Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection

Abstract

Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5°C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0°C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ~4.1°C of the 6.5°C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable. © 2014 Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.