An industrial-grade Nicotiana benthamiana line for the production of glycoproteins carrying fucose-free galactosylated N-glycans.

β1,4-galactosylation is a typical human N-glycan formation with functional impact on proteins, particularly known for IgGs. Therefore, the expression of recombinant proteins with controlled galactosylation is an important quality parameter in the biotech industry. Here we describe the establishment of a plant-based expression platform for the manufacturing of recombinant proteins carrying β1,4-galactosylated N-glycans.

Tip of the iceberg? Three novel TOPLESS-interacting effectors of the gall-inducing fungus Ustilago maydis.

Ustilago maydis is a biotrophic pathogen causing smut disease in maize. It secretes a cocktail of effector proteins, which target different host proteins during its biotrophic stages in the host plant. One such class of proteins we identified previously is TOPLESS (TPL) and TOPLESS-RELATED (TPR) transcriptional corepressors.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations.

A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated β1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing.

An oligosaccharyltransferase from increases the N-glycan occupancy on plant-produced IgG1.

N-Glycosylation of immunoglobulin G1 (IgG1) at the heavy chain Fc domain (Asn297) plays an important role for antibody structure and effector functions. While numerous recombinant IgG1 antibodies have been successfully expressed in plants, they frequently display a considerable amount (up to 50%) of unglycosylated Fc domain. To overcome this limitation, we tested a single-subunit oligosaccharyltransferase from the protozoan (LdOST) for its ability to improve IgG1 Fc glycosylation.

Plant-based biopharmaceutical engineering.

Plants can be engineered to recombinantly produce high-quality proteins such as therapeutic proteins and vaccines, also known as molecular farming. Molecular farming can be established in various settings with minimal cold-chain requirements and could thus ensure rapid and global-scale deployment of biopharmaceuticals, promoting equitable access to pharmaceuticals. State of the art plant-based engineering relies on rationally assembled genetic circuits, engineered to enable the high-throughput and rapid expression of multimeric proteins with complex post-translational modifications.

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors.

Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall-inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants. Using genetic, biochemical, cell-biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling.

Highly active engineered IgG3 antibodies against SARS-CoV-2.

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered plants.

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in .

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants that are identical in their antigen binding domain but differ in their subclass appearance.