“…Protein engineering is critical for developing protein-based therapeutics and diagnostics. Single-chain antibody variable domain fragments (scFvs) are important pharmaceutical molecules (Chan and Carter, 2010;Holliger and Hudson, 2005;Huang et al, 2010;Miller et al, 2010;Nelson and Reichert, 2009;Weatherill et al, 2012). Because the scFv structure is not stabilized by the constant domains, as in intact immunoglobulin, investigators have applied sequence fitness-searching principles based on random mutagenesis and screening (Jermutus et al, 2001;Jespers et al, 2004;Jung et al, 1999) or rational design with consensus-sequence profiles (Demarest and Glaser, 2008;Ewert et al, 2003;Honegger, 2008;Jordan et al, 2009;Kü gler et al, 2009;Miller et al, 2010;Monsellier and Bedouelle, 2006;Plü ckthun, 1998, 2001) to stability engineering of scFvs.…”