Engineering of bacterially expressed Erythropoietin by means of non-natural amino acids for improved therapeutic potential

Ich freue mich auf die nächsten 100 Jahre…. Summary 2 1. Summary The main incentive for the development of new techniques and biological tools is to understand nature or even improve it. In this thesis, approaches were evolved to realize this aim, working with the therapeutic protein Erythropoietin (EPO). EPO is highly glycosylated, hence stability, solubility and functionality of the protein depends on this posttranslational modification. Due to this glycosylation, production of EPO for therapeutic approaches has to be performed in eukaryotic systems, making EPO very expensive. As glycosylation is template-independent, glycosylation patterns can be very heterogeneous depending on the expression system and conditions. A complementary approach to stabilize EPO was investigated in the first part of this thesis. Incorporation of non-natural fluorinated amino acids into the protein backbone of EPO should clarify whether EPO is still properly folded and more resistant towards proteases and thermal denaturation like it was shown for other proteins and peptides. EPO was expressed in Escherichia coli (E.coli). As E.coli is not able to glycosylate proteins, isolated EPO is completely deglycosylated and must be recovered and refolded from inclusion bodies. Incorporation of fluorinated amino acids was performed via Selective Pressure Incorporation using auxotrophic bacterial strains. A variety of different hydrophobic, fluorinated amino acids were tested for incorporation into the backbone of EPO as well as evaluated for their influence on protein folding and stability. Unfortunately, the majority of the tested fluorinated amino acids could not be incorporated into EPO, or the synthesized protein was immediately degraded in cell. Nevertheless, EPO modified with fluorinated isoleucine could be expressed and purified. Refolding into its native structure and following purification led to complete loss of the fluorinated protein. It can be assumed that the incorporation of the fluorinated amino acids not only disturbed secondary and tertiary structure formation but also increased the hydrophobicity of the protein. As EPO already has a very hydrophobic surface, usually covered by glycans, increase in surface hydrophobicity is probably the reason for aggregation. However, enhanced resistance towards proteases and temperature, arising from increased core hydrophobicity, could not be studied. In the second part of this work EPO was again modified with a non-natural amino acid, para-azido phenylalanine (pAzF). In contrast to the first part, stabilization of EPO Summary 3 should not be based on the non-natural amino acid, but on the following site-specific modification with a simple glycan mimic, polyethylene glycol (PEG). The …