Affinity maturation of a TNFα‐binding Affibody molecule by Darwinian survival selection

Abstract

showed that the major factor for the improved affinity could be attributed to reduced off-rate constants.

Tumor Necrosis Factor-α Trimer Disassembly and Inactivation via Peptide-Small Molecule Synergy

“…The relative affinities of these compounds roughly correlated with the previously reported affinities for the full three-helix Z-domain peptides, as determined by SPR analysis ( Figure S5, left columns). 7,9,10 These results indicate that the two-helix α-peptide scaffold is fairly good at mimicking these other Z-domains; thus, these results highlight the generality of developing two-helix Z-domain derivatives based on three-helix prototypes identified via display/selection processes.…”

“…We wondered whether other Z-domain derivatives selected from combinatorial libraries to bind TNFα also displayed a detergent dependence. We chose seven peptides that have been previously selected from combinatorial libraries based on the Z-domain scaffold to bind TNFα 7,9,10 and designed α-peptides S15-S21 as two-helix, disulfide-constrained analogues of these three-helix peptides ( Figure S5). Peptides S15-S18 each contain a disulfide bond as well as five helix-stabilizing and solubility-enhancing substitutions, [4][5][6] while S19-S21 contain only the disulfide bond.…”

Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity

“…HER2 [7], 15-fold to HER3 [14], 8-fold to TNF-a [15] and 10-fold to PDGFR-b [16]. Our primary motivation was to investigate where affinity enhancing substitutions would appear, and what these would be, if the entire sequence was subjected to a 'free', step-wise in vitro evolution procedure.…”

Affinity proteins and their generation

“…PCA and hybrid systems have become valuable tools for discovery of protein–protein interactions, in particular for systems biology in order to identify biochemical networks . For combinatorial protein engineering purposes, PCA‐based methods utilizing either a split dihydrofolate reductase (mDHFR) gene or a split β‐lactamase gene have been reported . It has been speculated that unspecific binding to intracellular proteins and a difficulty to distinguish between binders with different affinity to the target may complicate the use of PCA for this purpose .…”

“…The fact that there is no prerequisite to have access to target proteins of high quality in terms of purity and stability is a key advantage for these systems. Indeed, the potential of the PCA selection platform was recently demonstrated in several studies where improved PCA systems using β‐lactamase as reporter protein were employed in successful selections of low nanomolar scFv fragments and affibody molecules from combinatorial libraries.…”