Arbabi-Ghahroudi Mehdi, Mackenzie Roger, Tanha Jamshid
Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada.
Methods Mol Biol. 2010;634:309-30. doi: 10.1007/978-1-60761-652-8_22.
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.
重组抗体片段是重要的治疗和诊断试剂。因此，它们的功效在很大程度上取决于其亲和力和生物物理特性。因此，诱变方法已被广泛应用于重组抗体，以提高其亲和力、稳定性和溶解性。在现有的重组抗体变体中，人V(H)结构域在其开发过程中的某个阶段通常需要进行溶解性工程改造，这一点很突出；这个溶解性工程步骤将V(H)转化为非聚集的功能性实体，使其作为治疗和诊断试剂有用。在这里，我们展示了几种用于开发非聚集人V(H)结构域的方法之一。我们通过拼接重叠延伸技术，将体外定点诱变方法应用于一个聚集性人V(H)结构域。与亲本野生型V(H)相比，所得的突变体V(H)不聚集，并且在热变性后不易聚集。
