Aggregation-resistant domain antibodies selected on phage by heat denaturation
Laurent Jespers1, 3, 4, Oliver Schon1, 3, 4, Kristoffer Famm2 & Greg Winter1, 2
1 Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, England, UK.
2 Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, England, UK.
3 Domantis Limited, 315 Cambridge Science Park, Cambridge CB4 0WG, England, UK.
4 Present address: Domantis Limited, 315 Cambridge Science Park, Cambridge CB4 0WG, England, UK.
Correspondence should be addressed to Greg Winter winter@mrc-lmb.cam.ac.uk, tel: +44-1223-402134
We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate1, 2. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases3, 4.
Nature Biotechnology 22, 1161 - 1165 (2004)
Published online: 08 August 2004; Corrected online: 15 August 2004 | doi:10.1038/nbt1000 |
