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Genetic engineering of Saccharomyces cerevisiae for efficient IgG-assembly and secretion
New Biotechnology · Volume 31S · July 2014
process. Optimisation of fermentation conditions to decrease cell stress has been shown to favour the accumulation of soluble and active recombinant proteins [1]. We have shown that shake-flask studies are an appropriate tool for the optimisation of fermentation conditions, reducing the time and cost involved in process development. This approach was used for the production of tumour necrosis factor ␣ (TNF␣) in E. coli using the arabinose-inducible T7 expression system. A volumetric yield of 3.82 g L−1 of TNF␣ was achieved by fermentation, 92% being soluble and active. The proven success of this approach can be now applied to a broader range of recombinant proteins. We will discuss the selection of adequate expression systems and approaches for the optimisation of cultivation conditions as key factors for the production of recombinant proteins. The main goal of this project is to test and integrate fermentation conditions allowing the design of platforms following a “toolbox approach” for protein production. Reference [1].Sevastsyanovich Y, Alfasi S, Overton T, et al. Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins. FEMS Microbiol Lett 2009;299:86–94.
http://dx.doi.org/10.1016/j.nbt.2014.05.953
PU-33 In vivo reconstitution of membrane protein by caveolin1 co-expression Jonghyeok Shin ∗ , Paul Heo, Joon-Bum Park, Myungseo Park, Younghun Jung, Da-Hyeong Cho, Byoung-jae Kong, Junghoon In, Jichun Lee, Dae-Hyuk Kweon Sungkyunkwan University/Bioengineering Department, South Korea Caveolae is a membrane-budding structure which exists in many animal vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicle. Recently, It was shown that caveolae could be formed in Escherichia coli by expressing caveolin-1. The heterologous caveolae may host other membrane proteins overexpressed inside the cell. We utilized this system for construction of proteo-liposome in Escherichia coli. SNARE proteins (Syntaxin1a, SNAP25, VAMP2) were introduced to prove our in vivo reconstitution system. Here, we show that the purified heterologous caveolae indeed contain the co-expressed membrane protein and the membrane proteins were facing outward. The size of the purified caveolae with membrane protein reconstituted were measured by dynamic light scattering. The presence of VAMP2 & Syntaxin1a on this proteoendosome was confirmed by Western blot analysis. Furthermore, membrane proteins (VAMP2 & Syntaxin1) embedded in caveolae retained the ability to form SNARE complex. Our study proposes an in vivo membrane protein reconstitution system. http://dx.doi.org/10.1016/j.nbt.2014.05.954
RECOMBINANT PROTEIN PRODUCTION
PU-34 Genetic engineering of Saccharomyces cerevisiae for efficient IgG-assembly and secretion Essi Koskela ∗ , Alexander Frey Aalto University, Finland IgG-antibodies are complex molecules that require multiple elements to assemble efficiently, including folding factors and an oxidative environment. The growing demand for biosimilars and alternative treatments makes the production process of human antibodies an intriguing area of research. Although simple microbial expression platforms such as Saccharomyces cerevisiae are not intrinsically suited for IgG-production, the tools to genetically modify the yeast cells for this purpose are versatile. To improve the secretion of produced protein, we focus on modifying ER luminal environment and protein folding process. One important approach is increasing the size of ER, which can be achieved with a single gene knock-out [1]. The extra space is supplemented with additional folding factors important in IgG-tetramer assembly including molecular chaperones, PDI and PPIases. These factors are tested in different amounts and combinations and the strains producing the highest yields are identified with high-throughput screening. Genetic engineering requires a substantial amount of laborintensive cloning work. To optimize this aspect, we recently discovered a new approach to molecular cloning, which outcompetes many of the existing cloning methods in simplicity and affordability. This novel protocol is presented along with preliminary results of genetic modifications on IgG-secretion. With suitable high-throughput methods in hand, the microbial platform for IgG-production is optimized at the level of the whole system. Reference [1].Schuck S, a Prinz W, Thorn KS, Voss C, Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 2009;187:525–36.
http://dx.doi.org/10.1016/j.nbt.2014.05.955
PU-35 Study on the domain function of Listeria monocytogenes p60 protein Minliang Guo ∗ , Hao Gu, Qian Xu, Jinrong Zuo College of Bioscience and Biotechnology, Yangzhou University, China Listeria monocytogenes p60 protein is an autolysin that can hydrolyze the peptidoglycans of bacterial cell walls. L. monocytogenes p60 protein was required for L. monocytogenes virulence. Besides the importance of p60 protein in bacterial pathogenesis, p60 protein can also be developed as a new proteinaceous antimicrobial if its activity to hydrolyze the peptidoglycans can be greatly improved. It contains two independent structural domains, Nterminal LysM domain and C-terminal NlpC/P60 domain, which www.elsevier.com/locate/nbt S197
process. Optimisation of fermentation conditions to decrease cell stress has been shown to favour the accumulation of soluble and active recombinant proteins [1]. We have shown that shake-flask studies are an appropriate tool for the optimisation of fermentation conditions, reducing the time and cost involved in process development. This approach was used for the production of tumour necrosis factor ␣ (TNF␣) in E. coli using the arabinose-inducible T7 expression system. A volumetric yield of 3.82 g L−1 of TNF␣ was achieved by fermentation, 92% being soluble and active. The proven success of this approach can be now applied to a broader range of recombinant proteins. We will discuss the selection of adequate expression systems and approaches for the optimisation of cultivation conditions as key factors for the production of recombinant proteins. The main goal of this project is to test and integrate fermentation conditions allowing the design of platforms following a “toolbox approach” for protein production. Reference [1].Sevastsyanovich Y, Alfasi S, Overton T, et al. Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins. FEMS Microbiol Lett 2009;299:86–94.
http://dx.doi.org/10.1016/j.nbt.2014.05.953
PU-33 In vivo reconstitution of membrane protein by caveolin1 co-expression Jonghyeok Shin ∗ , Paul Heo, Joon-Bum Park, Myungseo Park, Younghun Jung, Da-Hyeong Cho, Byoung-jae Kong, Junghoon In, Jichun Lee, Dae-Hyuk Kweon Sungkyunkwan University/Bioengineering Department, South Korea Caveolae is a membrane-budding structure which exists in many animal vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicle. Recently, It was shown that caveolae could be formed in Escherichia coli by expressing caveolin-1. The heterologous caveolae may host other membrane proteins overexpressed inside the cell. We utilized this system for construction of proteo-liposome in Escherichia coli. SNARE proteins (Syntaxin1a, SNAP25, VAMP2) were introduced to prove our in vivo reconstitution system. Here, we show that the purified heterologous caveolae indeed contain the co-expressed membrane protein and the membrane proteins were facing outward. The size of the purified caveolae with membrane protein reconstituted were measured by dynamic light scattering. The presence of VAMP2 & Syntaxin1a on this proteoendosome was confirmed by Western blot analysis. Furthermore, membrane proteins (VAMP2 & Syntaxin1) embedded in caveolae retained the ability to form SNARE complex. Our study proposes an in vivo membrane protein reconstitution system. http://dx.doi.org/10.1016/j.nbt.2014.05.954
RECOMBINANT PROTEIN PRODUCTION
PU-34 Genetic engineering of Saccharomyces cerevisiae for efficient IgG-assembly and secretion Essi Koskela ∗ , Alexander Frey Aalto University, Finland IgG-antibodies are complex molecules that require multiple elements to assemble efficiently, including folding factors and an oxidative environment. The growing demand for biosimilars and alternative treatments makes the production process of human antibodies an intriguing area of research. Although simple microbial expression platforms such as Saccharomyces cerevisiae are not intrinsically suited for IgG-production, the tools to genetically modify the yeast cells for this purpose are versatile. To improve the secretion of produced protein, we focus on modifying ER luminal environment and protein folding process. One important approach is increasing the size of ER, which can be achieved with a single gene knock-out [1]. The extra space is supplemented with additional folding factors important in IgG-tetramer assembly including molecular chaperones, PDI and PPIases. These factors are tested in different amounts and combinations and the strains producing the highest yields are identified with high-throughput screening. Genetic engineering requires a substantial amount of laborintensive cloning work. To optimize this aspect, we recently discovered a new approach to molecular cloning, which outcompetes many of the existing cloning methods in simplicity and affordability. This novel protocol is presented along with preliminary results of genetic modifications on IgG-secretion. With suitable high-throughput methods in hand, the microbial platform for IgG-production is optimized at the level of the whole system. Reference [1].Schuck S, a Prinz W, Thorn KS, Voss C, Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 2009;187:525–36.
http://dx.doi.org/10.1016/j.nbt.2014.05.955
PU-35 Study on the domain function of Listeria monocytogenes p60 protein Minliang Guo ∗ , Hao Gu, Qian Xu, Jinrong Zuo College of Bioscience and Biotechnology, Yangzhou University, China Listeria monocytogenes p60 protein is an autolysin that can hydrolyze the peptidoglycans of bacterial cell walls. L. monocytogenes p60 protein was required for L. monocytogenes virulence. Besides the importance of p60 protein in bacterial pathogenesis, p60 protein can also be developed as a new proteinaceous antimicrobial if its activity to hydrolyze the peptidoglycans can be greatly improved. It contains two independent structural domains, Nterminal LysM domain and C-terminal NlpC/P60 domain, which www.elsevier.com/locate/nbt S197