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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).ORCID iD: 0000-0003-4214-6991
2009 (English)In: New Biotechnology, ISSN 1871-6784, Vol. 26, no 5, 251-259 p.Article in journal (Refereed) Published
Abstract [en]

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.
Keyword [en]
protein fragment complementation assay (PCA), beta-lactamase, survival selection, affibody affinity proteins, tumor necrosis factor-alpha
National Category
Biocatalysis and Enzyme Technology Industrial Biotechnology
URN: urn:nbn:se:kth:diva-10964DOI: 10.1016/j.nbt.2009.06.980ISI: 000272773400008PubMedID: 19576305ScopusID: 2-s2.0-77950348500OAI: diva2:233325
Swedish Research Council

QC 20100729

Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2014-09-26Bibliographically approved
In thesis
1. On bacterial formats in protein library technology
Open this publication in new window or tab >>On bacterial formats in protein library technology
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii,102 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2009: 12
Affibody molecule, β-lactamase, combinatorial library, green fluorescent protein (GFP), protein engineering, protein fragment complementation assay (PCA), selection, TEVprotease, TNF-α
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology
urn:nbn:se:kth:diva-10993 (URN)978-91-7415-400-9 (ISBN)
Public defence
2009-09-25, AlbaNova Room FA 32, Roslagstullsbacken 21, Stockholm, 10:00 (Swedish)
QC 20100729Available from: 2009-09-08 Created: 2009-09-02 Last updated: 2011-12-08Bibliographically approved

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Löfdahl, Per-ÅkeNord, OlofNygren, Per-Åke
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