Optimized production of the Diels-Alderase antibody 1E9 as a chimeric Fab

Monoclonal antibody 1E9, which catalyzes the [4+2] cycloaddition between tetrachlorothiophene dioxide and N-ethylmaleimide, has been re-engineered for production as a chimeric human–murine Fab fragment in Escherichia coli. Stabilizing point mutations in the variable regions of the antibody were identified by replacing residues that rarely occur at individual positions in aligned immunoglobulin sequences with their consensus counterparts. By combining favorable substitutions, double (Met H87 Thr–Gly L63 Ser) and triple (Met H87 Thr–Gly L63 Ser–Phe L95 Pro) mutants were created, which can be produced in good yield (4 and 17 mg L –1 cell culture, respectively). The triple mutant exhibits a modest fourfold drop in the apparent k cat value for the cycloaddition reaction, but the kinetic properties of the double mutant are indistinguishable from those of the parent murine IgG. The availability of recombinant versions of this catalytic antibody will facilitate efforts to determine the origins of its selectivity and catalytic efficiency through mutagenesis.Key words: catalytic antibody, Fab fragment, bacterial production.