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Engineering of Affibody molecules targeting the Alzheimer’s-related amyloid β peptide
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-5192-7362
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2015. , s. x, 107
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:14
Emneord [en]
Affibody molecules, Alzheimer’s disease, AD, amyloid beta, Aß, combinatorial protein engineering, staphylococcal surface display
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-173864ISBN: 978-91-7595-663-3 (tryckt)OAI: oai:DiVA.org:kth-173864DiVA, id: diva2:855540
Disputas
2015-10-09, D3, Lindstedtsvägen 5, KTH, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20150922

Tilgjengelig fra: 2015-09-22 Laget: 2015-09-21 Sist oppdatert: 2015-09-22bibliografisk kontrollert
Delarbeid
1. Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
Åpne denne publikasjonen i ny fane eller vindu >>Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-ss peptide
Vise andre…
2013 (engelsk)Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 8, nr 1, s. 139-145Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
Wiley-VCH Verlagsgesellschaft, 2013
Emneord
Affibody molecules, Alzheimer's disease, Amyloid beta, Bacterial display, Combinatorial protein engineering
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-111860 (URN)10.1002/biot.201200228 (DOI)000312989500022 ()22987778 (PubMedID)2-s2.0-84871749509 (Scopus ID)
Forskningsfinansiär
Swedish Research Council, 2009-5758VINNOVA, 2009-00179
Merknad

QC 20130207

Tilgjengelig fra: 2013-01-14 Laget: 2013-01-14 Sist oppdatert: 2017-12-06bibliografisk kontrollert
2. A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
Åpne denne publikasjonen i ny fane eller vindu >>A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
2015 (engelsk)Inngår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, nr 11, s. 1707-1718Artikkel i tidsskrift, News item (Fagfellevurdert) 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.

Emneord
Affibody molecules, Affinity maturation, Amyloid beta, Bacterial display, Combinatorial protein engineering
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-173855 (URN)10.1002/biot.201500131 (DOI)000365129600007 ()2-s2.0-84947041962 (Scopus ID)
Merknad

Updated from E-pub to Published. QC 20151218

Tilgjengelig fra: 2015-09-21 Laget: 2015-09-21 Sist oppdatert: 2017-12-04bibliografisk kontrollert
3. Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease
Åpne denne publikasjonen i ny fane eller vindu >>Affibody-mediated Reduction of Amyloid Burden and Improvement of Cognitive Decline in an Animal Model of Alzheimer’s disease
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-173863 (URN)
Merknad

QS 2015

Tilgjengelig fra: 2015-09-21 Laget: 2015-09-21 Sist oppdatert: 2015-09-22bibliografisk kontrollert
4. Selection of binding proteins that specifically recognize protofibrillar aggregates of amyloid-β
Åpne denne publikasjonen i ny fane eller vindu >>Selection of binding proteins that specifically recognize protofibrillar aggregates of amyloid-β
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-173860 (URN)
Merknad

QS 2015

Tilgjengelig fra: 2015-09-21 Laget: 2015-09-21 Sist oppdatert: 2015-09-22bibliografisk kontrollert
5. Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
Åpne denne publikasjonen i ny fane eller vindu >>Development of a fluorescence-based intracellular method for function-based isolation of protein-based aggregation inhibitors
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-166644 (URN)
Merknad

QS 2015

Tilgjengelig fra: 2015-05-12 Laget: 2015-05-12 Sist oppdatert: 2015-09-22bibliografisk kontrollert

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