Glycoengineering of Aspergillus nidulans using an och1-GNT1 fusion protein

aspergillus;nidulans;glycoengineering;gnti;gnt1;mgat1;och1;filamentous;fungi;fungus;Chemical & Biomolecular Engineering

Paul, Jackson TylerBetenbaugh, Michael J ;

Johns Hopkins University

关键词: aspergillus;    nidulans;    glycoengineering;    gnti;    gnt1;    mgat1;    och1;    filamentous;    fungi;    fungus;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60164/Thesis.docx?sequence=2&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Glycoproteins are an integral type of treatment to modern medicine. Glycoproteins are proteins which are adorned with sugar chains known as glycans. Asparagine-linked glycans, also called N-glycans, are of particular importance. They are involved in many cellular processes such as cell signaling, protein folding, and secretion of products. While the beginning of the N-glycosylation pathway is mostly the same across eukaryotic organisms, the glycosylation pathways diverge in later steps, causing foreign N-glycans to be immunogenic to humans. This limits the possible production hosts for such glycoproteins used in medicine to hosts which produce similar enough glycans to humans that they will not cause immune rejection. In the industry today, most glycoproteins are produced by mammalian cell lines such as Chinese hamster ovary (CHO) cells. These cell lines produce glycoproteins containing glycans very similar to humans, however, they are very expensive to culture. Filamentous fungi such as Aspergillus nidulans produce and secrete glycoproteins at a much higher rate and grow in cheaper media, however, the glycans they produce are immunogenic to humans.In order to obtain more cost-effective glycoprotein production, A. nidulans can be engineered to produce human-like glycans. To achieve this end, this study uses an A. nidulans strain which has been engineered to produce glycans with fewer mannose residues and inserts an och1-GnTI fusion protein. The och1 section of this fusion protein is meant to direct the protein to the Golgi apparatus membrane, where it can then add N-acetylglucosamine (GlcNAc) to the glycans. This is a necessary step in the process to achieve humanized glycosylation in A. nidulans. Expression and activity of this fusion protein is analyzed using qRTPCR, Western, and lectin blotting. The qRTPCR andiiiWestern blot both show expression of GnTI, and the lectin blot indicates additional GlcNAcylation in the transformant strains compared to the parent strain. Both transformant strains produced in this study appear to express GnTI and actively GlcNAcylate glycoproteins.