Photolithographic surface functionalization for spatio-temporally controlled protein immobilization

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Titel: Photolithographic surface functionalization for spatio-temporally controlled protein immobilization
Autor(en): Bhagawati, Maniraj
Erstgutachter: Prof. Dr. Jacob Piehler
Zweitgutachter: Prof. Dr. Markus Haase
Zusammenfassung: Exploiting the functional diversity of proteins for fundamental research and biotechnological applications requires their functional organization into micro- and nanostructures while preserving their functional integrity to the highest possible level. My PhD research aimed to establish generic techniques based on photolithography which could be used to control the spatial as well as temporal organization of recombinantly expressed proteins on surfaces. My thesis describes in detail four strategies that I developed for achieving this goal. In the first approach a photo-induced Fenton reaction was used to selectively destroy tris(nitrilotriacetic acid) (tris-NTA) moieties on a surface. UV-irradiation through a photomask allowed localized photo-destruction and targeting of His-tagged proteins to non-irradiated regions. Photo-destruction could also be achieved by scanning selected regions with the UV laser of a confocal laser scanning microscope (CLSM) thus allowing flexible creation and modification of protein patterns. The second strategy was based on the photosensitive nitroveratryloxycarbonyl (NVOC) protection group, which was used to cage amine groups on a surface. Sequential uncaging by UV-irradiation through a photomask followed by reactions with biotin and coenzyme A was used to pattern streptavidin and ybbR-tagged proteins into microstructures. In the third approach a photo-fragmentable Histidine peptide was used to block tris-NTA surfaces against binding of His-tagged proteins. UV-irradiation through a photomask or by using a UV laser in a CLSM cleaved the peptide into short fragments which quickly dissociated from the surface due to loss in multivalency. His-tagged proteins could be efficiently targeted into irradiated regions even from a complex cell lysate. Sequential uncaging and immobilization allowed the construction of multiplexed protein patterns with a high degree of temporal control. The fourth strategy used combined peptide tags comprising of a His-tag as well as a Halo- or ybbR-tag to achieve rapid covalent immobilization of recombinant fusion proteins on surfaces functionalized with specific ligands. In combination with a photo-fragmentable histidine peptide as described above, stable spatio-temporal organization of proteins carrying these combined tags was possible. The techniques developed in this thesis enabled the photolithographical micropatterning of recombinant proteins carrying specific peptide or protein tags on surfaces in a functional manner. Owing to the generic nature of immobilization strategies, coupled with the ease of patterning, highly versatile applications of these methods both in fundamental research as well as bio-technological and analytical applications can be envisioned.
Schlagworte: protein patterning; protein-protein interactions; surface chemistry; solid-phase detection
Erscheinungsdatum: 27-Jan-2012
Enthalten in den Sammlungen:FB05 - E-Dissertationen

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