Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains

Recombinant antibody fragments are significant therapeutic and diagnostic reagents. As such, their efficacy depends heavily on their affinities and biophysical properties. Thus, mutagenesis approaches have been extensively applied to recombinant antibodies to improve their affinity, stability, and solubility. Among the existing recombinant antibody variants, human V_H domains stand out as the ones with the general need of solubility engineering at some point during their development; this solubility engineering step transforms V_H s into nonaggregating, functional entities, rendering them useful as therapeutic and diagnostic reagents. Here, we present one of several approaches that have been employed to develop nonaggregating human V_H domains. We apply an in vitro site-directed mutagenesis approach to an aggregating human V_H domain by means of a splice overlap extension technique. The resultant mutant V_H s are nonaggregating in contrast to the parent wild type V_H and less prone to aggregation following thermal unfolding.