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Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Molecular Biotechnology)
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Molecular Biotechnology)ORCID iD: 0000-0003-4214-6991
2010 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, 111-120 p.Article in journal (Refereed) Published
Abstract [en]

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.
Keyword [en]
protein fragment complementation assay (PCA), β- lactamase, affinity maturation, affibody affinity proteins, tumour necrosis factor-α
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology
URN: urn:nbn:se:kth:diva-10965DOI: 10.1042/BA20090274ISI: 000276792800001ScopusID: 2-s2.0-77953131705OAI: diva2:233326
Uppdaterad från manuskript till artikel: 20100729 QC 20100729Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2010-12-06Bibliographically 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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