisable and ready to be engaged in structural studies using X-ray crystallography.