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Biotechnology and Applied Biochemistry (2010) 55, (111–120) (Printed in Great Britain)
Affinity maturation of a TNFα-binding Affibody molecule by Darwinian survival selection
Per-Åke Löfdahl and Per-Åke Nygren1
Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden
Key words: Affibody affinity protein, affinity maturation, β-lactamase, protein fragment complementation assay (PCA), tumour necrosis factor α (TNFα). Abbreviations used: ABP, albumin-binding protein; Amp, ampicillin; Cml, chloramphenicol; HSA, human serum albumin; IPTG, isopropyl β-D-thiogalactoside; PCA, protein fragment complementation assay; Taz, tazobactam; Tc, tetracycline; TNFα, tumour necrosis factor α; TSB+Y, tryptic soy broth supplemented with yeast extract. 1To whom correspondence should be addressed (email perake@biotech.kth.se). The introduction of different methodologies for the construction and screening of complex protein libraries has provided powerful means in protein engineering for the development of molecules with desired traits. A challenge faced in many situations is to adapt a given methodology for efficient and rapid identification of the most interesting variants present in a library. In the present study, the concept of Darwinian selection based on a growth advantage for clones having the desired trait has been investigated. Using a β-lactamase-based PCA (protein fragment complementation assay), affinity maturation of a TNFα (tumour necrosis factor α)-binding Affibody molecule with an initial 2 nM affinity for the target has been performed. Initial characterization of the PCA system, based on the affinity-driven reconstitution of β-lactamase activity in the periplasm of cells harbouring a library member showing affinity for a co-expressed target protein, showed that the system was responsive to promoter induction level, interaction affinity and applied selection pressure. Using combinatorial protein engineering principles, a 107 library of second-generation Affibody molecules was constructed and subjected to selection of improved variants by library growth in liquid culture. The results show that, after a pre-selection step on semi-solid medium to eliminate non-binding variants, present in the majority, two rounds of selection in liquid culture resulted in an enrichment for binders showing up to 8-fold higher affinity for the TNFα target than the ancestral variant. Biosensor analyses showed that the major factor for the improved affinity could be attributed to reduced off-rate constants. Received 2 October 2009/13 January 2010; accepted 1 February 2010 Published as Immediate Publication 1 February 2010, doi:10.1042/BA20090274 © 2010 Portland Press Limited |
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