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N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
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2010 (English)In: Protein Science, ISSN 0961-8368, Vol. 19, no 12, 2319-2329 p.Article in journal (Refereed) Published
Abstract [en]

The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)

Place, publisher, year, edition, pages
2010. Vol. 19, no 12, 2319-2329 p.
Keyword [en]
amyloid, protein engineering, Alzheimer's disease, solid phase peptide synthesis, NMR spectroscopy
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-27658DOI: 10.1002/pro.511ISI: 000284793800006ScopusID: 2-s2.0-78649668010OAI: diva2:381081
Knut and Alice Wallenberg Foundation

QC 20101223

Available from: 2010-12-23 Created: 2010-12-20 Last updated: 2014-02-07Bibliographically approved
In thesis
1. Chemical Engineering of Small Affinity Proteins
Open this publication in new window or tab >>Chemical Engineering of Small Affinity Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS).

In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I).

In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III).

In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V).

Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. viii, 79 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:3
Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-141014 (URN)978-91-7595-004-4 (ISBN)
Public defence
2014-03-07, FR4 (Oskar Klein), AlbaNova Universitetscentrum, Stockholm, 10:00 (English)

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2014-02-07Bibliographically approved

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