Earticle

1980년대 유전공학 기술의 발달과 함께 쓰이기 시작한 용어인 생물의약품 (Biopharmaceuticals)은 유전공학적 방법이나 하이브리도마 기술을 이용하여 생산된 치료용 단백질을 일컬었으나 최근에는 유전자 요법이나 안티센스 테크놀로지에 사용되는 핵산, 세포를 이용한 치료제에도 생물의약품의 명칭이 붙게 되었다. 1980년대 초반 미국 FDA 승인을 획득한 재조합 인간 인술린 이후 수많은 제품들이 허가되었고 시장은 급속히 확대되어 2010년 이후에는 1000억 달러를 상회할 것으로 예상되고 있다. 또한, Biogeneric (Biosimilar) 제품 시장의 확대 및 경쟁 증가와 항체의약품 시장의 급격한 증가 그리고 유전자치료제/세포치료제 와 같은 신개념 제품들에 의한 단백질의약품 시장의 대체가 예상되고 있다. 국내에서는 LG생명과학이 1990년 국내최초로 인터페론 감마를 허가 받고 출시한 이후, 동아제약, 녹십자 등 대형 제약 기업 주도의 생물의약품과 수입 생물의약품이 시장을 장악하고 있으며, 정부주도의 위탁생산 시설 2곳과 기업주도의 위탁생산 전문 시설이 1곳 가동 중이며, 일부 중소제약기업 및 바이오벤처에서 생물의약품 제조관련 기술개발 및 제품화에 노력하고 있는 상황이나 세계 시장에 진입하기에는 역량이 턱없이 부족한 실정이다. 본 심포지움에서는 국내 생물의약 산업 현황 리뷰와 생물의약 관련기술 및 제품을 개발하고 있는 국내 비상장 바이오벤처들의 제품개발 노력 및 기업현황 발표를 통해 국내 생물의약 산업의 현황과 향후 모색해야 할 방향에 대해 음미해 보는 시간을 갖고자 한다.

2000년 광주과학기술원내 실험실 벤처로 시작된 애니젠(주)는 2004년 8월 광주테크노파크로 본사를 확장 이전하여, 신약개발 및 의약품용 펩타이드 원료생산 분야에 주력하고 있다. 최근 국내 바이오 관련 회사들의 수요에 부응하여 전임상 및 화장품 원료 펩타이드의 대량생산을 성공적으로 진행하였으며, 일본 Takeda 등 해외 영업에서도 높은 성장율을 기록하고 있다. 높은 품질과 기술력을 바탕으로 향 후 cGMP 시설 및 대량생산 시스템을 구축하여 펩타이드 의약품 원료시장을 주도하는 굴지의 바이오 회사로 성장하고자 한다. 또한 안정된 cash flow를 바탕으로 생체응답기작을 이용한 신약 후보물질을 창출함으로서 성공적 바이오벤처의 모델이되고자 한다. 애니젠(주)는 광주/전남권 최초의 바이오 벤처기업으로서 창사 이래 바이오 신약 개발 및 핵심소재의 개발에 부단의 노력을 경주해 오고 있다. 특히 펩타이드 공학, 단백질 공학, 의약화학 및 나노융합 기술을 연구개발의 핵심기술로 사용하여 펩타 이드 진통제, 항암제, 뇌졸중 치료제 및 MRI 조영제의 개발에 연구역량을 집중하고 있으며 현재 수종의 의약후보 물질을 도출하여 이들에 대한 전임상 시험을 진행 중에 있다. 애니젠(주)는 목표지향적 고부가가치 R&D를 통한 기업의 기술적 향상 및 경쟁력 강화를 목표로 하고 있으며 풍부한 기술 인프라와 인적 네트워크를 이용하여 적극적인 산학, 산연, 산산 공동연구를 수행하고 있으며 향후 이들 협동연구를 통해 도출된 핵심원천기술을 산업화로 가시화하여 비교우위적 기술경쟁력을 확보하고, 안정적 수익 구조를 갖춘 미래지향적 바이오기업으로 성장하고자 한다.

We studied charge engineered ubiquitin as a fusion partner in recombinant E.coli system. The ubiquitin sequence led fusion protein containing target protein to be expressed in a soluble form and/or over expression. The fusion protein is clearly cut at the junction between ubiquitin and target protein by ubiquitin specific protease, so that we could obtain target proteins with an exact N-terminal sequence. For the purpose of efficient purification, we changed the pI of fusion protein by adding polylysine or polyarginine to N-terminal end of modified ubiquitin in which three surface glutamic acids were substituted for arginine or lysine. The fusion proteins expressed in such a recombinant E. coli system were conveniently purified by using ion-exchange chromatographies. Especially in some cases of using on column cleavage system, the production yields of finally purified objective proteins were confirmed to be over 1.0 gram per 1.0 Liter fermentation broth.

Since 2001, ISU ABXIS is a biotech company specialized in developing and manufacturing biological therapeutics, esp., monoclonal antibodies. ISU ABXIS’ strategy is to position itself in the fast growing market of therapeutic antibodies, designed to address the unmet needs of currently marketed therapeutics realizing our fundemental goal of contributing to the development of more effective medicines and improvement of the quality of the life. The ISU ABXIS pipeline is well-focused in biological therapeutics. Based on world-class R&D expertise and GMP facility designed exclusively for mammalian cell production, the 1st biosimilar of antibody therapeutics (Clotinab® vs. ReoPro®) has been successfully launched in worldwide markets. Novel therapeutics has also been developed with best-in-class quality in the areas of cancer and inflammatory diseases. ISU ABXIS has KOL-networked, strong marketing power in domestic markets and manufacturing structure capable of sustaining a worldwide supply. The success record of marketing Clotinab® all around the world underlies in the companies’ capabilities in successful commercialization of R&D products with the best quality.

보툴리눔 톡신 타입 에이 (Botulinum Toxin Type A) 단백질은 보톡스라는 상품명으로 더 잘 알려진 주름 개선제이다. 이 단백질은 Clostridium botulinum이라는 균으로부터 정제되어 동결건조 과정을 거쳐 제품화 되며 원래는 사시나 안검경련과 같은 질병의 치료제로 개발되었으나, 2000년 초기부터 주름살 개선제로 사용되기 시작하였고 2003년에 미국 FDA에서 주름살 개선제로 임상허가를 획득하여 최근에는 미용목적으로 더 많이 사용되고 있는 제품이다. 휴젤㈜에서는 독소 단백질 정제과정과 동결건조 과정을 개선하여 기존 제품보다 Specific Activity가 약 5배에서 8배 높은 보툴리눔 타입 에이 단백질을 개발하였다. 이는 보툴리눔 톡신 단백질을 반복 주사하였을 경우에 생길 수 있는 항체반응의 가능성을 획기적으로 줄일 수 있는 특징을 가지고 있다고 사료된다. 휴젤㈜에서는 현재 GLP기관에서 독성시험을 마치고 임상용 제조시설을 구축 중에 있다.

인간 단백질을 식물에서 생산한다. 꿈같은 이야기 같지만 어느새 우리 앞에 현실로 다가와 있다. 1980년대 미국의 제넨테크사에서 인간성장호르몬을 유전자재조합으로 세균에서 생산하여 US FDA의 판매 승 인 획득 이후 30여년 만에 식물을 단백질 생산의 도구로 활용하는 산업의 패러다임을 바꾸는 새로운 생산수단이 대두되었다. 식물유전공학은 짧은 역사에도 불구하고 눈부신 발전을 하여 유전자조작에 의 한 형질전환 식물체 개발은 내병성, 내충성, 제초제내성 유전자 등의 도입에 의한 작물 생산성 증가 등의 농업적 응용에 집중되어 식물유전공학 1세대를 형성하였다. 최근에 들어서 식물을 다양한 산업 소재로서의 기능과 고등생물로서 지니는 장점 등을 이용하여 고부가가치의 유용한 재조합단백질의 생산을 위한 생산시스템으로의 개발에 관심이 집중되어 있다. 즉, 식물을 도입된 유전자로부터 유래하는 새로운 물질이나 기능을 이용하고자 하며, 이는 곧 2세대 식물유전공학의 기본개념이기도 하다. 식물은 의료용 및 산업용 단백질의 large-scale 생산 시스템으로 특히 매력적인 것은 기존의 확립되어 있는 전통적인 농업기반을 이용하여 쉽게 수확, 가공할 수 있으므로 원하는 biomass의 대량 확보가 가능하기 때문이다. 이러한 식물의 장점을 이용할 목적으로 식물에 유용 단백질 생산에 관여하는 유전자를 도입시켜 확보한 형질전환 식물체를 대상 단백질의 생산시스템으로 활용하는 방법을 Plant Molecular Farming(식물 분자농업)이라고 한다. 이 기술은 의학적으로 중요하고 산업적으로 유용한 단백질들을 식물체에서 생산하는 것으로 재조합 의료용 단백질들의 대량생산을 위한 저렴하고 안전한 방법을 제공하는 것 이다. 또한 형질전환된 식물에서 발현된 복잡한 동물유래의 단백질들이 원래의 단백질과 거의 동일하고 기능 면에서도 차이가 없으므로 field scale에서 의료용 단백질의 생산을 위하여 적합한 시스템으로 알려져 있다. 식물에서의 의료용 단백질 생산은 바이오의약품 (진단키트, 의약품, 백신, 치료제), 화장품소재 (미백, 주름제거), 농업소재 (동물의약품, 백신, 면역증강제), 산업용효소 (식품, 피혁) 등 광범위 한 단백질들을 대상으로 하고 있다.

Yeast is a good host for the expression of complicated mammalian proteins because yeast, like mammalian cells, has quality control system in the secretion pathway which can perform eukaryotic-specific proteolytic processing, chaperone-assisted folding and posttranslational modifications, such as disulfide-bond formation and certain types of glycosylation. Thus, yeast surface display system combined with high throughput screening techniques has been extensively employed as a platform technology for the protein and antibody engineering for affinity maturation with their ligands/antigens, higher stability and expression efficiency in yeast. However, yeast surface display system has been little explored to express cDNA library from some tissues/cells to isolate novel antigens or biomarkers. In this talk, I will present two topics related with antibody engineering and biomarker discovery using yeast surface display system. First, taking a specific example, I will introduce the construction of so called pseudo-immune antibody library to increase the probability of isolating a good binder antibody to a given antigen. Secondly, I will present a selection methodology to isolate patient sera-specific antigens from yeast surface-expressed cDNA library constructed from 15 patient lung tissues with non-small cell lung cancer. [This work was partially supported by grants from the National R&D Program for Cancer Control, Ministry of Health & Welfare (0520110-1 to YSK), Korea Research Foundation (R08-2004-000-10077-0 to YSK), and the "GRRC" Project of Gyeonggi Provincial Government (to YSK), Republic of Korea.]

Temperature is one of the key environmental parameters that affect the growth of Chinese hamster ovary (CHO) cells and the production of recombinant proteins. Generally, CHO cells are cultivated at 37°C to simulate the body environment where they are familiar. Recently, a number of studies have demonstrated that lowering culture temperature below 37°C is beneficial to specific productivity (q) in CHO cells although it decreases specific growth rate (µ). To investigate the effect of low culture temperature on cell growth and recombinant protein productions, CHO cells producing erythropoietin (EPO) and follicle-stimulating hormone (FSH) were cultivated between 28ºC to 37ºC with various culture modes such as batch, fed-batch and perfusion. Lowering the culture temperature below 37ºC suppressed cell growth but enhanced qEPO and qFSH. Also, the quality of EPO produced under different culture modes was investigated through isoform analysis. Among the culture modes tested, perfusion seems to be promising in terms of both quantity and quality of proteins.

The erythropoietin is 34-38kDa glycoprotein containing three N- and one O-glycosylation sites. It is known as a regulatory cytokine produced in adult kidney or fetal liver stimulating hematopoiesis 1). But its half-life is not so long thus it is necessary to enhance the stability of EPO in vivo. It is thought that fusion type of stable protein molecular linked EPO would increase in vivo half-life. In previous studies of other groups, an albumin was suggested as an ideal candidate for fusion protein to increase half-life and activity because it is the major protein and it has a long half-life in human blood stream2). We designed the human albumin gene fused N-terminal of EPO and inserted (GGSGG)4-repeated linker between albumin and EPO3). CHO-dhfr - was co-transfected with albumin-EPOs fusion genes and dhfr gene. Fused albumin-EPOs proteins were determined and quantified by Western blot and ELISA. Hematopoietic efficacy was estimated using EPO dependent cell line F-36E in vitro and normothaemic mice (B6D2F1) in vivo. And we also evaluated in vivo half-life of those proteins in SD-rat. As a result, albumin-EPOs showed better efficacy and longer half-life than the standard EPO.

For the production of therapeutic proteins, recombinant Chinese hamster ovary (rCHO) cells have been most widely used in industry. The popularity of rCHO cells is likely to persist as the demand for therapeutic proteins continue to increase. For any commercial process, high volumetric productivity and/or high product titer are desired for the reason of profit. This can be achieved by increasing the specific productivity and/or the time integral of viable cell concentration. Thus, cell engineering has been targeted mainly to increase either one of these factors. When cell engineering is considered, it is critical to understand the intracellular events of cells. For example, apoptotic cell death induced in mild suboptimal conditions is an active, genetically controlled process of cell suicide mediated by the activation of a series of caspases. Therefore, apoptotic cell death can be regulated to some extent by genetic modification. In contrast, necrotic cell death induced at high stress level is a passive, genetically uncontrollable death. Accordingly, overexpression of antiapoptotic gene for extending culture longevity is effective on the condition that cells die mainly by apoptosis, not necrosis. Various strategies such as the overexpression of antiapoptotic genes (mostly bcl-2), down-regulation of effector molecules (caspase-3 and caspase-7), down-regulation of CIRP, overexpression of pyruvate carboxylase, down-regulation of LDH-A, and overexpression of chaperones (ERp57, PDI, calreticulin, and calnexin) have been attempted in our laboratory and efficacy of each CHO cell engineering strategy will be discussed.

Glycerol carbonate is a key bifunctionalcompound employed as solvent, additive, monomer, and chemical intermediate. Commercial lipase, Novozym 435 was used to produce the benign monomer glycerol carbonate from renewable starting materials: glycerol and dimethyl carbonate. Tetrahydrofuran was used as the reaction medium to enhance the formation rate of glycerol carbonate and conversion approached quantitative yield. Removal of methanol produced as by-product duringreaction increased the yield of glycerol carbonate. Other by-products such as glycerol dicarbonate and diglycerol tricarbonate were found. However the total amount of by-products was minute compared with that of glycerol carbonate, which means that lipase-catalyzed reaction is very regioselective for the synthesis of glycerol carbonate.

Human blood is the source of a wide range of medicinal products, especially plasma derivatives, used for the prevention and treatment of diseases. However, the potential for blood-borne viral transmission is still considered to constitute a risk to patients. Viral removal/inactivation processes are introduced to the manufacturing process to assure the viral safety of human blood plasma products. This guideline pertains to the validation and assessment of the viral removal/inactivation steps. It also provides the checklist for inspection of viral clearance processes.

Since the supply of human organs does not meet demands of patients, xenotransplantation could be a good strategy to meet that urgent needs. However, there are many concerns regarding the safety aspects. KFDA published 3 guidelines in 2006 with regard to xenotransplantation1). We hope that the guidelines may help people in this field to get helpful answers.

Concerns about the pollution prevention (P2) and global warming, as well as the depletion of fossil fuel reservoir necessitate search for renewable energy sources. Mono-alkyl esters of long chain fatty acids, termed as biodiesel, are promising alternative to petroleum-based fuel. They are produced from triacylglycerides contained in plant oils, animal fats, and waste oils through transesterification with alcohols. Currently, biodiesel is commercially produced by chemical processes using acid or alkali as catalysts. However, there are several problems, mainly caused by the catalysts, such as generation of undesirable byproducts by saponification, corrosion, and contamination of products by catalysts. To avoid the negative effects and to make the process more economic, researchers have been trying to replace the chemical process with biological one using lipases as catalysts. Use of lipase is a great viable method for the production of esters from different oil sources. Research on this topic is still in progress due to the enzyme’s flexibility and adaptability to new processes. The bioprocess, however, is not yet competitive enough, mainly due to the enzyme’s vulnerability to alcohols, low productivity, and high cost. In this presentation, recent efforts to tackle these problems are reviewed and other technologies worth consideration are introduced.

A by-product in oleochemistry, glycerol is a three carbon compound produced by the saponification, hydrolysis and transesterification of lipid triacylglycerol. In biodiesel anufacturing process, the amount of glycerol accumulated is presumed by a tenth of that of biodiesel synthesized. Mass production of biodiesel in universe allowed a considerable decrease in glycerol price, of which crude type is estimated 40￠/kg at around 2010. To obtain the price competitiveness of biodiesel, the conversion of glycerol to high value-added chemical compounds should be necessary. Among glycerol derivatives such as glycerol carbonate, glycerol ester, 1,3-propanediol, the biosynthesis of 3-hydroxypropionic acid (3-HP), one of "the top-value added chemicals from biomass" chosen by U.S. Department of Energy1), is introduced in this presentation. 3-HP is a none-chiral organic acid with three carbons and can be used as a chemical intermediate to synthesize acrylic acid, acrylamide, malonic acid and 1,3-propanediol. Chemical and biotechnological approaches for 3-HP production have been proposed but commercially available processes were not developed to date2). Here some challenges and obstacles for the production of 3-HP from glycerol will be presented.

Recently there have been increasing demands for UV curing coating because public concern on volatile organic compounds (VOCs) is elevated. Since the consumption of VOCs can be significantly reduced when UV curing coating substitutes conventional coating, UV curing coating is being accepted as eco-friendly coating these days. A novel UV-curing coating material was developed by reacting a highly reactive acrylate compound with an glycerol derivatives using Candida antarctica lipase under mild reaction condition. Due to mild reaction condition, the cheap acrylic acid, methyl acrylate and methyl methacrylate can be used as acyl donors instead of expensive and highly reactive acryloy1 chloride. The resulting polymer has high transparency and hardness, which makes it possible that the product can be used as a novel coating material.

Biodiesel is the name applied to renewable fuels manufactured by the esterification of vegetable oils or animal fats which can be used a substitute for or as an additive to mineral diesel. The methyl ester produced using methanol and a basic catalyst is the predominant biodiesel available for compression ignition engines as its properties closely resemble those of No 2 mineral diesel. One of the major technical problems confronting the use of biodiesel fuels is their poor low-temperature properties in comparison with fossil-based diesel fuel. The crystallization temperature of biodiesel fuels is generally higher than that of fossil-based diesel, so the formation of crystals at relatively high temperastures can plug fuel lines and filters causing problems in fuel pumping and engine performance during winter operation. In order to solve the above mentioned problems, several additives such as pour point depressants (PPD), wax dispersant flow improver (WDFI) or wax anti-setting flow improver (WAFI) can be used. In this paper, we reviewed the low temperature properties of diesels (BD5 and BD20) containing several kinds of biodiesels and have tried to find a universal mechanism of PPDs to explain the change of the wax crystals.

Abstract - To registrate a drug, a developer should submit chemistry, manufacturing and controls(CMC) document to KFDA. This document provides guidance on the content and format of the chemistry, manufacturing, and controls(CMC) for therapeutic recombinant DNA-derived products and monoclonal antibody products for in vivo use. The submission document on CMC of biotechnological product follows generally 􋺵Provision for specifications and test procedures of drugs . This guideline is aimed to provide the distinct items of submission document on chemistry, manufacturing and controls(CMC) of biotechnological recombinant products, and the special consideration on the quality, safety and efficacy of monoclonal antibody of products. This guideline informs physicochemical characterization of reference standard, manufacture, method of manufacture, process control, reference standards, specification/analytical method, stability to anyone who intends to develop therapeutic recombinant DNA-derived products and monoclonal antibody products.

Biochip is defined as miniaturized test sites (microarrays) arranged on a solid substrate that permits many tests to be performed at the same time in order to achieve higher throughput and speed. Biochip came out through Bio-technology and Information-technology. Over the past few years, korean government has supported biochip industry as a key sector in korea economy. As a consequence, biochip technology and industry has been in bloom since 2000. A number of research groups developed various types of biochips which are applicable to pharmaceutics/medicine as well as basic science, including environmental and biological field. Noticeably, biochip has been proven to be a powerful tool in diagnosing several diseases and is now expanding its share of market rapidly. KFDA has classified biochip as in-vitro diagnostic devices since 2003. While many kinds of diagnostic chips have been reviewed, HPV genotyping DNA chip is the first device to be approved by KFDA for its commercial use in July, 2004. Since then, other similar or upgraded HPV DNA chips has been approved. As with virus diagnostic chips, cancer diagnostic chips have been under developing and we hope these products will be on market in near future. Based on our experience on evaluating Specification and Test Methods for HPV chips, we now published the new guideline which is intended as a reference for both developer and reviewer. This guideline basically propose the minimum requirement to evaluate the quality of HPV chips and several other documents may be required by reviewer when needed. While US FDA prepares the draft guideline for In-vitro multi-variate index assay, this guideline is the first one specific for HPV. KFDA hope that this guideline is a help for the companies, academic, research institute, and pharmaceutical company which are involved in In-Vitro diagnostic devices. Since this guidance represents the current thinking of KFDA, we are open to comment or advice from related areas.

우리나라는 2016년까지 세계 생물의약품 시장의 10%에 해당하는 약 200억불의 생물의약품수출을 달성함으로써 세계 5위의 생물의약품 수출국이 되는 동시에 바이오산업 국가 경쟁력 7위를 달성하는 것을 국가적 비전으로 삼고 있다. 그러나, 선진국을 포함한 많은 국가들이 의약품의 수입허가 시 CGMP 또는 이에 준하는 규정(EU-GMP 등)에 따른 제조 및 관리를 필수적으로 요구하고 있어 개발된 의약품의 해외시장 진출을 위해서는 CGMP 규정을 만족시키는 기술 및 체제를 시급히 갖추는 것이 필수적이다. 본 강연에서는, 이와 같은 의약품 관련 CGMP 기반 마련의 시급성에 부응하여 한국생물공 학회가 산업자원부가 지원하는 표준기술력향상사업의 일환으로서 수행하고 있는 ‘생물의약의 생산시설(CGMP)표준화기반 구축‘ 과제에 대한 소개를 할 예정이다.

Enzymes are necessary for maintaining all living systems and also can catalyze a number of chemical reactions of possible biotechnological importance. Some major research and technology areas are needed to develop the biotechnology-based production of valuable products. These areas include, for example, techniques for the screening, expression, evolution, production, immobilization and application of enzymes. Among a variety of enzymes as biocatalysts, lipases, which hydrolyze and synthesize the ester bond of a large number of natural and synthetic ester substrates, are ubiquitous enzymes found in animals, plants, and microorganisms. They can catalyze the hydrolysis of triglycerides to glycerol and fatty acids at the lipid-water interface and the synthetic applications in non-aqueous medium such as transesterification of triglycerides and resolution of racemic mixture. We will provide discussions concerning above-mentioned major research and technology areas to develop the in-house lipase biocatalysts for successful industrial applications; isolation of novel lipases, directed evolution of lipase-secreting apparatus, mass-production of extracellular lipase, immobilization of lipase to synthetic polymeric carriers and lipase-catalyzed reactions.

Biosilicification is an evolutionary old and widespread type of biomineralization both in unicellular and multicellular organisms including sponges, diatoms, radiolaria, choanoflagellates, and higher plants. In the last few years combined efforts in molecular biology, cell biology, and inorganic and analytical chemistry allowed first insights in the molecular mechanisms by which these organisms form an astonishing structural variety of siliceous structures not reached by chemical methods. The skeletal elements of two classes of sponges, Demospongiae and Hexactinellida, the siliceous spicules, consist of glassy amorphous silica (biosilica). The demosponges exhibit the unique ability to synthesize biosilica using a novel group of enzymes which have been termed silicateins. Silicateins have been isolated, cloned and sequenced both from marine sponges and freshwater sponges. Sponges also possess a silicase, which mediates the dissolution of amorphous silica. Diatoms, which belong to the Protista, have used an independent strategy to utilize silica for the construction of their skeletal elements. The recombinant silica enzymes (protected by patents worldwide [W.E.G. Müller and H.C. Schröder]) are of high interest and potential importance for a variety of medical and technical applications, e.g. surface modification of glasses and other materials including biomaterials, the preparation of resins, insulators, mesoporous molecular sieves and catalysts. First, strategies have been designed towards the application of these enzymes for surface modification (coating) of biomaterials. The advantage of these bio-based approaches compared to conventional technical procedures is the fact that the enzymatic silicatein reaction occurs under mild physiological conditions, whereas physical–chemical methods require high temperatures or pressures, and the use of caustic chemicals which possibly damage the organic (bio)materials used. Such biomaterials may include collagen used in tissue engineering and in bone replacement materials. The use of biocatalytically (silicatein) formed silica may also be a suitable approach for coating metal implants to increase biocompatibility or to attach bioactive substances to the relatively inert metal surface. The same strategy could be employed for the encapsulation of drugs, hormones and other bioactive molecules and the controlled release of these compounds. In addition, the recombinant silica enzymes may also be applied for the synthesis of the nanostructures of amorphous silica. In industry, micro- and mesoporous silicas are used as reinforcing fillers in plastics, paints, sealants and rubber materials, as adsorbents and catalyst supports, as desiccant agent and as filters in separation technologies. Such applications require silicas with specific mechanical strength, pore volume, surface area and pore-size distribution. Finally, the application of techniques in lithography based on enzyme (silicatein)-mediated biosilicification may represent an innovative approach in the field of fabrication of microelectronics. Besides the use of the recombinant enzymes, biomimetic approaches have been undertaken to exploit the biosynthetic potential of the natural iosilicification mechanisms, using either naturally occurring polyamines and their analogues (spermine, spermidine and putrescine homologues, mimicking the biomineralization process in diatoms) or bifunctional molecules acting as catalysts for silica formation at neutral pH. In addition, block copolypeptides have been used to mimic catalytic activity of silicatein.

Marine biofilm is composed of complex communities of microorganisms that interact with each other and their environment. The marine biofilms causeindustrial and economical problems. However, the developments of bacterial community in biofilm have not been studied in detail so far. The goals of this study were to assess the diversity and succession of bacterial communities involved in marine biofilm on three surfaces (acryl, glass, and steel substratum). The test surface differed in physicochemical properties was submerged in seawater for 36 hours with the sampling interval of 3 or 12 hours. We investigated the structure of bacterial communities of marine biofilms by terminal restriction fragment length polymorphism (T-RFLP) and the nucleotide sequences of 16S rRNA genes. The succession from the initial to late communities was observed at 12 hours on three surfaces by T-RFLP analysis. The diversity index appeared that the value of late stage was higher than that of initial stage in the acryl surface. The analysis of the nucleotide sequences of 16S rRNA genes identified from the biofilm showed that γ-proteobacteria were predominant in the community of initial stages, and the ratio of α-proteobacteria were increased as the biofilm develops into late stages. The major clones of 16S rRNA library in the biofilm on the acryl surface were affiliated with Pseudomonas sp. and Acinetobacter sp. of γ-proteobacteria and appeared to be involved in the initial stage within the 12 hours, although the distinct structure of biofilm on the steel surface was not observed. In conclusion, γ-proteobacteria and α-proteobacteria play an important role in development and succession of biofilm on artificial surfaces. [Supported by grants from MarineBio21]

Bacterial adhesion is an initial stage of biofilm formation. As biofilm has strong resistance to the conventional disinfectants and antimicrobials, control of bacterial adhesion is important to prevent biofilm formation. Bacteria have various surface groups such as flagella, flimbriae, lipopolysaccharides and so on. When bacteria adhere on surfaces, these surface groups bind specifically. Three major forces, van der Waals force, electrostatic force, and acid base interaction, are known to be related to adhered bacteria on solid surfaces. Bacterial adhesion can be controlled by changing hydrophobicity, surface free energy, and roughness of solid surfaces. Applying electrical energy on the surface can be one of the most effective ways since it is not only applicable extensively to any conductive surfaces but also environmental-friendly. This talk will cover the overview of bacterial adhesion or detachment studies by diverse electrical methods. This technology has been studied for antifouling of marine underwater structures ( e.g. ship hulls) as well as medical implants. In our laboratory, we setup real-time bacterial adhesion control and observation system using transparent ITO (Indium Tin Oxide) electrode and developed the important concept for controlling bacterial adhesion. These results will be introduced with bacterial behavior movies. we demontrated that the suitable combination of negative and positive current promotes bacterial detachment and inactivation on conductive surfaces.

KBCC(Korea Biotechnology Commercialization Center, located in Songdo Technopark, Incheon city), a part of KITECH(Korea Institute of Industrial Technology), is a government funded initiative set to become an Asia’s leading biopharmaceutical CMO(contract manufacturing organization), capable of manufacturing a wide variety of biopharmaceuticals for clinical and early stage of commercial products. KBCC was established to meet the recent rising demand for cGMP multi-purpose and multi-product biopharmaceutical contract manufacturing capacity in Korea and other areas. KBCC will be a state of the art manufacturing facility that complies with the latest US and EU-GMP regulation and guideline and due to the opening in 2008. The basic engineering design was performed by US engineering company in accordance with the US and EU GMP regulation and guideline, and based on the type C meeting with USA-FDA. The process concepts are campaigned production in one suite, concurrent productions in plant, flexible design (minimal fixed piping & movable equipment), unidirectional personnel/material flows, and two purification rooms (pre and post viral removal room). KBCC quality system and qualification program for cGMP facilities and equipment, supported by US cGMP consulting company, has been designed to meet U. S. Code of Federal Regulations, Parts 210, 211, 820 as well as all other sections applicable, European Union Directives 2003/94/EC and 93/42/EEC, Health Canada, Health Protection Branch (HPB) Pharmaceutical and Medical Device regulations, and ISO Standard 13485:2003.

Naturally existing functional modules are considered as building blocks for complex structures, and novel functions result from the assembly of different combinations of these building blocks. The identification and characterization of these functional building blocks is relevant to the elucidation of questions regarding protein evolution, folding, and protein engineering for tailored functions. In this study two pyridoxal-5’-phosphate enzymes, Damino acid aminotransferase (D-AAT) and β-tyrosinase (TPL), were characterized about the functional modality after engineered for the higher activity and stability. In D-AAT, a recruitment of the catalytic loop structure (LRcD) from a highly active homolog distinctively changed a weakly active Geobacilli D-AAT: a 68% increase in catalytic activity. Homology modeling suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus enzyme had a π/π-interaction that was replaceable with the salt-bridge interaction between the arginine and aspartate residues in the LRcD sequence. TPL catalyzes α,β-elimination and β-replacement of L-tyrosine and its related amino acids, with pyridoxal 5'-phosphate (PLP) as the cofactor. Random mutagenesis of the TPL gene resulted in the generation of mutations on the N-terminal arms, which exhibited a higher stability and activity towards L-DOPA as the substrate. The N-terminal arm of the TPL structure was intertwined and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. When considering the common structural features of α-family PLP enzymes, the N-terminal arm may have increased the rigidity of the cofactor binding architecture of C. freundii TPL through adjusting the quaternary interfaces. Therefore, an enzyme with improved N-terminal arm could be used for the development of a new bioconversion strategy for the efficient production of L-DOPA at high temperatures, where it can catalyze the reaction more actively. Ideally, the ultimate goal of protein engineering is to create an enzyme for any given chemical reaction. However, our current knowledge of proteins is far from ideal for de novo design of protein function. To overcome this obstacle, two alternate solutions have been extensively pursued: directed evolution based on high throughput screening of mutant library and rational design based on molecular modeling of protein structure. Although evolutionary technologies have accomplished fabulous contribution in biocatalyst developments, it has been compromised by the limited coverage of genetic diversity. Thus, finding the modularity in proteins and limiting the genetic diversity on a particular module could reliably help to combine these two strategies to be most successful and provide further guidance for the improvement of biocatalyst function.

The role of periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC) and thiol peroxidase (Tpx), of the Shiga Toxin-producing Escherichia coli O157:H7 (STEC) in biofilm formation was investigated. Proteomic analyses have shown that expression of both periplasmic antioxidant systems (SodC and Tpx) were significantly increased in STEC cells grown under biofilm conditions when compared to planktonic cells. Analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene is highly induced only during the exponential growth phase. The aerobic growth of STEC sodC and tpx mutants were reduced more than that of their parental strain by exogenous hydrogen peroxide. The two mutants also displayed significant reductions in attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth although the growth rates of both wild type and mutants are similar during aerobic growth conditions. The STEC biofilm formation was only observed with wild type STEC cells in glass capillary tubes under continuous flow-culture system conditions compared to the STEC sodC and tpx mutants. The data presented here suggest that biofilms are physiologically heterogeneous and that oxidative stress defenses from both exponential and stationary growth stages play important roles in STEC biofilm formation. Further analysis of the role of DsbA (disulfide bond forming periplasmic enzyme) in E. coli O157:H7 on attachment, biofilm formation and virulence will be discussed.

The purpose of any analytical measurement is to get consistent, reliable and accurate data. There is no doubt that incorrect measurement results can lead to tremendous costs. If pharmaceuticals with incorrectly measured specfications (eg. inadequate assay, efficacy) are marketed, it may have to recalled. If pharmaceuticals with undetected impurities are distributed, they can have negative impact on people"s health. In addition, reporting incorrect analytical results can leads to loss of a laboratory"s confidence in the validity of future results. Therefore, any laboratory should report the results using accurate and reliable data with art state technology. GMP system including validation and qualification in analytical laboratory will help to achieve this goal. In GMP(Good manufacturing practice), GCP(Good Clinical practice), GLP(Good Laboratory Practice), validation of analytical method, equipment qualifiacition, adequate documentation system and traceability should be required.

t is a common belief that some residues of a protein are more important than others. In some cases, point mutations of some residues make butterfly effect on the protein structure and function, but in other cases they do not. In addition, the residues important for the protein function tend to be not only conserved but also coevolved with other interacting residues in a protein. Motivated by these observations, we propose that there is a network composed of the residues, the residue-residue interaction network (RRIN), where nodes are residues and links are set when the interaction strengths between residues are sufficiently large. We build the RRIN for the 40 diverse protein families. The interaction strengths are calculated by using McBASC algorithm. After constructing the RRIN, we identify residues that have high degree of connectivity (hub nodes), and residues that play a central role in network flow of information (information centrality nodes). We show that these residues are likely to be functionally important residues. Unlike other similar methods, our method is solely based on sequences. Therefore, the method can be applied to the function annotation of a wider range of proteins.

Inhabitation on a boundary surface brings various advantages for organisms; such organisms therefore have developed a variety of molecular systems to hold themselves on boundary surfaces during their evolution. Marine sessile organisms are one of these that possess underwater attachment capability as an indispensable physiological function that allows them to live on a liquid-solid boundary during most of their life cycle. Recent advances in underwater holdfast studies on the mussel1] and barnacle,2] which represent two typical organisms demonstrating this kind of activity, have indicated that the biological adhesion is, in general, mediated by an insoluble multi-protein complex. Underwater attachment is a multi-functional process, including displacement of the bound-water layer on a foreign substratum by the adhesive, as well as spreading, coupling of the adhesive with a variety of material surfaces, self-assembly of the adhesive, curing to make the holdfast stiff and tough, and protection from microbial degradation. It remains unachievable technology and is considered to be based on a completely different approach from that used in developing man-made adhesives in air. The barnacle, a unique sessile crustacean, has long been noted for its underwater adhesion capability. The underwater adhesive material that is used, called cement, joins two different materials, the animal’s own calcareous base and the foreign substratum, together in water as a molecular event. The holdfast system of the barnacle has no similarity to that of mussel; no sequence similarity has been found among the protein components between the two systems. The mussel holdfast system depends on several protein modifications, including typical 3,4-dihydroxy phenylalanine (DOPA); however, no involvement of DOPA in the barnacle cement has been found. This presentation summarizes the characteristics of barnacle cement, and then compares the molecular systems between the barnacle and mussel to bring the natural concept into sharp relief. The design of self-assembling peptide material inspired by a cement protein, and the application of cement proteins as an adsorbent are also reported.