11:10-12:00, Rm 103

Biotechnology and bioengineeringK-18-15Catechol type siderophore production by Bacillus lentimorbusproducing phenazine 1-carboxylic acid and glucosidase inhibitorKyoung-Ja Kim, Kyoung-Min Kwak and Jae-Hun LeeDepartment of Biotechnology, Soonchunhyang University, Asan City 336-745, KoreaCatechol type siderophore was produced by Bacillus lentimorbus under iron limitedculture condition. In this culture condition α-glucosidase inhibitor and β-glucosidaseinhibitor were also produced by this strain. Addition of iron into culture medium inhibitedthe production of siderophore and α-glucosidase inhibitor But production of β-glucosidaseinhibitor was not inhibited by iron addition. β-glucosidase inhibitor showed temperatureand pH stability, while α-glucosidase inhibitor was unstable at high temperature, 80℃, butstable at various pH Siderophore activity was unstable at high temperature, 80℃.Antioxidative activity was also observed from culture supernatant of this strain. Thisactivity was stable at 80℃. Because this strain was reported as phenazine 1-carboxylicacid producer, the relationship between PCA production, siderophore and α-, β-glucosidase inhibitor production was investigated.K-18-18Targeted modification in high eukaryote cells using tal effectornucleasesYongsub Kim, Hye Joo Kim, Jin-Soo KimDepartment of Chemistry, Seoul National University, Seoul 151-747, KoreaTargeted genome editing with zinc finger nucleases (ZFNs) is broadly useful in research,biotechnology, and molecular medicine. But functional ZFNs are technically challengingand time-consuming to make, and have sequence-bias toward guanine-rich sites, whichoften hampers precise manipulation of the genome. Here, we report that the DNA-bindingmodules of Transcription activator-like (TAL) effectors derived from plant pathogens cansubstitute for zinc fingers to make TAL Effector Nucleases (TENs) and that bothTEN/ZFN hybrid pairs and TEN pairs induce bona-fide genome modifications atendogenous sites in cultured human cells. TENs which can be designed to recognize anyDNA sequence may have reduced off-target effects and cellular toxicity compared toZFNs.K-18-16Substrate specificity of OsGSTU3 and OsGSTU4 for detoxification ofherbicidesJin-Ju Lee, Ji-Na Kong, Mi-Jin Beak and Kwang-Hoon KongBiomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences,Chung-Ang University, 221, Huksuk-Dong, Dongjak-Ku, Seoul 156-756, KoreaGlutathione S-transferases (GSTs, EC 2.5.1.18) catalyze the conjugation of GSH toreactive endogenous and exogenous compounds (herbicides particularly in the case ofplant). Although GSTs have continuously been studying since 1980s, the study on plantGSTs is still insufficient to understand both substrate specificity and catalytic mechanism.In this study, we have investigated the biochemical properties of tau type OsGSTUs. Thetau type GST genes from Oryza sativa were successfully cloned from cDNA library byPCR using oligonucleotide primers based on the OsGSTU3 and OsGSTU4 sequence.They were subcloned into the pET26 vector and transformed in Escherichia coli.Recombinant proteins were purified by GSH affinity chromatography. The amino acidsequences of OsGSTU3 and OsGSTU4 showed approximately 64% and 70% sequenceidentity with the sequences of the tau class ZmGSTU6 and ZmGSTU19, respectively.The OsGSTU4 showed high activities toward herbicides, acetochlor (1.91 nkat/mg) andalachlor (1.74 nkat/mg). These values for the OsGSTU4 were higher than reported forother tau and phi.class OsGSTs. From these results, we suggest that OsGSTU4 wouldplay important role in the herbicides detoxification.K-18-19ProteoChip-based screening of phage display library for interactingpeptides of bFGFChang-ki Choi¹, Hyun Park², Mi-Hyang Nam², SeulKi Lee¹, Yoonsuk Lee¹, IlJunPark¹, Yong-wan Cho¹, Eun Kyoung Lee¹, Mi Sook Kim¹, Hyejin Lim¹, Soo-IkChang²and Moon Hi Han¹¹Protein Chip Research Center, Proteogen, Inc., Chuncheon, Korea, ²Department of Biochemistry,Chungbuk National University, Cheongju, KoreaBasic fibroblast growth factor (bFGF) induced angiogenesis in tumor formation andinvolved proliferation in tumor growth. Recently, anti-bFGF neutralizing antibody inhibitedproliferation, migration and tube formation in human umbilical vein endothelial cells.Peptides have been known to be potential drug compounds due to their specific biologicalactivity and low toxicity. Biologically active peptide can be screened from either syntheticpeptide library or phage display library. Here we report on-chip screening methods fromphage display dodecapeptide library with bFGF in application of ProteoChip technologyand selected positive phage clones. The peptide identified by DNA sequencing andsynthesized 12-mer peptide (BFP-6). Binding of the bFGF to BFP-6 was quantified bymeasuring the fluorescence resonance energy transfer (KD = 50nM). Functionally, BFP-6inhibited bFGF induced phosphorylation of ERK in HeLa cells. Our results demonstratedthat ProteoChip can well be applied for phage display library screening and that BFP-6can be useful anti-bFGF peptide for further studies of anti-tumor activity.K-18-17Expression and Purification of human T1R2-T1R3 sweet receptor inEscherichia coliDong-Hyeon Jo, Sug-Young Yoon, Hyun-Joo Jo and Kwang-Hoon KongBiomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Science, Chung-Ang University, Seoul, KoreaThe human sweet-sensing T1R2-T1R3 receptors are T1R family of Class C G proteincoupledreceptors in the peripheral gustatory system. Family C GPCRs are hetero- orhomodimeric receptors with a large extracellular region composed of two domains: theVenus flytrap module (VFTM) and the cysteine-rich domain (CRD). The VFTM is a twolobedclamshell-like structure . The CRD lies between the VFTM and the heptahelicaltransmembrane domain. T1R2 combines with T1R3 to form a heterodimer that binds withsugars and other sweeteners. Tasr2 and Tasr3 genes encoding T1R2 and T1R3 wassynthesised only extracellular region without transmembrane domain after optimizing thecodon usage of genes to increase its expression level in Escherichia coli. The optimizedT1Rs genes in the pGEX-KG system were transformed into E. coli BL21. RecombinantT1R2 and T1R3 proteins were expressed and purified by GST affinity columnchromatography. They were collected for measuring interaction with sweet-taste proteinswhich are brazzein and brazzein variants. By studies on interactions between sweet-tastereceptors and brazzein, the tasting mechanism of human will be elucidated.K-18-20GSK3βactivity assay and its inhibitor screening on Kinase ChipHyo Sil An, Eun Kyoung Lee, Yoonsuk Lee, Yong-Wan Cho, IlJun Park, SeulKi Leeand Moon Hi HanProtein Chip Research Center, Proteogen, Inc., ChunCheon, KoreaGlycogen synthase kinase 3 (GSK3) is a serine/threonine kinase associated withimportant regulatory functions such as glycogen metabolism and Wnt signal pathways.Currently, GSK3 has been highly implicated in the incidence and progression of a numberof diseases including Type II diabetes, Alzheimer’s disease, and cancers. RecognizingGSK3 as a potential drug target for such diseases, GSK3 inhibitors has been intensivelystudied for their potential therapeutic applications. Previously, we have developed KinaseChip providing a novel method of kinase assay and inhibitor screening in application of amicro-well chip immobilized with intact protein substrates and enzymes in a minutequantity (1 ul & ng proteins). Here we report further application of Kinase Chip for GSK3βassay and its inhibitor screening from various libraries. GSK3βkinase chip wasconstructed by immobilization of glycogen synthase (GS) as a substrate and successfullyapplied for screening of GSK3 inhibitors by measuring signal reduction of GSphosphorylation. With this Kinase Chip system, we were able to identify 6 hit compoundswith strong inhibitory activity of GSK3βfrom 2000 chemical libraries. The inhibitoryactivities of these hit compounds were confirmed by the subsequent dose-responseanalysis.274 Korean Society for Biochemistry and Molecular Biology