Production and functional characterization of recombinant Human transferrin

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http://urn.fi/URN:NBN:fi:hulib-202012094812
Title: Production and functional characterization of recombinant Human transferrin
Author: Zeinoddin, Narjes
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2020
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202012094812
http://hdl.handle.net/10138/322599
Thesis level: master's thesis
Discipline: perinnöllisyystiede
Genetics
genetik
Abstract: Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.
Subject: Transferrin
Actin
Recombinant protein
CME
SpyCatcher/SpyTag


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