Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection.
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.
American Society for Engineering Education Postdoctoral Fellow, Naval Research Laboratory, Washington, DC 20375, USA.
Kendrick B
KB
Turner
Dan
D
Zabetakis
Ellen R
ER
Goldman
George P
GP
Anderson
D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World; D004768:Enterotoxins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D004768:Enterotoxins; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; C031818:enterotoxin B, staphylococcal
differential scanning calorimetry; phage display; random mutagenesis; single domain antibody; thermal stabilization