Aggregation-resistant domain antibodies selected on phage by heat denaturation.
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 aggregate. 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 diseases.
Jespers
Laurent
L
Schon
Oliver
O
Famm
Kristoffer
K
Winter
Greg
G
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006358:Hot Temperature; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000302:isolation & purification* / Q000528:radiation effects; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000302:isolation & purification* / Q000528:radiation effects; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000528:radiation effects; D019151:Peptide Library; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D046912:Multiprotein Complexes; D019151:Peptide Library