Discovery of extracellular respiration of NADH and its application in novel NADH regeneration
1
understanding of biophysical properties of glycoproteins and protein– glycan conjugates for biotechnological purpose. References 1. Shental-Bechor, D. and Levy, Y.: Effect of glycosylation on protein folding: a close look at thermodynamic stabilization, Proc. Natl. Acad. Sci. USA, 105, 8256-8261 (2008). 2. Choi, Y., Lee, J.H., Hwang, S., Kim, J.K., Jeong, K., and Jung, S.: Retardation of the unfolding process by single N-glycosylation of ribonuclease A based on molecular dynamics simulations. Biopolymers. 89., 114-123 (2008). 3. Wyss, D.F. and Wagner, G.:, The structural role of sugars in glycoproteins. Curr. Opin. Biotechnol., 7., 409-416 (1996). doi:10.1016/j.jbiosc.2009.08.178 BE-O11 Discovery of extracellular respiration of NADH and its application in novel NADH regeneration Chong Zhang, 1 Yo Hirose, 1,2 Xi Wu, 1 Kun Ma, 1 Ichiro Okura, 2 and Xin-Hui Xing 1 Department of Chemical Engineering, Tsinghua University, Bejing, China 1 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Tokyo, Japan 2 Soluble substrates can be utilized extracellularly by extracellular respiration. But no study has showed NADH could be respired outside the cells. The present work have interestingly discovered that NADH could be extracellularly respired by many organisms. The extracellular NADH oxidase activities (ENOAs) of whole cells of Enterobacter aerogenes, Pseudomonas putida and Bacillus cereus were 0.1, 0.5 and 1.2 U/OD 600 , respectively. When the whole cells of the above three strains were treated with proteinase K, the ENOAs were lost, suggesting that the NADH oxidase was located outside of the cells. To clarify the extracellular NADH oxidization system, the general secretion pathway II with the function for protein secretion was knocked-out for analyzing its influences on the ENOAs. Moreover, the extracellular NADH oxidase was purified and sequenced. And the function of the target enzyme was confirmed through its overexpression. As far as we know, this is the first report on the discovery of extracellular respiration of NADH. The extracellular NADH oxidation is considered as a promising tool for developing novel NADH regenera- tion system. Acknowledgments: This work was supported by the Project of the Natural Science Foundation of China (Grant No. 20806046) and the National Basic Research Program of China (Grant No. 2009CB724702). doi:10.1016/j.jbiosc.2009.08.179 BE-O12 Biomolecular engineering of human lectin galectin-2 Hui Wang, 1 Lizhong He, 1 Martin Lensch, 2 Hans-Joachim Gabius, 2 Conan J. Fee, 3 and Anton P.J. Middelberg 1 The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Centre for Biomolecular Engineering, St Lucia, QLD 4072, Australia 1 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, 80539 Munich, Germany 2 and Department of Chemical and Process Engineering, University of Canterbury, Christchurch 8140, New Zealand 3 The activity of galectins as potent regulators of cell growth and adhesion, e.g. in malignancy and autoimmune diseases, make them attractive drug candidates (1). In this work, we demonstrate that these endogenous lectins can be transformed into pharmaceutically stable forms, using human galectin-2 (Gal2) as a proof-of-concept example. We constructed three mutants of Gal2 (C57A, C57M and C57S) by introducing mutations at the site of one of the two Cys residues. Only the C57M variant was expressed in E. coli in highly soluble form. Mutant C57M retained its binding ability to lactose, facilitating a single-step affinity purification using lactose-agarose. The modified protein showed no detectable aggregation following three weeks of storage, in contrast to significant aggregation for the wild type protein. The C57M mutation enabled site-specific chemical modification as exploited by conjugation with poly- ethylene glycol at the remaining sulfhydryl group (Cys75). Ion exchange chromatography was used to separate homogenous PEG- Gal2 from the reaction solution. The results thus demonstrate the feasibility of combined genetic and chemical modifications to enhance the suitability of a human lectin as a pharmaceutically relevant protein, in addition to the known physiological benefits of PEGylation. Reference 1. Gabius, H.-J.: The sugar code. Fundamentals of glycosylation. Wiley-VCH. Weinheim (2009). doi:10.1016/j.jbiosc.2009.08.180 BE-O13 Construction of a novel detection system for protein–protein interactions using yeast G-protein signaling Nobuo Fukuda, 1 Jun Ishii, 2 Tsutomu Tanaka, 2 and Akihiko Kondo 1 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan 1 and Organization of Advanced Science and Technology, Kobe University, Kobe, Japan 2 In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein gamma subunit (Ggamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of Ggamma is required for signal transduction. In our system, mutated Ggamma (Ggamma cyto ) lacking membrane locali- zation ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when Ggamma cyto was expressed in the cytoplasm of yeast cells from which the endogenous Ggamma gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to Ggamma cyto . The Fc-Ggamma cyto fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore S61 Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S57–S74
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Transcript of Discovery of extracellular respiration of NADH and its application in novel NADH regeneration
S61Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S57–S74
understanding of biophysical properties of glycoproteins and protein–glycan conjugates for biotechnological purpose.
References
1. Shental-Bechor, D. and Levy, Y.: Effect of glycosylation on protein folding: a close lookat thermodynamic stabilization, Proc. Natl. Acad. Sci. USA, 105, 8256-8261 (2008).
2. Choi, Y., Lee, J.H., Hwang, S., Kim, J.K., Jeong, K., and Jung, S.: Retardation of theunfolding process by single N-glycosylation of ribonuclease A based on moleculardynamics simulations. Biopolymers. 89., 114-123 (2008).
3.Wyss, D.F. and Wagner, G.:, The structural role of sugars in glycoproteins. Curr. Opin.Biotechnol., 7., 409-416 (1996).
doi:10.1016/j.jbiosc.2009.08.178
BE-O11
Discovery of extracellular respiration of NADH and its applicationin novel NADH regeneration
Chong Zhang,1 Yo Hirose,1,2 Xi Wu,1 Kun Ma,1 Ichiro Okura,2 andXin-Hui Xing1
Department of Chemical Engineering, Tsinghua University, Bejing, China1
Graduate School of Bioscience and Biotechnology, Tokyo Institute ofTechnology, Tokyo, Japan2
Soluble substrates can be utilized extracellularly by extracellularrespiration. But no study has showed NADH could be respired outsidethe cells. The present work have interestingly discovered that NADHcould be extracellularly respired by many organisms.
The extracellular NADH oxidase activities (ENOAs) of whole cells ofEnterobacter aerogenes, Pseudomonas putida and Bacillus cereus were0.1, 0.5 and1.2U/OD600, respectively.When thewhole cells of the abovethree strains were treated with proteinase K, the ENOAs were lost,suggesting that the NADH oxidase was located outside of the cells. Toclarify the extracellular NADHoxidization system, the general secretionpathway II with the function for protein secretionwas knocked-out foranalyzing its influences on the ENOAs. Moreover, the extracellularNADH oxidase was purified and sequenced. And the function of thetarget enzyme was confirmed through its overexpression.
As far as we know, this is the first report on the discovery ofextracellular respiration of NADH. The extracellular NADH oxidation isconsidered as a promising tool for developing novel NADH regenera-tion system.
Acknowledgments: This work was supported by the Project of theNatural Science Foundation of China (Grant No. 20806046) and theNational Basic Research Program of China (Grant No. 2009CB724702).
doi:10.1016/j.jbiosc.2009.08.179
BE-O12
Biomolecular engineering of human lectin galectin-2
Hui Wang,1 Lizhong He,1 Martin Lensch,2 Hans-Joachim Gabius,2
Conan J. Fee,3 and Anton P.J. Middelberg1
The University of Queensland, Australian Institute for Bioengineering andNanotechnology, Centre for Biomolecular Engineering, St Lucia,QLD 4072, Australia1 Institute of Physiological Chemistry, Faculty of
Veterinary Medicine, Ludwig-Maximilians-University, 80539 Munich,Germany2 and Department of Chemical and Process Engineering,University of Canterbury, Christchurch 8140, New Zealand3
The activity of galectins as potent regulators of cell growth andadhesion, e.g. in malignancy and autoimmune diseases, make themattractive drug candidates (1). In this work, we demonstrate thatthese endogenous lectins can be transformed into pharmaceuticallystable forms, using human galectin-2 (Gal2) as a proof-of-conceptexample. We constructed three mutants of Gal2 (C57A, C57M andC57S) by introducing mutations at the site of one of the two Cysresidues. Only the C57M variant was expressed in E. coli in highlysoluble form. Mutant C57M retained its binding ability to lactose,facilitating a single-step affinity purification using lactose-agarose.The modified protein showed no detectable aggregation followingthree weeks of storage, in contrast to significant aggregation for thewild type protein. The C57M mutation enabled site-specificchemical modification as exploited by conjugation with poly-ethylene glycol at the remaining sulfhydryl group (Cys75). Ionexchange chromatography was used to separate homogenous PEG-Gal2 from the reaction solution.
The results thus demonstrate the feasibility of combined geneticand chemical modifications to enhance the suitability of a humanlectin as a pharmaceutically relevant protein, in addition to the knownphysiological benefits of PEGylation.
Reference
1. Gabius, H.-J.: The sugar code. Fundamentals of glycosylation. Wiley-VCH. Weinheim(2009).
doi:10.1016/j.jbiosc.2009.08.180
BE-O13
Construction of a novel detection system for protein–proteininteractions using yeast G-protein signaling
Nobuo Fukuda,1 Jun Ishii,2 Tsutomu Tanaka,2 and Akihiko Kondo1
Department of Chemical Science and Engineering, Graduate School ofEngineering, Kobe University, Kobe, Japan1 and Organization ofAdvanced Science and Technology, Kobe University, Kobe, Japan2
In the current study, we report the construction of a novelsystem for the detection of protein-protein interactions using yeastG-protein signaling. It is well established that the G-protein gammasubunit (Ggamma) is anchored to the inner leaflet of the plasmamembrane via lipid modification in the C-terminus, and that thislocalization of Ggamma is required for signal transduction. In oursystem, mutated Ggamma (Ggammacyto) lacking membrane locali-zation ability was genetically prepared by deletion of the lipidmodification site. Complete disappearance of G-protein signal wasobserved when Ggammacyto was expressed in the cytoplasm ofyeast cells from which the endogenous Ggamma gene had beendeleted. In order to demonstrate the potential use of our system, weutilized the Staphylococcus aureus ZZ domain and the Fc portion ofhuman immunoglobulin G (IgG) as a model interaction pair. Todesign our detection system for protein-protein interaction, the ZZdomain was altered so that it associates with the inner leaflet of theplasma membrane, and the Fc part was then fused to Ggammacyto.The Fc-Ggammacyto fusion protein migrated towards the membranevia the ZZ-Fc interaction, and signal transduction was therefore
