Plant Galactosylation
Abstract
The majority of therapeutic proteins are glycoproteins and require the attachment of sugar structures (i.e. glycosylation) to attain full therapeutic activity. Current manufacturing methods based on mammalian cell culture do not allow for the control of glycosylation and produce a mixture of different glycoforms; some of which are more active then others and some of which have no activity at all. This proposal aims at the generation of a plant based expression system that allows an efficient production of recombinant proteins with high glycoform uniformity. We aim at the generation of proteins with consistent beta 1,4-galactosylation. The presence of this terminal mammalian-type N-glycan residue, naturally absent in plants, may not only influence the efficacy of a protein, galactosylated structures also serve as acceptor substrate for sialylation, the final step of human glycosylation. Recently we generated a glycosylation mutant of Nicotiana benthamiana dXT/FT (a tobacco related species) that lacks unwanted plant specific N-glycan residues and allows for the generation of mammalian proteins with a human like glycan structure required for efficient beta 1,4-galactosylation. Preliminary results obtained by transient expression of the mammalian enzyme beta 1,4 galactosyltransferase (GalT) in dXT/FT exhibit a quantitative galactosylation of a reporter protein. Based on these results dXT/FT will serve as parental line for the stable transformation of various GalT constructs along with other endogenous glycosylation enzymes that might negatively interfere with efficient galactosylation, to generate a line enabling the production of recombinant glycoproteins with consistent galactosylated structures. To this end a highly galactosylated therapeutic glycoprotein (a monoclonal antibody) will be expressed in these lines and tested with regard to its biological activity. The availability of glyco-engineered plants enabling the generation of recombinant mammalian glycoproteins with a consistent galactosylation proved an alternative for mammalian cell culture and allow for the production of therapeutic glycoproteins in plants with increased in vivo efficacy. Moreover, consistent galactosylation of glycoproteins, which is not achieved by mammalian cell based expression systems, will expand our ability to conduct other biological important studies e.g. structure-function investigations and will be a further step towards the reconstruction of protein sialylation in plants.
Publikationen
Project staff
Herta Steinkellner
Ao.Univ.Prof. Mag.rer.nat. Dr.nat.techn. Herta Steinkellner
herta.steinkellner@boku.ac.at
Tel: +43 1 47654-94370
Project Leader
01.05.2009 - 30.06.2013