Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection
2010 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, 111-120 p.Article in journal (Refereed) Published
The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.
Place, publisher, year, edition, pages
2010. Vol. 55, 111-120 p.
protein fragment complementation assay (PCA), β- lactamase, affinity maturation, affibody affinity proteins, tumour necrosis factor-α
Industrial Biotechnology Biocatalysis and Enzyme Technology
IdentifiersURN: urn:nbn:se:kth:diva-10965DOI: 10.1042/BA20090274ISI: 000276792800001ScopusID: 2-s2.0-77953131705OAI: oai:DiVA.org:kth-10965DiVA: diva2:233326
Uppdaterad från manuskript till artikel: 20100729
QC 201007292009-09-022009-08-312010-12-06Bibliographically approved