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