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Surface display of a single-domain antibody library on Gram-positive bacteria
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
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2013 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 70, no 6, 1081-1093 p.Article in journal (Refereed) Published
Abstract [en]

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

Place, publisher, year, edition, pages
2013. Vol. 70, no 6, 1081-1093 p.
Keyword [en]
Bacterial display, Combinatorial protein engineering, Nanobodies, Phage display, Recombinant antibodies
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-111859DOI: 10.1007/s00018-012-1179-yISI: 000315343600009PubMedID: 23064703OAI: diva2:587395
Swedish Research Council, 2009-5758Vinnova

QC 20130411

Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2014-12-03Bibliographically approved
In thesis
1. Bacterial display systems for engineering of affinity proteins
Open this publication in new window or tab >>Bacterial display systems for engineering of affinity proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. 89 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:18
Combinatorial protein engineering, staphylococcal display, Affibody, biparatopic, VEGFR2, nanobody, E. coli display, autotransporter
National Category
Engineering and Technology
Research subject
urn:nbn:se:kth:diva-156420 (URN)978-91-7595-374-8 (ISBN)
Public defence
2014-12-19, FD5, AlbaNova Universitetscentrum, KTH, Stockholm, 10:00 (English)
Swedish Research CouncilVinnovaSwedish Foundation for Strategic Research

QC 20141203

Available from: 2014-12-03 Created: 2014-11-28 Last updated: 2014-12-03Bibliographically approved

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