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A method to stably integrate multiple genetic elements into Chinese hamster ovary (CHO) cells
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
New Biotechnology · Volume 31S · July 2014
markers and two cloning sites. The incorporation of the genetic elements of choice is mediated by RMCE at two specific genomic locations. The second system established combines the RMCE system with the R4 Integrase System to enable integration of up to four genes at a single specific genomic location. The stable integration of DNA elements of choice at specific genomic locations and the testing of candidate genes for phenotypic effects without disturbance by clone specific variation is getting feasible with these methods. http://dx.doi.org/10.1016/j.nbt.2014.05.959
PU-39 Construction of pH-sensitive Her2 binding antibody fragment by directed evolution using yeast display Elisabeth Lobner ∗ , Michael Traxlmayr, Florian Rüker, Christian Obinger University of Natural Resources and Life Sciences, Vienna, Austria The half-life of therapeutic antibodies, which are internalized together with their antigenic receptor, can be increased by decreasing their affinity at acidic endosomal conditions. Here, a directed evolution protocol was developed for construction of pHdependent binding sites by using yeast display. The C-terminal structural loops of an antigen binding crystallizable fragment of immunoglobulin G1 (Fcab) [1] have been engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A library based on a Her2-ECD binding lead Fcab was constructed by parsimonious mutagenesis and displayed on yeast. Alternating selections for binding at pH 7.4 and non-binding at pH 6.0 were performed by FACS probing the binding to the antigen as well as a structurally specific ligand. The three best performing variants (P1, P2, P3) were selected. Displayed on yeast they showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4). Additionally, solubly expressed P1, P2 and P3 exhibited pH-dependent interactions with Her2positive cells whereas their conformational and thermal stability was pH-independent. The interaction of P1, P2 and P3 with the neonatal Fc receptor remained wild-type like showing the inverse pH-dependence compared to Her2-ECD binding. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. Reference [1].Wozniak-Knopp, et al. Protein Eng Des Sel 2010;23:289–97.
RECOMBINANT PROTEIN PRODUCTION
PU-40 The employment of a heterologous yeast expression system for production of VP1-derived virus-like particles originated from novel human polyomaviruses Alma Gedvilaite ∗ , Milda Norkiene, Rita Lasickieke Vilnius University Institute of Biotechnology, Lithuania Virus-like particles (VLPs) resemble their parent virion in structure, immunogenicity, tropism and transduction efficiency, but do not contain any viral genetic material. They can be used for diagnostic purposes, vaccination and gene therapy. Polyomaviridae is a growing family of naked, double-stranded DNA viruses that infect birds and mammals. The major capsid protein VP1 of all polyomaviruses (PyV) is sufficient for assembly of VLPs and represents the major immunogenic protein of PyV. In the last few years, the human polyomavirus (HPyV) family has expanded to 12 members. Serological studies are the primary tool to investigate the prevalence of various polyomaviruses in human populations. The recombinant VP1 VLPs are particularly valuable for the serological detection of these viruses as many PyV cannot be easily cultured. The earlier discovered PyV VP1-derived VLPs were successfully produced using different eukaryotic and prokaryotic expression systems including yeast. Here, we report that the galactoseinducible yeast S. cerevisiae expression system is efficient for high-level production and self assembly of VP1 derived from new HPyV: KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7. The formation of empty VP1-derived VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Yeast-generated VP1 VLPs were free of toxins, host cell DNA and proteins. The purified VP1 VLPs originating from KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7 were successfully used for generation of monoclonal antibodies and might be useful for the generation of new diagnostic tools, antiviral vaccines or gene delivery systems. http://dx.doi.org/10.1016/j.nbt.2014.05.961
PU-41 Purification by affinity chromatography of recombinant L-asparaginase I from Saccharomyces cerevisiae expressed in Escherichia coli Adalberto Pessoa-Jr 1,∗ , Gisele Monteiro 1 , Joao Santos 2 , Johanna Oses 1 , Albert Peixoto 3 , Juan Santos 1 , Joao Molino 1 , Laura Oliveira 4 , Joao Coutinho 2 , Sonia Ventura 2 , Andre Lopes 1 1
University of Sao Paulo, Brazil University of Aveiro, Brazil 3 Universidade Estadual do Sudoeste da Bahia, Brazil 4 University of Campinas, Brazil 2
http://dx.doi.org/10.1016/j.nbt.2014.05.960
L-Asparaginase is known by its capacity to catalyse the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This enzyme has been clinically acceptable as an anti-tumour agent for treatment of acute lymphoblastic leukemia and lymphosarcoma. This biopharmaceutical is produced by fermentation processes and subsequently it needs to be correctly separated from the contaminants www.elsevier.com/locate/nbt S199
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
New Biotechnology · Volume 31S · July 2014
markers and two cloning sites. The incorporation of the genetic elements of choice is mediated by RMCE at two specific genomic locations. The second system established combines the RMCE system with the R4 Integrase System to enable integration of up to four genes at a single specific genomic location. The stable integration of DNA elements of choice at specific genomic locations and the testing of candidate genes for phenotypic effects without disturbance by clone specific variation is getting feasible with these methods. http://dx.doi.org/10.1016/j.nbt.2014.05.959
PU-39 Construction of pH-sensitive Her2 binding antibody fragment by directed evolution using yeast display Elisabeth Lobner ∗ , Michael Traxlmayr, Florian Rüker, Christian Obinger University of Natural Resources and Life Sciences, Vienna, Austria The half-life of therapeutic antibodies, which are internalized together with their antigenic receptor, can be increased by decreasing their affinity at acidic endosomal conditions. Here, a directed evolution protocol was developed for construction of pHdependent binding sites by using yeast display. The C-terminal structural loops of an antigen binding crystallizable fragment of immunoglobulin G1 (Fcab) [1] have been engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A library based on a Her2-ECD binding lead Fcab was constructed by parsimonious mutagenesis and displayed on yeast. Alternating selections for binding at pH 7.4 and non-binding at pH 6.0 were performed by FACS probing the binding to the antigen as well as a structurally specific ligand. The three best performing variants (P1, P2, P3) were selected. Displayed on yeast they showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4). Additionally, solubly expressed P1, P2 and P3 exhibited pH-dependent interactions with Her2positive cells whereas their conformational and thermal stability was pH-independent. The interaction of P1, P2 and P3 with the neonatal Fc receptor remained wild-type like showing the inverse pH-dependence compared to Her2-ECD binding. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. Reference [1].Wozniak-Knopp, et al. Protein Eng Des Sel 2010;23:289–97.
RECOMBINANT PROTEIN PRODUCTION
PU-40 The employment of a heterologous yeast expression system for production of VP1-derived virus-like particles originated from novel human polyomaviruses Alma Gedvilaite ∗ , Milda Norkiene, Rita Lasickieke Vilnius University Institute of Biotechnology, Lithuania Virus-like particles (VLPs) resemble their parent virion in structure, immunogenicity, tropism and transduction efficiency, but do not contain any viral genetic material. They can be used for diagnostic purposes, vaccination and gene therapy. Polyomaviridae is a growing family of naked, double-stranded DNA viruses that infect birds and mammals. The major capsid protein VP1 of all polyomaviruses (PyV) is sufficient for assembly of VLPs and represents the major immunogenic protein of PyV. In the last few years, the human polyomavirus (HPyV) family has expanded to 12 members. Serological studies are the primary tool to investigate the prevalence of various polyomaviruses in human populations. The recombinant VP1 VLPs are particularly valuable for the serological detection of these viruses as many PyV cannot be easily cultured. The earlier discovered PyV VP1-derived VLPs were successfully produced using different eukaryotic and prokaryotic expression systems including yeast. Here, we report that the galactoseinducible yeast S. cerevisiae expression system is efficient for high-level production and self assembly of VP1 derived from new HPyV: KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7. The formation of empty VP1-derived VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Yeast-generated VP1 VLPs were free of toxins, host cell DNA and proteins. The purified VP1 VLPs originating from KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7 were successfully used for generation of monoclonal antibodies and might be useful for the generation of new diagnostic tools, antiviral vaccines or gene delivery systems. http://dx.doi.org/10.1016/j.nbt.2014.05.961
PU-41 Purification by affinity chromatography of recombinant L-asparaginase I from Saccharomyces cerevisiae expressed in Escherichia coli Adalberto Pessoa-Jr 1,∗ , Gisele Monteiro 1 , Joao Santos 2 , Johanna Oses 1 , Albert Peixoto 3 , Juan Santos 1 , Joao Molino 1 , Laura Oliveira 4 , Joao Coutinho 2 , Sonia Ventura 2 , Andre Lopes 1 1
University of Sao Paulo, Brazil University of Aveiro, Brazil 3 Universidade Estadual do Sudoeste da Bahia, Brazil 4 University of Campinas, Brazil 2
http://dx.doi.org/10.1016/j.nbt.2014.05.960
L-Asparaginase is known by its capacity to catalyse the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This enzyme has been clinically acceptable as an anti-tumour agent for treatment of acute lymphoblastic leukemia and lymphosarcoma. This biopharmaceutical is produced by fermentation processes and subsequently it needs to be correctly separated from the contaminants www.elsevier.com/locate/nbt S199