The cloning, characterisation and engineering of an IGF-I-BINDING single chain Fv

摘要: This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) utilisation this scFv as a model protein for study application DNA shuffling ribosome display to antibody engineering. The variable domain genes were isolated from hybridoma cell line producing monoclonal antibody successfully joined by PCR scFv, named anti-GPE. Sequencing gene revealed unusually short heavy CDR2 region. The cloned was expressed in E. coli purified. Expression levels low has poor solubility, most likely due reduction folding efficiency caused abbreviated CDR2. purified monomeric form analysed binding IGF-I using surface plasmon resonance on BIAcore 1000 with specificity IgG version three N-terminal residues - Gly-Pro-Glu reproduced. scFv's calculated dissociation constant 3.68 µM is affinity approximately 36-fold weaker than Fab version antibody, but it concluded that apparent may be underestimation true presence non-functional or misfolded species within gel-filtration monomer peaks. A mutant anti-GPE inserted restore normal length produced improved expression solubility characteristics while retaining IGF-I-binding. It short CDR2 deletions generated during somatic mutation process is described. A method rabbit reticulocyte lysate source ribosomes developed specific selection against IGF-I. Error prone used produce random point mutated library (EPGPE). taken through several cycles non-specifically scFvs commonly observed. probably ribosome-displayed proteins system used, possibly DTT and/or capacity framework tolerate stability. assumed misfolds, exposing hydrophobic regions, would have tendency interact surface. Of 64 EPGPE clones screened four rounds selection, many shown non-specific binding, one characterised matured 2.6-fold over wild type IGF-I. A libraries chimaeric between anti-GPE and NC10 (anti-neuraminidase scFv) objective isolating functional IGF-I-binding chimaeras. had its CDRs replaced prior to increase likelihood binders. Ribosome chimaera libraries. Selection strategies included elution binders GPE peptide 10-mer peptide. also performed immobilised sensorchip a selection Again, much observed seen EPGPE, what expected same reasons. Selected genuinely mostly their binding. One selected chimaera, made up segments each parental scFvs, bind specifically BIAcore. Steps improve been identified and are discussed.