Abstract
As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response. Nevertheless glycoproteins with an identical polypeptide backbone mostly differ in their glycosylation patterns. Due to this heterogeneity, the mapping of different glycosylation patterns to their associated function is nearly impossible. In the last years, glycoengineering tools including cell line engineering, chemoenzymatic remodeling and site-specific glycosylation have attracted increasing interest. The therapeutic hormone erythropoietin (EPO) has been investigated in particular by various groups to establish a production process resulting in a defined glycosylation pattern. However commercially available recombinant human EPO shows batch-to-batch variations in its glycoforms. Therefore we present an alternative method for the synthesis of active glycosylated EPO with an engineered O-glycosylation site by combining eukaryotic cell-free protein synthesis and site-directed incorporation of non-canonical amino acids with subsequent chemoselective modifications.
Highlights
The glycosylation moieties of hEPOs cover up to 40% of the final molecular weight and they have a significant influence on its stability and solubility[1]
Glycosylated EPO has been already synthesized in cell free systems[17,18,23], the process of N-glycosylation in Sf21 lysates and the resulting glycan structures attached to the protein backbone have not been investigated yet
Previous studies demonstrated the activity of the ER-resident α-glycosidase I/II and mannosidase I in insect cells, which are important for the initial trimming steps from the precursor glycan Glc3Man9GlcNAc2 to Man8GlcNAc24
Summary
The glycosylation moieties of hEPOs cover up to 40% of the final molecular weight and they have a significant influence on its stability and solubility[1]. It is the mammalian cell expression that is mostly used to produce recombinant hEPO due to the presence of highly complex glycosylation patterns, which are a crucial factor influencing the pharmacokinetic activity of hEPO in vivo[8]. In addition a specific glycosylation site might be associated with different glycoforms and not all glycosylation sites must be necessarily occupied. Such a high degree of glycan micro and macro heterogeneity gives rise to a set of glycoforms which expand the proteome diversity far beyond the genetic code and reflects functional diversity required in complex organisms. As a promising alternative to in vivo produced and chemically synthesized hEPO, we present the potential of cell-free glycoprotein synthesis. The synthesis of the protein of interest starts immediately after addition of a template, amino acids, energy and an energy regeneration system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
