This title appears in the Scientific Report :
2014
Please use the identifier:
http://hdl.handle.net/2128/5960 in citations.
Identifizierung von artifiziellen Liganden eines in Nanodiscs inkorporierten integralen Membranproteins
Identifizierung von artifiziellen Liganden eines in Nanodiscs inkorporierten integralen Membranproteins
Membrane proteins play a role in a variety of cellular processes. They are involved in the transport of substances across the membrane, in signal transduction, energy generation, metabolic reactions and much more. Membrane proteins are of special interest in the pharmaceutical industry, since 60 % o...
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Personal Name(s): | Pavlidou, Marina (Corresponding author) |
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Contributing Institute: |
Strukturbiochemie; ICS-6 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2014
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Physical Description: |
106 S. |
Dissertation Note: |
Universität Düsseldorf, Diss., 2013 |
ISBN: |
978-3-89336-942-3 |
Document Type: |
Dissertation / PhD Thesis |
Research Program: |
Structural Biology |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien / Key Technologies
80 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
Membrane proteins play a role in a variety of cellular processes. They are involved in the transport of substances across the membrane, in signal transduction, energy generation, metabolic reactions and much more. Membrane proteins are of special interest in the pharmaceutical industry, since 60 % of all clinical drugs target membrane proteins (Overington et al., 2006). Identifying ligands that modulate the activity of membrane proteins is of great interest in the development of novel pharmaceuticals. Phage display is an effective method for the selection of peptides binding specifically a target protein by using peptide libraries displayed on the surface of phage (Smith, 1985). The use of membrane proteins in phage display selections, however, is a challenge, since membrane proteins require due to the strong hydrophobicity of their transmembrane regions a membrane environment. Additionally, a membrane environment is necessary to retain correct folding and epitope formation of the protein. In this work, bacteriorhodopsin (bR) as a model of a polytopic membrane protein was used as target to screen a phage library for potential ligands. Nanodiscs were used for the first time as a model membrane in a phage display selection. Nanodiscs consist of a lipid bilayer encircled by two copies of a membrane scaffold protein and show compared to other model membranes high stability and homogeneity. In this work, expression of bR was achieved in a cell-free protein synthesis approach in the presence of empty preformed nanodiscs. Insertion of bR into the nanodiscs occurred cotranslationally and was proven by affinity and size exclusion chromatography. Correctly folded and functionally active bR was verified by an absorbance spectrum due to the bR characteristic absorbance maximum. Screening a 12-mer phage display peptide library against bR incorporated into nanodiscs led to the isolation of phage clones binding specifically to bR. Epitope mapping revealed that the loop AB and EF of bR form a discontinuous binding site for the binding of the selected phage clones. |