Protein Engineering vol. 16 no. 12 pp. 1089-1097, 2003
© 2003 Oxford University Press
Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody
1VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland 3Present address: Orion Pharma, PO Box 65, FIN-02101 Espoo, Finland
2 To whom correspondence should be addressed. e-mail: tarja.nevanen@vtt.fi
The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.
Received April 23, 2003; revised September 25, 2003;; accepted October 21, 2003