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Nanobodies as new tools for studying large cargo transport and lamina organization

dc.contributor.advisorGörlich, Dirk Prof. Dr.
dc.contributor.authorGebura, Myroslav
dc.date.accessioned2018-06-26T08:17:14Z
dc.date.available2018-06-26T08:17:14Z
dc.date.issued2018-06-26
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-002E-E42F-E
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-6938
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-6938
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc572de
dc.titleNanobodies as new tools for studying large cargo transport and lamina organizationde
dc.typedoctoralThesisde
dc.contributor.refereeWienands, Jürgen Prof. Dr.
dc.date.examination2017-10-09
dc.description.abstractengNanobodies are the smallest recombinantly expressed antibody fragments, which show outstanding performance in numerous applications. In this work, we set out to generate nanobodies as tools to study nucleo-cytoplasmic transport of large cargoes, such as ribosomes. We also generated a set of nanobodies targeting the X. laevis lamina. To meet these objectives we have optimised phage-display and designed an M13 phage that presents a nanobody library in SUMO-protease cleavable form. This allows a very specific phage-elution from immobilised antigens, namely by adding the SUMO-protease in a mild physiological buffer, which releases the phages from the still antigen-bound nanobody. Using our newly developed SUMO-cleavable phage M13 we have successfully selected and characterized six orthogonal nanobodies against ribosomes from Thermus thermophilus. This includes the very first nucleic acid-binding nanobody targeting the Asite of the large ribosomal subunit. So far, all attempts to produce lamins in bacteria lead to insoluble aggregates. We now discovered, however, that certain solubility-enhancing tags combined with appropriate protease cleavage modules allow E. coli to produce lamins in a soluble and correctly folded form. This not only established that lamins do not require any specialised eukaryotic chaperones for folding, but also allowed to isolate a set of orthogonal anti-lamin B3 nanobodies. We show that anti-lamin nanobodies perform excellently in purifying native protein complexes as well as in imaging.de
dc.contributor.coRefereeBohnsack, Markus Prof. Dr.
dc.subject.engrecombinant expression of soluble lamin; SUMO-protease cleavable bacteriophage M13; nanobodies, heavy chain only antibodyde
dc.identifier.urnurn:nbn:de:gbv:7-11858/00-1735-0000-002E-E42F-E-8
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.identifier.ppn1025240421


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