Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation |
| |
Authors: | Luginbühl Béatrice Kanyo Zoltan Jones R Mark Fletterick Robert J Prusiner Stanley B Cohen Fred E Williamson R Anthony Burton Dennis R Plückthun Andreas |
| |
Affiliation: | Biochemisches Institut, Universit?t Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. |
| |
Abstract: | Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations. |
| |
Keywords: | BoPrP, bovine prion protein BSE, bovine spongiform encephalopathy Cam, chloramphenicol chFab, mouse/human chimeric Fab fragment (V domains are murine, C domains are human) CDI assay, conformation-dependent immunoassay CDR, complementarity-determining region CJD, Creutzfeldt-Jakob disease CWD, chronic wasting disease dNTPs, deoxynucleoside triphosphate 8-oxo-dGTP, 8-oxo-2′-deoxyguanosine triphosphate dPTP, 6(2-deoxy-β- smallcaps" >D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]-oxazin-7-one-triphosphate GSS, Gerstmann-Sträussler-Scheinker syndrome IPTG, isopropylthiogalactoside KinExA, kinetic exclusion assay PDEA, 2-(2-pyridinyldithio)ethaneamine hydrochloride MBo2Mo, mouse/bovine chimeric prion protein (residues 23-231 are mouse, except for residues 90-144, which are bovine) MoPrP, mouse prion protein PrPC, cellular prion protein PrPSc, disease-associated isoform of prion protein scFv, single-chain Fv fragment ELISA, enzyme-linked immunosorbent assay SPR, surface plasmon resonance RIA, radioimmunoassay RT, room temperature TSE, transmissible spongiform encephalopathy VL, variable light chain domain VH, variable heavy chain domain |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|