Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
2009 (English)In: New Biotechnology, ISSN 1871-6784, Vol. 26, no 5, 251-259 p.Article in journal (Refereed) Published
Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naive 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the P-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira (R)), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PICA is a promising methodology for stringent selection of binders from complex naive libraries to yield high affinity reagents with selective target binding characteristics.
Place, publisher, year, edition, pages
Amsterdam: Elsevier , 2009. Vol. 26, no 5, 251-259 p.
protein fragment complementation assay (PCA), beta-lactamase, survival selection, affibody affinity proteins, tumor necrosis factor-alpha
Biocatalysis and Enzyme Technology Industrial Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-10964DOI: 10.1016/j.nbt.2009.06.980ISI: 000272773400008PubMedID: 19576305ScopusID: 2-s2.0-77950348500OAI: oai:DiVA.org:kth-10964DiVA: diva2:233325
FunderSwedish Research Council
QC 201007292009-09-022009-08-312014-09-26Bibliographically approved