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Efficient blocking of VEGFR2-mediated signaling using biparatopic Affibody constructs
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-9282-0174
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0001-9423-0541
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
URN: urn:nbn:se:kth:diva-156532OAI: oai:DiVA.org:kth-156532DiVA, id: diva2:767028
Anmärkning

QS 2014

Tillgänglig från: 2014-11-28 Skapad: 2014-11-28 Senast uppdaterad: 2014-12-03Bibliografiskt granskad
Ingår i avhandling
1. Bacterial display systems for engineering of affinity proteins
Öppna denna publikation i ny flik eller fönster >>Bacterial display systems for engineering of affinity proteins
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins.

Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro.

In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application.

The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules.

In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2014. s. 89
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:18
Nyckelord
Combinatorial protein engineering, staphylococcal display, Affibody, biparatopic, VEGFR2, nanobody, E. coli display, autotransporter
Nationell ämneskategori
Teknik och teknologier
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-156420 (URN)978-91-7595-374-8 (ISBN)
Disputation
2014-12-19, FD5, AlbaNova Universitetscentrum, KTH, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
VetenskapsrådetVinnovaStiftelsen för strategisk forskning (SSF)
Anmärkning

QC 20141203

Tillgänglig från: 2014-12-03 Skapad: 2014-11-28 Senast uppdaterad: 2014-12-03Bibliografiskt granskad

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Personposter BETA

Ståhl, StefanLöfblom, John

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Av författaren/redaktören
Fleetwood, FilippaStåhl, StefanLöfblom, John
Av organisationen
Proteinteknologi
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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