| dc.contributor.advisor | Görlich, Dirk Prof. Dr. | |
| dc.contributor.author | Gebura, Myroslav | |
| dc.date.accessioned | 2018-06-26T08:17:14Z | |
| dc.date.available | 2018-06-26T08:17:14Z | |
| dc.date.issued | 2018-06-26 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-002E-E42F-E | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-6938 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-6938 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Nanobodies as new tools for studying large cargo transport and lamina organization | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Wienands, Jürgen Prof. Dr. | |
| dc.date.examination | 2017-10-09 | |
| dc.description.abstracteng | Nanobodies 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.coReferee | Bohnsack, Markus Prof. Dr. | |
| dc.subject.eng | recombinant expression of soluble lamin; SUMO-protease cleavable bacteriophage M13; nanobodies, heavy chain only antibody | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E42F-E-8 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1025240421 | |