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GlycoDelete technology: shortcutting mammalian cell N-glycosylation
New Biotechnology · Volume 31S · July 2014
RECOMBINANT PROTEIN PRODUCTION
can be separated at the amino acid residue 270. However, the active functions of these two domains in p60 protein and their influences on the substrate recognition and catalytic activity of p60 protein are unknown. Here we identified both of the functional hot spot and the mutational hot spot amino acid residues in these two structural domains by means of the amino acid sequence alignment of different p60 variants, including two p60 variants screened in our lab. The functional hot spot and the mutational hot spot amino acid residues were substituted to alanine (A) by using site-directed mutation to construct p60 variants. These p60 variants in combination with some truncated p60 proteins were used to unveil the molecular mechanism of substrate recognition and catalysis of p60 protein in the domain level. Results confirmed that the N-terminal LysM domain in p60 protein could bind to the bacterial cell wall tightly, whereas the C-terminal NlpC/P60 domain showed slight ability to hydrolyze the cell walls. These fundamental studies on p60 protein variants will provide strong support for engineering the p60 protein molecular. http://dx.doi.org/10.1016/j.nbt.2014.05.956
PU-36 GlycoDelete technology: shortcutting mammalian cell Nglycosylation Francis Santens ∗ , Leander Meuris, Morgane Boone, Nico Callewaert VIB Ghent University, Belgium Mammalian complex-type N-glycan synthesis is a multi-step process that results in heterogeneous glycosylation of proteins. Heterogeneity in therapeutic glycoproteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. Here we report engineered mammalian cell lines that have a shortened Golgi N-glycosylation pathway, which leads to the expression of proteins with small, sialylated trisaccharide N-glycans. This glycoengineering strategy, which we call GlycoDelete [1], results in proteins with substantially reduced glycan heterogeneity. To assess the potential of these GlycoDelete glycans and their influence on glycosylated pharmaceutical proteins, human GM-CSF and an anti-CD20 antibody were produced in 293s and 293sGlycoDelete cells. Both proteins were purified and thoroughly analysed. For hGM-CSF we did not see a significant influence of the GlycoDelete sugars on the activity of the protein. GlycoDelete anti-CD20 on the other hand has a significantly reduced Fc␥R affinity and an increased circulation times in mice compared to 293S produced anti-CD20. Reference [1].Meuris L, Santens F, Elson G, et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 2014, advance online publication.
http://dx.doi.org/10.1016/j.nbt.2014.05.957
S198
www.elsevier.com/locate/nbt
PU-37 Extracellular transaminases for biocatalysis Katrin Weinhandl 1,∗ , Margit Winkler 1 , Anton Glieder 1 , Andrea Camattari 2 1 2
Austrian Center of Industrial Biotechnology (ACIB), Austria TU Graz, Austria
Branched chain aminotransferase (BCAT, EC 2.6.1.42) of Escherichia coli is an intracellular protein and an interesting tool for the production of chiral amines or amino acids. Secretion of BCAT to the culture supernatant was the method of choice to facilitate industrial enzyme applications and downstream processing by whole cell applications while counteracting limited cell permeability for target substrates. Pichia pastoris was chosen as expression host because of its positive characteristics, such as the ability to reach high biomass levels as well as the lack of background proteins in the extracellular environment during expression. Although secretion of intracellular proteins was reported to be problematic in the past, we were able to secrete BCAT in Pichia pastoris and obtained a maximum activity level in the supernatant of 150 mol/min/mg total protein (L-leucine conversion in a coupled enzymatic assay [1]). In order to improve the expression level, several approaches were investigated: on the one hand we examined different Pichia strains. On the other hand, alternative signal peptides and different promoters were evaluated for improved expression and secretion of BCAT. In our hands methanol-induced expression lead to a higher activity in the supernatant, compared to constitutive expression which still allowed satisfying BCAT secretion. Reference [1].Weinhandl, et al. Tetrahedron 2012;68(37):7586–90.
http://dx.doi.org/10.1016/j.nbt.2014.05.958
PU-38 A method to stably integrate multiple genetic elements into Chinese hamster ovary (CHO) cells Sabine Vcelar 1,∗ , Martina Baumann 1 , Nicole Borth 2 1
ACIB - Austrian Centre of Industrial Biotechnology, Austria Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
2
CHO cells are the preferred host cells for the production of therapeutic proteins and the most commonly used mammalian expression system. Advantages such as an easy cultivation, fast growth, complex protein folding and human-like posttranslational modifications are in part set of by slow cell line and process development. These constraints lead to an increased requirement for CHO cell line modification tools. The present work focuses on the integration of up to four genetic elements into CHO cells. Two different approaches were established. The principle of both systems is the Recombinasemediated cassette exchange (RMCE). Systems A is engineered on the basis of transfection vectors comprised of different resistance
RECOMBINANT PROTEIN PRODUCTION
can be separated at the amino acid residue 270. However, the active functions of these two domains in p60 protein and their influences on the substrate recognition and catalytic activity of p60 protein are unknown. Here we identified both of the functional hot spot and the mutational hot spot amino acid residues in these two structural domains by means of the amino acid sequence alignment of different p60 variants, including two p60 variants screened in our lab. The functional hot spot and the mutational hot spot amino acid residues were substituted to alanine (A) by using site-directed mutation to construct p60 variants. These p60 variants in combination with some truncated p60 proteins were used to unveil the molecular mechanism of substrate recognition and catalysis of p60 protein in the domain level. Results confirmed that the N-terminal LysM domain in p60 protein could bind to the bacterial cell wall tightly, whereas the C-terminal NlpC/P60 domain showed slight ability to hydrolyze the cell walls. These fundamental studies on p60 protein variants will provide strong support for engineering the p60 protein molecular. http://dx.doi.org/10.1016/j.nbt.2014.05.956
PU-36 GlycoDelete technology: shortcutting mammalian cell Nglycosylation Francis Santens ∗ , Leander Meuris, Morgane Boone, Nico Callewaert VIB Ghent University, Belgium Mammalian complex-type N-glycan synthesis is a multi-step process that results in heterogeneous glycosylation of proteins. Heterogeneity in therapeutic glycoproteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. Here we report engineered mammalian cell lines that have a shortened Golgi N-glycosylation pathway, which leads to the expression of proteins with small, sialylated trisaccharide N-glycans. This glycoengineering strategy, which we call GlycoDelete [1], results in proteins with substantially reduced glycan heterogeneity. To assess the potential of these GlycoDelete glycans and their influence on glycosylated pharmaceutical proteins, human GM-CSF and an anti-CD20 antibody were produced in 293s and 293sGlycoDelete cells. Both proteins were purified and thoroughly analysed. For hGM-CSF we did not see a significant influence of the GlycoDelete sugars on the activity of the protein. GlycoDelete anti-CD20 on the other hand has a significantly reduced Fc␥R affinity and an increased circulation times in mice compared to 293S produced anti-CD20. Reference [1].Meuris L, Santens F, Elson G, et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 2014, advance online publication.
http://dx.doi.org/10.1016/j.nbt.2014.05.957
S198
www.elsevier.com/locate/nbt
PU-37 Extracellular transaminases for biocatalysis Katrin Weinhandl 1,∗ , Margit Winkler 1 , Anton Glieder 1 , Andrea Camattari 2 1 2
Austrian Center of Industrial Biotechnology (ACIB), Austria TU Graz, Austria
Branched chain aminotransferase (BCAT, EC 2.6.1.42) of Escherichia coli is an intracellular protein and an interesting tool for the production of chiral amines or amino acids. Secretion of BCAT to the culture supernatant was the method of choice to facilitate industrial enzyme applications and downstream processing by whole cell applications while counteracting limited cell permeability for target substrates. Pichia pastoris was chosen as expression host because of its positive characteristics, such as the ability to reach high biomass levels as well as the lack of background proteins in the extracellular environment during expression. Although secretion of intracellular proteins was reported to be problematic in the past, we were able to secrete BCAT in Pichia pastoris and obtained a maximum activity level in the supernatant of 150 mol/min/mg total protein (L-leucine conversion in a coupled enzymatic assay [1]). In order to improve the expression level, several approaches were investigated: on the one hand we examined different Pichia strains. On the other hand, alternative signal peptides and different promoters were evaluated for improved expression and secretion of BCAT. In our hands methanol-induced expression lead to a higher activity in the supernatant, compared to constitutive expression which still allowed satisfying BCAT secretion. Reference [1].Weinhandl, et al. Tetrahedron 2012;68(37):7586–90.
http://dx.doi.org/10.1016/j.nbt.2014.05.958
PU-38 A method to stably integrate multiple genetic elements into Chinese hamster ovary (CHO) cells Sabine Vcelar 1,∗ , Martina Baumann 1 , Nicole Borth 2 1
ACIB - Austrian Centre of Industrial Biotechnology, Austria Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
2
CHO cells are the preferred host cells for the production of therapeutic proteins and the most commonly used mammalian expression system. Advantages such as an easy cultivation, fast growth, complex protein folding and human-like posttranslational modifications are in part set of by slow cell line and process development. These constraints lead to an increased requirement for CHO cell line modification tools. The present work focuses on the integration of up to four genetic elements into CHO cells. Two different approaches were established. The principle of both systems is the Recombinasemediated cassette exchange (RMCE). Systems A is engineered on the basis of transfection vectors comprised of different resistance