Improvement of proteolytic stability through in silico engineering.

VHHs usually display high physical and proteolytic stability, but in some cases stability needs to be increased further for their intended applications. The high thermal stability is due to the stable 3D structure of VHHs, which consists of a sandwich of nine beta-strands with a high number of intramolecular interactions, resulting in a very compact structure. Because of this compact structure, relatively low numbers of (basic) amino acids are accessible for proteases, explaining their usually high proteolytic stability. The high stability of VHHs is required when used as therapeutics given orally and nasally or when used as microbicides given, e.g., intra-vaginally. When given orally, VHHs should be stable at the low pH in the stomach and be resistant against all proteases in the intestines. Here a method is described to predict the proteolytic susceptibility of VHHs and to subsequently increase the proteolytic stability through genetic engineering.