11:10-12:00, Rm 103

Biotechnology and bioengineeringK-18-02Development of electrochemical impedance detection based cancerdiagnostic biochip using matrix-metalloproteinases (MMPs)Seung Yong Lee¹, Chansoo Park¹, Je-Sik Jeong¹, Hee Bong Kim², Seung YongHwang³ , *¹Department of Bio-Nanotechnology, Graduate School, Hanyang University, Ansan 426-791,Korea, ²Department of Mechanical Engr., Graduate School, Hanyang University, Seoul 133-791,Korea, ³Department of Biochemistry, Graduate School, Hanyang University, Ansan 426-791,KoreaA protease is any enzyme that catalyses the hydrolysis of proteins into smaller peptidefraction and amino acids, a process known as proteolysis. They are involved in amultitude of normal biological processes as well as in diseases. As a preliminaryexperiment for a microfluidic-based protease assay system to detect proteolytic activityusing electrochemical impedance sepctroscopy (EIS) by peptide conjugates immobilizedon the gold electrode. In this study, we experiment the reactivity between proteases andits peptide substrates We decided two matrix-metalloproteinases, MMP-2, MMP-7 forbiomaker of oavrian cancer and colorectal cancer, respectively. In the presence of matrixmetalloprotease-7 (MMP-7) and matrix metalloprotease-2 (MMP-2), peptide bond in thepeptide conjugates was digested, therefore some changes in electrochemical impedance.The designed biosensor consists of two electrode system (gold working electrode,platinum counter and reference electrode) and microchannel-based onpolydimethylsiloxane (PDMS)-glass. This technology is not limited to sensing MMP-7,MMP-2, can be used as an alternative to currently used methods. Our method could beused as an effective tool for other molecular diagnostic systems.K-18-03Improvement of the accessibility for protease to its substrate by surfacePEGylation of the Quantum dotsChansoo Park¹, Seung Yong Lee¹, Je-Sik Jeong¹, Seung Yong Hwang²¹Department of Bio-Nanotechnology, Graduate School, Hanyang University, Ansan 426-791,Korea, ²Department of Biochemistry, Graduate School, Hanyang University, Ansan 426-791,KoreaA Steric hindrance or steric resistance occurs when the size of groups within a moleculeprevents chemical reactions that are observed in related smaller molecues. In ourprevious study, We demonstrated a microfluidic-based assay system to detect proteolyticactivity using fluorescence resonance energy transfer (FRET) by quantum dot (QD)-peptide conjugates immobilized on microbeads. However, the matrix metalloproteinase-7(MMP-7), as a model protease, could not be accessed to the substrate peptide as muchas we expected due to lack of accessibility. Here we present a improved model probesurface-modified with polyethylene glycol (PEG) to minimize steric hindrance and providebetter accessibility for MMP-7 to its substrate peptide. PEGylated QD and normal QDmixture was filtrated using centrifugal filter. As a results, PEG was successfully attachedonto the surface of QD and PEGylated QD was confirmed using electrophoresis andPEG staining. This technology is not limited to sensing MMP-7, but could be liable tomonitor other protease activity.K-18-05Screening of phage library for peptides that selectively bind toMyoglobin- Dynabeads M-270 epoxyGuruprasath Padmanaban, Napolean Bonaparte, Bodhraj Acharya, Hye KyungPark, So-Yeon Lee, Kai Wang, In-San Kim, Byung-Heon LeeDepartment of Biochemistry and Cell Biology and Cell & Matrix Research Institute, School ofMedicine, Kyungpook National University, Daegu, KoreaMyoglobin, a heme protein found in both heart and skeletal muscle. Myoglobin is one ofthe first markers to rise above normal levels. Solid phase enzyme-linked immunosorbentassay were currently used for quantitative analysis of Myoglobin in the serum. Here wetry to replace the monoclonal antibodies with peptides. A M13 phage library containingCX7C random peptides (5×1010 plaque forming units) was incubated with 10 µg ofMyoglobin coated on to an Elisa Plate. Five rounds of panning were performed using themyoglobin. The phages that bound to myoglobin were collected and amplified for the nextround of screening. A 5-fold enrichment in phage titer was obtained after the fifth roundscreening compared to that of the first round. The phage clones that specifically bind tomyoglobin were collected. Inserted phage clones were identified using PCR. Twenty fiveindividual clones from the forth and fifth screening round were picked up and the insertswere amplified by PCR for sequencing. The selected phage clones demonstrated highlyspecific affinity to myoglobin. Further Biotinylated peptides were synthesized and coatedon a plastic solid phase for performing ELISA. The use of a streptavidin-biotinylatedpeptide system for coating microplates with peptide markedly increased both thesensitivity and the specificity compared to a standard ELISA based on synthetic peptides.These findings suggest that the peptide is a useful tool for quantitatively measure theamount of myoglobin present in the serum.K-18-07Expression and purification of SOX2 and identification of its highaffinitypeptides by phage displayKyoung-jin Lee and Moon-Young YoonDepartment of Chemistry, Hanyang University, Seoul 133-791, KoreaSOX2 is a transcription factor, necessary to human embryonic stem (hES) cells from thecell differentiating. Their function as embryonic stem (ES) cells can self-renewmaintaining the undifferentiated state. SOX2 can act synergistically with othertranscription factors, which regulate the expression of stem cell and self renewal ofembryonic stem (ES) cells. It was also reported that human cancer is frequentlyassociated with cells expressing the cancer stem cell marker, SOX2. Therefore, theSOX2 has been a fascinating biomarker in prevention and vaccinization for variouscancers. In the present study, we have amplified SOX2 gene from the Human lung smallcell carcinoma leaves by polymerase chain reaction and cloned into pET28a expressionvector. Nucleotide sequence analysis revealed that SOX2, a 954bp gene encodes aprotein of 318 amino acids. The expression plasmid was transformed into E. coliRosetta2 (DE3) cells, induced by 0.5 mM IPTG at 18℃ overnight and purified. Thepurified recombinant SOX2 provides a basis to screen novel peptides which binds withhigh affinity and specificity to cancer stem cell biomarker, SOX2.K-18-04Ethanol production from citrus peel waste by Sacchromyces cerevisiaeByoung-Jun Yoon¹, Han-Na You², Jae-Hoon Kim¹ , ²¹Subtropical and Tropical Organism Gene Bank, Jeju National University, ²Faculty ofBiotechnology, Jeju National University, Jeju 690-756, KoreaApproximately 60 thousand metric tons of citrus were processed into citrus juice fromfarmhouse by-product. In this study, we measured the pre-treatment effect and differentloadings of cellulase, pectinase and beta-glucosidase enzymes to hydrolyze citrus peelwaste to produce sugars. When the loadings of commercial cellulase, pectinase andbeta-glucosidase enzymes were 40EGU, 400PGU, 15Cbu to 1% peel dry matter with50mM sodium acetate buffer pH 4.8 at 45℃ for 24h. we can obtained the highestconcentration of fermentable sugar. The obtain monomeric sugar medium was used forethanol production by fermentation using a yeast strain, Saccharomyces cerevisiae.Acknowledgment : This work was supported from Renewable Energy R&D Programthrough the Korea Insititue of Energy Techonolgy Evolution and Planning (KITEP)fundedby the Ministry of Knowledge Economy (2010T100100573).K-18-08Detection of metastasis-associated transcripts using on-chip DNApolymerizationEung-Sam Kim¹, Bong Jin Hong², Chang-Wook Park³, Youngkyu Kim¹, Ji-hyeShin³, Joon Won Park¹ , ²and Kwan Yong Choi¹ , ³¹School of Interdisciplinary Bioscience and Bioengineering, National Core Research Center forSystems Bio-Dynamics, ²Department of Chemistry, Division of Integrative Biosciences andBiotechnology, ³Department of Life Science, Division of Molecular and Life Sciences, PohangUniversity of Science and Technology, San 31 Hyoja-dong, Pohang 790-784, KoreaWe demonstrated the spacing-controlled surface showing a high efficiency of theenzymatic on-chip DNA polymerization (OCP) could detect metastasis-associatedtranscripts from the liver cancer cells. The surface was modified with the 9-acid and 27-acid dendrons to provide the uniform spacing for surface-bound DNA primer as 3.4 and6.2 nm, respectively, in the lateral direction, whereas the lateral spacing of the highdensitysurface was ca. 0.45 nm. The 9-aicd dendron-coated surface showed the 8-foldhigher efficiency of OCP compared to the high-density surface. Unlike the conventionalDNA microarray that required fluorophore-labeled cDNA prior to hybridization, theunlabeled cDNA from SK-Hep1 was hybridized to the gene probes on the 9-acid dendronsurface followed by the OCP to extend and label capture probes. The fluorescence signalwas quantified to compare the expression levels of metastasis-related genes and theirlevels revealed high correlation with those of the real-time PCR (R2=0.81). Our resultssuggested that the OCP on the spacing-controlled surface could detect the genetranscripts of interest without pre-labeling cDNA and the optimum spacing facilitated theaccess of DNA-manipulating enzymes to their substrates on the surface. [Supported byNCRC, WCU, and KIAT]272 Korean Society for Biochemistry and Molecular Biology