Improved production and function of llama heavy chain antibody fragments by molecular evolution

The aim of this study was to improve production level of llama heavy chain antibody fragments (V_HH) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V_HH fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V_HH with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V_HH fragments were selected. We found that these shuffled V_HH fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V_HHs point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90°C were compared with parental V_HHs. One shuffled V_HH was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V_HH displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90°C. Structural analysis suggested that improved antigen binding is associated with the A24→V24 substitution, which reduces the size of the hydrophobic pit at the llama V_HH surface. We demonstrate that it is possible to improve desired characteristics of the same V_HH fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.