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

Abstract

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 (MetH87Thr–GlyL63Ser) and triple (MetH87Thr–GlyL63Ser–PheL95Pro) 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 kcat 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.

Immunological Optimization of a Generic Hydrophobic Pocket for High Affinity Hapten Binding and Diels–Alder Activity

“…The discrepancy between the SPR and fluorescence titration measurements probably reflects the different nature of interactions between the antibody and a solid support on the one hand and a ligand free in solution on the other. [25,26] In contrast to the results obtained with MetH100bPhe, a reduction in ligand affinity and catalytic efficiency is observed when an even bulkier tryptophan residue is introduced at position H100b (Table 1). Although the complexity of the overall reaction sequence makes the interpretation of structure±activity relationships in this system difficult, the activity of the 1E9 variants thus appears to correlate roughly with affinity for the transition-state analogue.…”

“…[16] This mutation has no effect on hapten binding or catalysis in 1E9. [26] CDR: complementarity-determining region. tene derivative 7 and catalyzes an unrelated Diels±Alder reaction, [16,23] but it possesses a distinctively shaped active site.…”

“…Site-Directed Mutagenesis: Plasmid p4xH-1E9(MetH87Thr/ GlyL63Ser), which was previously constructed for optimized production of the chimeric 1E9 Fab fragment in E. coli, [26] served as a template for site-directed mutagenesis. Substitutions were generally chosen to exploit the most frequently used codon for a particular amino acid among highly expressed genes in E. coli (Wisconsin Sequence Analysis package, version 8, Genetics Computer Group, Inc.).…”

“…Constructs were confirmed by DNA sequencing with the HSEQF, HSEQB, and LSEQB primers, as previously described. [26] Production, Purification, and Characterization of Chimeric Fab Fragments: Large-scale antibody production was achieved by transforming the TOPP2 E. coli strain with p4xH-1E9 (MetH87Thr/ GlyL63Ser) and its mutagenized derivatives, followed by high-density fermentation (2 L) in a BIOFLO 3000 Bioreactor (New Brunswick Scientific, Edison, NJ) as previously described. [26,36] The chimeric Fab fragments were purified from crude periplasmic lysates by protein G affinity chromatography, followed by Mono S cation-exchange chromatography.…”

“…[b] 1E9 wt is a recombinant chimeric Fab, which contains two point mutations (MetH87Thr and GlyL63Ser) for high level production and exhibits kinetic parameters within experimental error of those reported for the original IgG; [26] all mutations were introduced into this background.…”