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Engineering of Affibody molecules targeting the Alzheimer’s-related amyloid β peptide
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-5192-7362
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , x, 107 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:14
Keyword [en]
Affibody molecules, Alzheimer’s disease, AD, amyloid beta, Aß, combinatorial protein engineering, staphylococcal surface display
National Category
Other Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-173864ISBN: 978-91-7595-663-3 (print)OAI: oai:DiVA.org:kth-173864DiVA: diva2:855540
Public defence
2015-10-09, D3, Lindstedtsvägen 5, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150922

Available from: 2015-09-22 Created: 2015-09-21 Last updated: 2015-09-22Bibliographically approved
List of papers
1. Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
Open this publication in new window or tab >>Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
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2013 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 8, no 1, 139-145 p.Article in journal (Refereed) Published
Abstract [en]

We have previously generated an affibody molecule for the disease-associated amyloid beta (A beta) peptide, which has been shown to inhibit the formation of various A beta aggregates and revert the neurotoxicity of A beta in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the A beta-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and A beta binding. Overall, the detailed engineering and characterization of this promising A beta-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2013
Keyword
Affibody molecules, Alzheimer's disease, Amyloid beta, Bacterial display, Combinatorial protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-111860 (URN)10.1002/biot.201200228 (DOI)000312989500022 ()22987778 (PubMedID)2-s2.0-84871749509 (Scopus ID)
Funder
Swedish Research Council, 2009-5758VINNOVA, 2009-00179
Note

QC 20130207

Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically approved
2. A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
Open this publication in new window or tab >>A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
2015 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, no 11, 1707-1718 p.Article in journal, News item (Refereed) Published
Abstract [en]

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.

Keyword
Affibody molecules, Affinity maturation, Amyloid beta, Bacterial display, Combinatorial protein engineering
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173855 (URN)10.1002/biot.201500131 (DOI)000365129600007 ()2-s2.0-84947041962 (Scopus ID)
Note

Updated from E-pub to Published. QC 20151218

Available from: 2015-09-21 Created: 2015-09-21 Last updated: 2017-12-04Bibliographically approved
3. Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease
Open this publication in new window or tab >>Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173863 (URN)
Note

QS 2015

Available from: 2015-09-21 Created: 2015-09-21 Last updated: 2015-09-22Bibliographically approved
4. Selection of binding proteins that specifically recognize protofibrillar aggregates of amyloid-β
Open this publication in new window or tab >>Selection of binding proteins that specifically recognize protofibrillar aggregates of amyloid-β
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-173860 (URN)
Note

QS 2015

Available from: 2015-09-21 Created: 2015-09-21 Last updated: 2015-09-22Bibliographically approved
5. Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
Open this publication in new window or tab >>Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-166644 (URN)
Note

QS 2015

Available from: 2015-05-12 Created: 2015-05-12 Last updated: 2015-09-22Bibliographically approved

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