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

von Myroslav Gebura
Dissertation
Datum der mündl. Prüfung:2017-10-09
Erschienen:2018-06-26
Betreuer:Prof. Dr. Dirk Görlich
Gutachter:Prof. Dr. Jürgen Wienands
Gutachter:Prof. Dr. Markus Bohnsack
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-6938

 

 

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Zusammenfassung

Englisch

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.
Keywords: recombinant expression of soluble lamin; SUMO-protease cleavable bacteriophage M13; nanobodies, heavy chain only antibody
 
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