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

Kendrick B Turner, Dan Zabetakis, Ellen R Goldman, George P Anderson
Protein Engineering, Design & Selection: PEDS 2014, 27 (3): 89-95
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.

Full Text Links

Find Full Text Links for this Article


You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read

Save your favorite articles in one place with a free QxMD account.


Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"