Earticle

Monoclonal antibodies have already established a secure position as a very promising strategy for therapeutics. Advances in genetic engineering have enabled to generate human antibodies which circumvent the problems of using murine antibodies in human therapy. High specificity and high affinity are attractive characteristics of antibodies which make them suitable for clinical applications. Neutralization of antigens, immunological effects, specific delivery of antibody conjugate to target cells, and direct effects through receptor engagement by antibodies have been exploited as mechanisms which generate antibody activity. Here, data will be presented on a series of human antibodies generated using the KM MouseTM that demonstrates desirable characteristics for therapeutic use including robust biological activity and low toxicity. The preclinical data for agonistic and antagonistic antibodies to TNF receptor super family members including TRAIL-R2 and CD40, will be presented. In addition, neutralizing antibody against influenza virus M2 protein will be also presented.

We constructed a large human naive Fab library by phage display. Human B cells were isolated from PBL, bone marrow, lymph node, and spleen. The cDNAs encoding the light chain variable regions (VL) or heavy chain variable regions (VH) were synthesized by PCR from the B cells using the primers matching each of the functional germline alleles. The VL library was subcloned into the BstXI sites of pKB-Fab phagemid vector to generate a light chain library (1 X 108), and the VH library was subcloned into the SfiI sites of the light chain library to construct a Fab library (5 X 1010). For evaluation of the resulting Fab library, 200 VL (100 VK, 100 Vlamda) and 192 VH clones were randomly selected and analyzed for the sequences and expression in E.coli. Therapeutic antibodies were screened by panning against target antigens and characterized for their antigen binding affinities and biological activities.

The pharmaceutical industry has traditionally focused on developing small molecule therapies. However, the dynamics of the pharmaceutical industry are changing as a result of an R&D productivity crisis and several regulatory affairs. However novel and innovative biological therapies are expected to outshine small molecule therapies over the next 10 years and become the major sources of growth for the pharmaceutical industry. In contrast of smole molecule therapies, the biological drug targets are a key dimension of pharma industry. In this presentation, I will discuss the novel and efficient method of target discovery using the SOLUBLE RECEPTOR LIBRARY.

After 30 years of development, therapy with monoclonal antibodies has started to realize its promise. Therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry. There are currently 20 monoclonal antibodies in the market that have been approved by the FDA and over 150 in clinical developments. Monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of cancer, autoimmune diseases, viral infections, asthma, and other diseases. Hepatitis B virus (HBV) is one of the main pathogens of hepatitis and hepatocarcinoma. Human plasma-derived Hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it is required to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBV antibody titer and possible contamination of human pathogens. In order to meet this requirements, we selected human antibody to the HBsAg from an anti-HBs Ab-enriched phage-display library and a Chinese hamster ovary (CHO) cell line, HBC7A, was established which produces a fully human IgG1 antibody against HBV. Mass production and purification processes were established and this mAb was characterized in terms of HBV neutralization and safety for the purpose of human trial for liver transplantation.

Vascular cell adhesion molecule-1 (VCAM-1) is a member of the diverse cellular adhesion molecule family which includes intergrins, selectins, and the immunoglobulin superfamily and is a critical component of the leukocyte-endothelial adhesion cascade. VCAM-1 is upregulated on the endothelial under inflammatory conditions including autoimmune inflammatory diseases, atherosclerosis and organ transplantation rejection. Transendothelial migration of inflammatory cells is a key step in autoimmune inflammatory diseases (asthma, rheumatoid arthritis, IBD (inflammatory bowel disease)) and is mediated by the presence of cell adhesion molecule, including VCAM-1, on the vascular endothelium. Activated vascular endothelial cell expresses VCAM-1 which can be bound by inflammatory cells expressing very late antigen-4 (VLA-4), the ligand of VCAM-1. VCAM-1-VLA-4 interaction plays a vital role in the passage of the inflammatory cell between endothelial cells. Currently, increasing attention is being paid to VCAM-1-VLA-4 interaction as targets for therapeutic interventions in inflammatory diseases. The goal of this study is to develop a novel cross-species specific anti-VCAM-1 therapeutic antibody for inflammatory disease treatments, which has better advantages for the evaluation of therapeutic efficacy in preclinical animal models.

Therapeutic antibodies can be generated from many routes. At early stage of research, as many candidate antibodies as possible should be evaluated for optimal efficacy and manufacturability. For anti-cancer treatment, very high dose of antibody therapeutics are administered. Successful antibody development must overcome the challenge of high titer cell culture, maximum yield at down stream process, long-term stability in highconcentration formulation and optimal pharmacokinetic, pharmacodynamic property and minimum immunogenecity in human. How these issues could be managed at early stage of research will be discussed.

There have been many attempts to develop sensitive and accurate techniques for the detection and diagnosis of pathogenic bacteria using nucleic acid-based technology. To achieve efficient simultaneous detection of eleven selected pathogens, we constructed oligonucleotide microarray containing double specific capture probes and developed a statistical pattern mapping analytical tool for pathogenic bacteria detection. The two capture probes were designed from the total variable regions of 16S rDNA for precise detection. This microarray system harboring double capture probes and pattern recognition showed precise subtype discrimination between two closely related species. Therefore, using the proposed oligonucleotide microarray, we could successfully classify species and even subtypes of food-borne pathogens simultaneously.

Protein chip technology has shown its great potential in basic research, diagnostics and drug discovery to replace the classical assay. Protein chips, compared to DNA chips, have developed slowly because protein is complex and difficult for preparation. Recently various kinds of protein chips were practical used in. In diagnostic application, protein chips can replace immunoassay. Due to their high throughput, they have great potential to reduce the assay time and to get much information in a time. But immunoassay protocols were different depend on the target material and diseases. Competitive ELISA, antibody capture ELISA, direct ELISA and indirect ELISA have been used in diagnostic immunoassay. They could not be assayed in a well. Determination of various items but similar molecules to be assayed in a single protocol, such as autoimmune disease or allergy can be easily applicable area in protein chip based diagnostics. In a practical use of protein chip in diagnostic area, grouping of assays is necessary and introduced.

The covalent coupling of antibodies to solid surfaces or other biological/chemical molecules is a key step for the development of immune-based assays such as biosensors and antibody arrays. The choice of coupling method significantly affects subsequent antibody-antigen interactions. In order to fully maintain antigen binding abilities of modified antibodies, modifications must be site-selective and away from antigen binding sites, providing well-oriented antibodies. Antibody binding protein, protein G specifically binds the Fc region of an antibody and therefore provides proper orientation of the bound antibody, resulting in the antigen binding sites optimally-exposed to the assay solution without the need of any antibody modifications. Here we introduce several novel linker systems which utilize engineered protein G constructs for antibody immobilization. Various genetic and/or chemical modifications of protein G allowed controlled and efficient immobilization of antibodies on the surfaces of different assay platforms, such as those of glass/gold chips and nanoparticles.

PNA (Peptide Nucleic Acid) is synthetic DNA analogues with neutral polypeptide backbone. The PNA probes enhance thermal stability, resistance to the nuclease and protease, stronger and faster binding affinity to the complementary DNA or RNA. These remarkable hybridization properties of PNA suggest that PNA probes may be efficiently incorporated into microarrays. We have developed a PNA array platform technology for HPV genotyping, detection of lamivudine-resistant HBV, CYP 450 SNP genotyping, and miRNAs expression profiling. PNA arrays have a greatly increased specificity and sensitivity, and extended shelf-life relative to DNA arrays. We have also developed FLASH (Fluorescent Labeling After Speedy Hybridization) technology on PNA array. The FLASH allows that only hybridized target DNAs can be labeled with fluorescence dye after PNA-DNA hybridization. Specificity of the PNA array was achieved by combining FLASH technology into the assay. PNA array platform technology provides a powerful and reliable assay for SNP genotyping and gene expression profiling. Therefore, PNA array is expected to play a vital role in diagnostic applications.

As biological technology is rapidly developing, the drugs which use the BT technology is also emerging. But these drug are difficult to be applied the classical evaluation method which used to evaluate of the safety and efficacy. So many person says that regulatory process by NRA(National Regulatory Authority) is the bottleneck of the commercialization of the new bio-drugs. To break the bottleneck, KFDA has published the guideline for the evaluation in recombinant biological products since 2005. In this presentation, first the status of guideline and how to make a guideline will be introduced. Secondly, the guideline about quality evaluation and how to get an information about guideline will be showed. Ⅰ. The status of guideline about biologicals Ⅱ. The Process of the making a guideline Ⅲ. The guideline for evaluation of the quality in biologicals 1. Vaccines 2. Recombinant Products 3. Gene & Cell therapy 4. Others Ⅳ. On-line information about guideline Ⅵ. Conclusion

전체 바이오산업 중 생물의약품이 차지하는 비율이 약 50% 수준이며 우리나라 10대 차세대 성장 동력 산업인 생물제약 산업은 신약개발 자체가 최첨단 과학기술인 동시에 생물활성을 갖는 제제의 특성상 상업화를 위한 제조에 있어서는 엄격한 제조 및 품질관리기준이 요구됩니다. 이에 우리나라를 비롯한 세계 각 국은 일반 의약품의 GMP 기준 이외에 생물의약품을 위한 별도의 GMP 기준을 마련․준수하고 있습니다. 오늘은 우리나라 생물의약품 GMP 기준에 대한 개략적인 설명과 향후 생물의약품 GMP 관련 식약청 정책 추진 방향에 대해 설명하도록 하겠습니다. 우선 일반적인 GMP(Good Manufacturing Practice) 정의 및 KGMP가 제정된 경위를 설명하고, KGMP 제도의 근거 약사법령을 설명합니다. 그리고 생물의약품의 연구개발, 전임상시험단계, 임상시험단계, 품목허가 신청, 시판까지 생물의약품의 제품화 과정 각 단계 중 GMP 적용시점과 그 이유에 대해 말씀드리겠습니다. 생물의약품과 화학물질을 주성분으로하는 일반적인 의약품의 적용법규의 차이점을 알고, 생물의약품 GMP 기준의 범위와 그 적용 대상을 말씀드리고, 생물의약품이 일반의약품의 GMP 기준에 비하여 추가적으로 또는 강화되는 사항, 실제 GMP 업소 점검시 중점 점검사항, 주요 지적 사항 및 ‘08년도 식약청 사후관리 계획에 대해서도 말씀드리겠습니다. 마지막으로 저희 생물의약품관리팀에서 추진하고 있는 생물의약품 GMP 관련 정책 추진사항과 진행사항에 대하여 소개하도록 하겠습니다.

The validation is the one of key factors of the biopharmaceutical industry. From the research to commercial lunching of the biopharmaceutical product, the validation of the process, analytical method, and product itself is the one of key factors and basic requirement to prove the safety and efficacy of the target product. Recently, the overall validation regarding current Good Manufacturing Process (cGMP) has been become common requirement and also required by the laws. Topics will be covered including personnel qualification, equipment qualification, process validation, cleaning validation, computerized system validation, and also method validation.

The Common Technical Document(CTD) is part of the ICH(International Conference on Harmonization) program for developing global, harmonized guidelines for the contents and format of new drug and biologic products applications to be submitted to regulatory agencies in the ICH regions. The CTD is divided into 5 modules : 1. Region specific administrative and prescribing information 2. High level summary 3. Quality -chemical, pharmaceutical and biological documentation 4. Non-clinical study reports 5. Clinical study reports. The potential advantages of CTD can be extensive. A pharmaceutical companies could save in resources and time, so that they could facilitate simultaneous submissions in ICH regions. Also, CTD could provide a more efficient evaluation process with regulators, as well. Finally, a faster availability of new medicines is expected in the future. The CTD was first agreed upon in 2000 and now required in the EU, Japan, Canada and Switzerland and is strongly encouraged in the US. In Korea, CTD submissions will be accepted by KFDA for New Drug Application in March, 2009. In this session, a general overview of the CTD documents and a detailed introduction with each modules will be presented. The background, scientific and regulatory aspects of CTD documents will be focused.

일반 공산품과 달리 상처의 치유나 생명 연장을 위해 복용하는 의약품이 그 목적을 상실하는 것을 막도록 하기 위해 GMP라는 법을 만들었다. 이러한 GMP (Good Manufacturing Practice) 규정은 오늘날 국제적으로 의약품의 생산과 품질관리의 기본적인 규범으로 자리 매김하고 있다. GMP의 기반을 구축하기 위해서는 GMP 규정에 대한 이해가 필수적이다. 특히 글로벌 시장 공략을 위해서는 각 국의 GMP 규 정에 대한 이해를 토대로 GMP의 방향을 수립해야 한다. 이러한 목적을 달성하고자 각 국의 GMP 규정에 대한 비교와 나아가 GMP의 발전 방향을 모색하는 기회를 가지고자 한다. GMP의 시발은 미국이다. 1963년 미국이 GMP를 제정․공포하면서 GMP시대가 열렸다. 1969년 WHO (World Health Organization, 세계보건기구)가 제22차 WHO총회에서 WHO-GMP를 가결하고 회원국에게 GMP 제도의 실시를 권고하므로써 각 국의 GMP가 제정되어 실시하기에 이르렀다. 우리나라도 WHO의 권고에 따라 1977년 3월􋺳우수의약품제조관리기준􋺴을 공포함으로써 GMP실시증명제도에 참가하게 되었다. 일본은 1974년에 정부의 GMP가 제정․공포되었다. 유럽에서는 EFTA (European Free Trade Association, 유럽자유무역연합)가 1970년 EFTA-GMP를 제정하였고, 의약품 제조의 사찰에 관한 정보의 교환 및 상호 승인을 목적으로 EFTA가맹 7개국을 포함한 14개국이 1983년 PIC (Pharmaceutical Inspection Convention, 제약사찰협회)를 체결하고 PIC-GMP를 제정하였다. 유럽공동체는 1989년 EU-GMP를 제정하였다. 우리나라의 GMP 규정은 각 국의 GMP 규정과 비교시 GAP을 가지고 있다. 이러한 GAP을 극복하고 국제수준의 GMP 운영시스템을 구축하기 위해 2008년 1월 15일 약사법시행규칙이 개정되어 새로운 GMP (NEW GMP) 시대를 맞이하게 되었다. 밸리데이션이 의무화되고 품목별 사전 GMP 제도가 도입되면서 의약품 등의 품질 수준과 대외 경쟁력 제고를 위한 GMP 규정의 국제적 조화를 추진하는 계기를 마련 하게 되었다. 궁극적으로 각 국의 GMP 규정은 ICH (International Conference on Harmonization, 국제조화회의) 규정으로 통일화될 전망이다.

PAT is a system for designing, analysing, and controlling manufacturing process through timely (i.e., during processing) measurements of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality. It is a recently-developed tool for process validation together with quality by design (QbD). In this presentation, basic components of PAT and its practice will be discussed.

Single-use disposable systems offer increased flexibility and versatility while mitigating the risk of cross-contamination during manufacturing in a multi-product facility. In this presentation, the rationales for use of disposables in biopharmaceutical manufacturing, suitable applications for single-use manufacturing, and its practice examples in KBCC will be discussed.

CGMPs와 Modern Quality Systems은 확립된 품질의 특성 (Quality), 설계에 녹아있는 품질 (Quality by Design), 위험관리 및 평가 (Risk Management and Risk Assessment), CAPA, 변경관리(Change Control), 품질부서 (Quality Unit: QC & QA), 그리고 Six-System Model (Quality System + 5 Manufacturing Systems)의 6가지 요소가 언급된다. CAPA는 다른 요소와 마찬가지로 Quality System의 효과적인 운영을 위해서 필수적인 것으로 일탈 (Failure)에 대한 관리, 변경(Change)관리절차 등을 포함하여 Quality의 확립(Establishment) 및 관리 (Management)를 효율적으로 운영하기 위하여 다른 요소들과 상호 연관되어 있다. 이는 PAT (Process Analytical Technology: 공정분석기술)가 목적하는 것과 직접적으로 연관되어 있다. PAT는 작업이 원하는 방향과 속도로 진행되고 있는 지를 관찰하고, 만약 일탈이 발생하였을 경우 이를 적절한 제어장치를 통해서 원하는 공정궤도로 복귀시킬 수 있도록 관리하는 총체적인 시스템을 의미한다. 이와 같이 QbD, PAT, CAPA 등이 상호 연관되어 Quality System을 구축하는데 기여하는데, CAPA의 적용예를 바탕으로 설명하고자 한다.

Water is the most widely used raw material in the pharmaceutical industry. The use of the water dictates the quality attributes, the design of the system, and the level of control in the system. The design, construction, and validation of water systems for the pharmaceutical industry represent key opportunities for manufacturers, engineering professionals, and equipment suppliers. This system is required to meet current Good Manufacturing Practice cGMP regulations while remaining in compliance with all other laws and regulations. The cost of bringing this system on line is highly variable, owing to interpretation of regulatory requirements and overly conservative design approaches. The purpose of this Guide is to focus on engineering issues, and provide cost effective pharmaceutical water systems.

Enzyme-linked immunosorbent assay (ELISA) using enzyme-conjugated antibody is the most popular detection method for biochemical materials and can be utilized for a wide variety of analytes. In recent years, rapid, automatic, and sensitive detection methods of ELISA have been required because of the increasing number of biological samples in clinical, environmental, and bioindustrial fields. For these purposes, several methods, including one-step ELISAs and membrane blotting immunoassays, have been proposed. Among them, an open sandwich assay, a one-step ELISA using affinity tag and an immunoliposome blotting assay will be introduced. The open sandwich method depends on the antigen-dependent re-association of antibody variable domains, VH and VL [1]. Each domain is coupled with a moiety of an interacting pair, and re-association of VH and VL domains completes the formation of the pair. For example, mutants of N- and C-terminal deleted β-galactosidase were genetically conjugated to VH and VL domains, and appearance of β-galactosidase activity was detected. In one-step ELISA using affinity tag, an antibody was conjugated with an affinity peptide tag (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene [2]. This tag-conjugated antibody and an enzyme-conjugated antibody were used in sandwich immunoassay and resulted in a substantial reduction in operational time with a higher sensitivity compared with the conventional ELISA. Immunoliposomes encapsulating color markers were used for membrane blotting immunoassay [3]. Multipoint interaction between antigen and antibody and higher signal intensity increased the sensitivity of the membrane blotting immunoassay by two orders of magnitude even at the blotted sample volume of 2 μl.

The design and fabrication of scaffolds have received much attention with regard to functional vital engineered tissues. A provisional scaffold serves as a temporal cell adhesion and proliferation substrate or platform until natural extracellular matrix (ECM), which consists of a nanoscale fibrous network of proteins and proteoglycans, is produced by inoculated cells to form matrix architecture or tissue morphology, which resembles that of a native tissue. Recently, the fabrication and design of submicronto nanoscale structural architectures, which geometrically or topologically mimic the native state of ECM, have received much attention in regenerative medical applications. Electrospinning (ELSP) is a fiber spinning technique driven by a high-voltage electrostatic field using a polymeric solution or liquid that produces polymer fibers with diameters ranging from several micrometers down to 100 nm or less. The fibers are typically deposited in the form of a non-woven fabric onto a target metallic collector through a random deposition of a projected jet of polymer solution, the so-called instable jet. To fabricate a geometrically biomimetic scaffold composed of nanoscale fibers, electrospinning has been proven to be an effective method. Furthermore, the major advantages over conventional wet spinning methods include simplicity, convenience, and inexpensiveness. In this talk, the principle and method of ELSP, and also medical applications especially for artificial vascular graft will be introduced.

The genotype identification method to measure nucleic acid using is one of the interesting tools to detect E.coli in environmental samples. The four independent processes, that is, cell lysis, nucleic acid purification/concentration, nucleic acid amplification, and detection are required to detect E.coli. If micro chip format is utilized in genotype identification method, it also requires all processes required for the detection of E.coli because the volume reduction issue is key process to increase the detection sensitivity in environmental sample that has relative large volume, considering microchip format. However, the washing issue might be emerged in nucleic acid isolation process, one of the four essential processes, when applying it to micro chip format because the most popular nucleic acid isolation process, called boom technology, utilizes chaotropic salt and ethanol as known as PCR inhibitors, which requires long washing step to remove chaotropic salt and high power usage such as centrifugal force to dry ethanol. In this study, new method to isolate and concentrate nucleic acid is introduced for micro chip format to detect E.coli using microbead. Proposed method was optimized using RT-PCR and was well-demonstrated by measurement of ultra low concentration of E.coli without special reagent. Acknowledgments : This research was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-D00099) and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government(MOST) (No.2007-04373)

Enzymatic steps from two different biosynthetic pathways were combined in E. coli, directing the synthesis of a new class of biomolecules – ubiquinones with desaturated prenyl side chains. This was achieved by the activity of a C30 carotenoid desaturase CrtN from S. aureus, which exhibited an inherent flexibility in substrate recognition when compared to other carotenoid desaturases. By utilizing the known plasticity of the E. coli ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with desaturated prenyl side chains were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and NMR analysis. In vivo 14C- labeling and in vitro activity studies showed that CrtN desaturates octaprenyl dipohosphates but not the ubiquinone compounds directly. Antioxidant properties of desaturated side-chain ubiquinones were analyzed in an in vitro β-carotene-linoleate model system and found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks classes of compounds not observed in nature can be synthesized, and structural motifs that are functionally important can be combined or enhanced

Nanotechnology is rapidly growing as a cutting-edge technology that will fundamentally change our future life. Given the physical limitations of conventional lithography technology, bottom-up approaches motivated by biological principles are burgeoning as a major trend, i.e. bio-inspired nanotechnology.1-4 Biomolecules, although they have evolved to fulfill natural needs, are recently reengineered to perform specific nanoscale tasks that nature has not tried yet. DNA is a versatile construction material that can be programmed to self-assemble into nanoscale structures based on specific complementarity between bases.5-6 DNA-programmed protein nanostructures responsive to external signals are promising for biomedical applications including biosensors and drug delivery materials. We designed and constructed stimuli-responsive nanostructured biomaterials consisting of proteins and DNA, which lead to smart materials of which self-assembly is controlled in response to the presence of target signals such as DNA oligonucleotides and biochemicals. We investigated two such biomaterials of which structural changes are controlled by elaborate sequence design of DNA crosslinkers: 1) SNP sensor materials of which dissociation rate constants are dramatically changed by single nucleotide mutations in target DNA sequences and 2) smart materials switchable by adenosine for programmable drug release. These examples illustrate how DNA programmability can be harnessed to construct smart biomaterials displaying new functionalities and structural designability for biomedical applications.

For the purpose of developing a high-level expression system for production of a human kringle fragment (LK8), secretion pathway engineering was applied by modifying a number of cellular regulations involved in the endoplasmic reticulum homeostasis in a recombinant Saccharomyces cerevisiae strain. Effects of unfolded protein response (UPR) on LK8 production was investigated to see if the coexpression of the HAC1 gene which encoded a UPR transcription activator enhanced the total expression of LK8. Using the Tn7 transposon yeast library, the ubc6Δ strain was selected as an over-secreting mutant. Thus, partial blocking of polyubiquitination was assumed to accelerate the LK8 secretion since the protein encoded by the UBC6 gene played a role of an ubiquitin conjugation enzyme. On the other hand, in an attempt to develop cost-effective process of LK8 production, both the GAL1 and HXK2 were disrupted and a pattern for LK8 expression was compared in continuous cultures. As a result, the secretion pathway in S. cerevisiae was modified for the enhanced production of LK8. Furthermore, an economic process for LK8 production was developed through strain improvement and fermentation technology.

Living organisms use simple or complex types of diverse biomolecule interactions (i.e. protein-protein, proteincompound, protein-carbohydrate, and carbohydrate-carbohydrate). Such interactions initiate biologically meaningful events and consequently, cell or organism can maintain its metabolism. Carbohydrate-protein interaction plays critical roles in living organisms; cell-cell communication, signaling, cell adhesion, fertilization, and immunological process. These interactions also initiate infection of host cells by bacterial toxin protein and viruses. Therefore, understanding of the molecular relation for carbohydrate-protein interaction not only provides useful information on biological processes in living organisms but also is helpful for development of potent biomedical agents. Interaction between ganglioside GM1 and Vibrio cholera toxin proteins has been generally regarded as a good model for carbohydrateprotein interaction. Pentameric B subunits of Vibrio cholera toxin are well known to interact individually with pentasaccharide of ganglioside GM1. Monomeric A subunit interacts with the membrane surface through pentameric B subunit and is known as enhancing avidity of whole interaction. In the present work, we employed three nanobio-analytical techniques, surface plasmon resonance (SPR), electrochemical detection, and atomic force microscopy (AFM), to detect and analyze more accurate interactions among three biomolecule components, ctxA, ctxB, and GM1 pentasaccharide. To analyze interaction, biomolecules should be immobilized on solid surface. Since ganglioside GM1 that includes ceramide portion having highly hydrophobic carbon chain was selected as a target biomolecule, it was difficult to immobilize this molecule covalently onto solid surface. Therefore, we designed novel carbohydrate modification method to introduce thiol-group to reducing sugar and successfully modified mono- and pentasaccharide using this method. The proposed modification method has several advantages over previously reported methods, including direct and rapid one-step immobilization onto a gold surface without surface pretreatment(s) by thiol group coupling in mild reaction environment, and exposure of functional carbohydrate moieties through oriented immobilization of the terminal reducing sugar. We found that kinetic result of equilibrium constant (2.71 x 10-10 M) for ctxB-GM1 from SPR analysis was in good agreement with other reports even though we used novel immobilization method for GM1 pentasaccharide on gold surface which is more facile method. This result indicated that ctxB-GM1 has the highest affinity among the carbohydrate-lectin interaction. From the comparison analyses to reveal the role of ctxA, ctxAB-GM1 is expected to have high interaction compared with other receptor-ligand interactions. However, in the relative interaction analyses including SPR and electrochemical method, ctxAB-GM1 showed lower degree of binding than due to steric hindrance from different protein size than can affect the exact interaction values. However, cholera toxin ctxA increased the binding affinity through the complexation with ctxB from the unbinding force measurement using AFM. From structural analyses using the GM1 analogues, we conclude that sialic acid and terminal galactose residues are mostly important in cholera toxin-GM1 pentasacchariede interaction and reorganizing in the binding sites occurred in the absence of its complementary fragments. Finally, interaction strength of several carbohydrates with complex cholera toxin showed following order: GM1 >> LST-b > LST-a > asialo GM1 > GM3. Collectively, we obtained interaction information about kinetics and affinity from SPR, highly sensitive specificity and affinity from electrochemical detection, and direct interaction unbinding force from AFM. These analytical systems have individual advantages and disadvantages for interaction analysis. We noted that specificity can not simply decide affinity because other binding factors can affect binding affinity. In addition, it is hard to say that high affinity interaction decides high specificity. Therefore, the data from all three analytical methods should be integrated and collectively analyzed to obtain more accurate information about the interaction.

A pPT-BRpi expression vector for the production of human insulin was constructed and transformed into E. coli JM109. E. coli JM109/pPT-BRpi has the P2 promoter, lac operator, and ColE1 origin of replication. E. coli JM109/pPT-BRpi was cultured by a three-step temperature shift fermentation method. The induction of human preproinsulin expression was made by a temperature shift from 30℃ to 37℃. The dry cell weight and preproinsulin content in the three-step temperature shift fermentation with E. coli JM109/pPT-BRpi were higher than those of the constant temperature fermentation. The three-step temperature shift fermentation gave the final dry cell weight of 45.5 g/L and expression content of 69.0%. Preproinsulin was expressed as inclusion body in recombinant E. coli. The inclusion bodies were solubilized with β-mercaptoethanol and refolded to restore biological activity. The folded preproinsulin was transformed into human insulin by the enzyme reactions with trypsin and carboxypeptidase B. The most cumbersome problem with conventional downstream processes was the formation of many human insulin derivatives at the enzymatic modification step. Desthreonine (B30) insulin and insulin fragments were synthesized during the enzyme reaction. Des-threonine (B30) human insulin which is human insulin without B-30 threonine was formed when the C-terminal of B29-lysine of preproinsulin was cleaved by trypsin. The insulin fragments were formed by β-mercaptoethanol during the enzyme reaction as well. To minimize the formation of human insulin fragments by β-mercaptoethanol, hydrogen peroxide was used during the enzyme reaction with trypsin and carboxypeptidase B. To block the formation of desthreonine human insulin, citraconic anhydride was used during the enzyme reaction. Combination of H2O2 addition and citraconylation improved remarkably the insulin production yield to 50%. The overall yield of the purified human insulin from the enzyme reaction with citraconylated preproinsulin to crystallization was about 67%. In a 200 L fed-batch fermentation, 0.485 g human insulin could be produced per 1 L fermentation broth at cell concentration of 45.5 g dry cell weight/L. The purity of purified human insulin was above 98.5%.

p-Aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthease starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. Homologous AurF proteins are expected to be also involved in the biosynthesis of other aromatic nitro compounds such as spectinabilin, alloaureothin, griseulin and SNF4435C/D. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy1,2. Here we report the in vitro reconstitution of the enzymatic activity by both biological and chemical methods, the crystal structure of AurF in oxidized state, and the co-crystal structure of AurF with product pNBA. Our combined biochemical and structural analysis unequivocally indicate that AurF is a nonheme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitrosoamine intermediates.

Bacteria of the genus Leuconostoc represent a diversified group of heterofermentative organisms commonly found in the fermented foods. Among lactic acid bacteria (LAB), Leuconostoc plays important role for the fermentation of plant materials including kimchi and sauerkraut producing organic acids and carbon dioxide. For the molecular level study and genetic modulation of Leuconostoc, development of effective gene transformation systems is of great necessity. For construction of plasmid shuttle vectors auto-replicating in Leuconostoc, plasmid-harboring Leuconostoc strains were screened from kimchi. pCB18 had one open reading frame (ORF) potentially encoding replicase protein and pCB42 revealed two ORFs with transposase and DNA-binding protein. Using pCB18 a Leuconostoc-E. coli shuttle vector (pLeuCM) was constructed with pUC19 and the staphylococcal chloramphenicol acetyltransferase (CAT) gene. Also, pLeuCM42 was constructed using pCB42. The autoreplicative Leuconostoc-E. coli shuttle showed a segregational stability in Leuconostoc over 100 generation of cell division under nonselective condition. Gene of β-galactosidase was obtained by PCR amplification from Lb. plantarum. It was subcloned into pLeuCM and pLeuCM42 to construct pLeuCMgal and pLeuCM42gal, and introduced into E. coli and Ln. citreum 95. Successful expression of β-gal in hosts were confirmed by enzyme activity assays. However, the transformant containing pLeuCMgal lost the plasmids after 50 generations of growth under non-selective medium revealing low segregational stability. The insertion of foreign DNAs in pLeuCM was supposed to result in disturbing plasmid replication and accumulation of ssDNA intermediate. Meanwhile, pLeuCM42 and pLeuCM42gal showed a high segregational stability in Leuconostoc over 100 generation. The high stability of pLeuCM42 is an advantage when a food-grade vector is to be constructed from pLeuCM42 since no antibiotic marker genes are allowed for food-grade vectors. In this respect, pLeuCM42 might be useful for construction of a food-grade vector. The systems developed and applied in this study would be useful tools for the genetic manipulation of Leuconostoc and they will facilitate the genomic study of the strains.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) fusion protein was produced from transgenic rice cell suspension cultures using RAmy3D promoter as a novel immunosuppressive agent. Since RAmy3D promoter was strongly induced by sugar starvation, both cell viability and protein productivity were reduced remarkably after induction. To overcome these obstacles, various strategies were contemplated to develop the production processes of hCTLA4Ig. The expression of proteases and repressors was reduced by siRNA. To apply siRNA technology into transgenic rice cell cultures, three steps should be optimized, to put it briefly, 3D steps: 1) Design of siRNA sequences, 2) Delivery of siRNA into rice cells, and 3) Detection of the inhibited level of target proteins. siRNA sequences were designed by web-based programs like siRNA Target Finder, siDESIGN Center, and BLOCK-iT RNAi Designer. siRNA was combined with polyethyleneimine (PEI) to form siRNA complexes for the delivery into plant cells. Moreover, sonoporation was practically treated to enhance the transport of siRNA complexes.