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Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro
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2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, 93-103 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For Z(TNF alpha:185), subnanomolar affinity (K-D = 0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the Z(TNF-alpha:185) affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dinners with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Place, publisher, year, edition, pages
2009. Vol. 54, 93-103 p.
Keyword [en]
affibody molecule, albumin-binding-domain-based affibody-screening ELISA (ABAS ELISA), biospecifc interaction analysis (BIA), immobilized-metal-ion affinity chromatography (IMAC), phage display, tumour necrosis factor-alpha (TNF-alpha)
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-127468DOI: 10.1042/BA20090085ISI: 000270769000003OAI: oai:DiVA.org:uu-127468DiVA: diva2:330218
Available from: 2010-07-15 Created: 2010-07-13 Last updated: 2010-07-15Bibliographically approved

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