Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolution
(English)Manuscript (preprint) (Other academic)
The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.
affibody molecule, ribosome display, error-prone PCR, library, evolution
IdentifiersURN: urn:nbn:se:kth:diva-33427OAI: oai:DiVA.org:kth-33427DiVA: diva2:415406
QS 20112011-05-062011-05-062011-05-06Bibliographically approved