Development of bispecific filamentous bacteriophages for the generation of a novel automated screening system based on phage display technology

Phage display is a powerful tool for screening large protein libraries in order to identify novel therapeutics, diagnostics or vaccines. In the emerging field of protein based drug development, there is an urgent need for high-throughput technologies for automated library handling and phage display selection. Currently, suitable technologies have been developed by adapting the basic selection steps of the phage display panning procedure to robotics. This thesis presents an innovative strategy for the generation of an even faster high-throughput screening system, called Protein Nanochip. The basis for this novel protein microarray device was established within this thesis circumventing the entire panning procedure through direct laser-based detection of singular binding partners, combined with FACS, and final identification of the interacting partners by duplex-PCR and sequencing. This technology is suitable for screening of an immobilized phage library against a second fluorescent labeled phage library and therefore allows the identification of new targets and their specific ligands within one single automated screening step. For the development of this highly innovative screening devise a bispecific filamentous bacteriophage and a suitable technology for side-directed immobilization of these phages onto the surface of the Protein Nanochip had to be developed. Two different strategies were investigated for the generation of bispecific filamentous bacteriophages. First of all, a modified phage vector based on the fd-tet vector was established. The final bispecific phage vector was generated by modifications of geneVI and geneIX. Multivalent display of the extracellular domain of the human CD30 receptor fused to the C-terminus of the pVI coat protein was shown to be feasible but reduced the assembly efficiency of the phages. Multivalent display of single chain Fv antibodies fused to pIX coat protein showed a lethal effect on the bacterial host, whereas multivalent display of small peptides on the same coat protein was shown to be feasible and provides a tool for the generation of recombinant phages not inhibiting the propagation of the bacterial host. The second strategy was based on the establishment of a novel bispecific phagemid vector system. The final phagemid vector was generated by insertion of a second expression cassette encoding geneIX into the pHENHi phagemid vector. A single chain Fv antibody encoding DNA sequence was fused to geneIII and the Strep-tag® encoding DNA to geneIX. Functional coexpression of the resulting scFvCWP/pIII- and Strep-tag®/pIX-fusion proteins as well as their incorporation into correctly assembled bispecific phage particles was verified by phage ELISA and immunogold assay with subsequent electron microscopy. These results provided evidence for functional display of both the single chain Fv antibody on pIII coat protein and the Strep-tag® on pIX coat protein on the same phage particle and prove the concept of the novel phagemid vector system. In addition, the Strep-tag® technology was shown to provide a universal tool for purification, detection and side-directed immobilization of Strep-tag®-displaying filamentous bacteriophages. In conclusion, a novel phagemid system for the generation of bispecific filamentous bacteriophages, and suitable technologies for purification, detection and side-directed immobilization of these phages was successfully developed allowing the immobilization of any kind of phage libraries on the biotinylated surface of the Protein Nanochip or comparable devices. Therefore, the bispecific phagemid vector system in combination with the adapted Strep-tag® technology represents an initial step toward the establishment of novel HTS systems allowing the automated screening of two different protein libraries against each other.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2005

Author dc:creator

• Stolle, Tim Oliver

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Microarray
• Phagen-Display-Bibliothek
• High throughput screening
• Biowissenschaften, Biologie
• bispecific filamentous bacteriophages
• phage display
• pIX-display
• high-throughput screening
• Strep-tag

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123721%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62128