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PHD THESiS: Bacterial display systems for engineering of affinity proteins
(Department of Biotechnology, KTH)
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.