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Nagels B.,Ghent University | Nagels B.,Bayer BioScience N.V. | Weterings K.,Bayer CropScience | Callewaert N.,Unit for Medical Biotechnology | And 2 more authors.
Critical Reviews in Plant Sciences | Year: 2012

In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided. © 2012 Copyright Taylor and Francis Group, LLC. Source


De Schutter K.,Unit for Medical Biotechnology
Methods in molecular biology (Clifton, N.J.) | Year: 2012

Yeast surface display is being employed as an efficient tool for the isolation and engineering of traditional antibody fragments, both scFv and Fab, as well as single domain antibodies. Here we describe the protocols for a yeast surface display system developed in the methylothrophic yeast Pichia pastoris, the most commonly used yeast species for protein production. In this system the immune or maturated library of single domain antibodies is fused to the C-terminal domain of Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed on the surface of P. pastoris cells. Labeling with ligands enables rapid and quantitative analysis in conjunction with isolation of single domain antibodies with the desired characteristics. Source


Nagels B.,Ghent University | Nagels B.,Bayer BioScience N.V. Technologiepark 38 | Van Damme E.J.M.,Ghent University | Callewaert N.,Unit for Medical Biotechnology | And 3 more authors.
Plant Science | Year: 2012

Because the pathway for protein synthesis is largely conserved between plants and animals, plants provide an attractive platform for the cost effective and flexible production of biopharmaceuticals. However, there are some differences in glycosylation between plants and humans that need to be considered before plants can be used as an efficient expression platform.In the presented research the human genes encoding α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) were introduced in the fast cycling model plant Arabidopsis thaliana to synthesize tri-antennary N-glycans. The GnT-IV and -V enzymes were targeted to the Golgi apparatus with plant-specific localization signals. The experiments were performed both in a wild type background, as well as in plants lacking β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) activity. Glycan analysis of endogenous proteins in the transgenic lines using CE-LIF showed that tri-antennary N-glycans could be produced in the XylT/FucT deficient line, while these structures were not found in the wild type background. Since β-N-acetylhexosaminidases, that remove terminal GlcNAcs, are active in A. thaliana plants, the specificity of these enzymes for different GlcNAc linkages was tested. The results showed that there is no pronounced preference of the A. thaliana hexosaminidases for human-type GlcNAc-linkages. © 2011 Elsevier Ireland Ltd. Source


Nagels B.,Ghent University | Nagels B.,Bayer BioScience N.V. | van Damme E.J.M.,Ghent University | Pabst M.,University of Natural Resources and Life Sciences, Vienna | And 4 more authors.
Plant Physiology | Year: 2011

In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions. © 2011 American Society of Plant Biologists. Source


Guerfal M.,Unit for Medical Biotechnology | Guerfal M.,Ghent University | Claes K.,Unit for Medical Biotechnology | Claes K.,Ghent University | And 6 more authors.
Microbial Cell Factories | Year: 2013

Background: Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results: We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the {increment}pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the {increment}pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions: We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells. © 2013 Guerfal et al.; licensee BioMed Central Ltd. Source

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