Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity.
The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.
Division of Molecular Biotechnology, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
Sebastian
S
Grimm
Samaneh
S
Salahshour
Per-Åke
PÅ
Nygren
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002942:Circular Dichroism; D019020:Directed Molecular Evolution; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf