Recent Progress in Biomolecular Engineering

Ryu, Dewey D. Y.,Nam, Doo-Hyun 영남대학교 약품개발연구소 2000 영남대학교 약품개발연구소 연구업적집 Vol.10 No.-

During the next decade or so, there will be significant and impressive advances in biomolecular engineering especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology. DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques. DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific desclplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins. Including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Heroeptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.

Biomolecular engineering : a new frontier in biotechnology 생명공학의 새로운 개척분야

Ryu, Dewey D.Y.,Nam, Doo-Hyun 영남대학교 약품개발연구소 2001 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-

The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at an ever-accelerating rate will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. The biomolecular engineering will no doubt become one of the most important scientific disciplines in that it will enable us to comprehensively analyze gene expression pattems in both normal and diseased cells and to discover many new biologically active molecules rationally and systematically. As an applied molecular evolution technology. DNA shuffling will play play a key role in biomolecular engineering. In contrast to the point mutation techniques. DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many more novel proteins, due to the relative ease of screening and identifying desirable proteins. Its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics including EST-based or SAGE-tag-based functional genomics and proteomics will continue to advance rapidly. Its biological knowledge base will expand the scope of biomolecular engineering, and the impact of well-coordinated biomolecular engineering research will be very significant on our understanding of gene expression, upregulation and downregulation, and posttranslational protein processing in healthy and diseased cells. The bioinformatics for genome and proteome analysis will contribute substantially toward over more accelerated advances in pharmaceutical industry. When the functional genomics database. EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis are all put to good use, the biomolecular engineering research will yield new drug discoveries, improved therapies, and new or significantly improved bioprocesses. With the advances in biomotecular engineering, the rate of finding new high-value peptides or proteins including antibodies, vaccines, enzymes, and therapcutic peptides will continue to be accelerated. The targets for rational design of biomolecules will be bery broad, diverse, and complex, but many application goals can be achieved throught the expansion of knowledge base on biomolecules of interest and their roles and functionsl in cells and tissues. In the near future, more therapeutic drugs and high-value biomolecules will be designed and produced for the treatment or prevention of not-so-easily-cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Also anticipated are many more industnal enzymes that will be engineered to confer desirable properties for the process improvement and manufacturing of many high-value biomolecular products. Many more new metabolites including novel antibioties that are active agains resistant strains will be also produced by recombinant organisms having de novo engineered biosynthetic pathwy enzyme systems. The biomolecular engineering era is here and a great deal of benefits can be derived form this field of scientific research for many years to come if we are willing to put it to good use. ⓒ 2000 Elsevier Science B.V All rights reserved.

Recent Advances and Trends in Antibiotics Fermentation Technology

Dewey Doo-Yung Ryu,Kang Man Lee 대한약학회 1977 약학회지 Vol.21 No.3

BIOENERGY AND CHEMICAL FEEDSTOCK FROM RENEWABLE RESOURCES

유두영 ( Dewey Ryu ) 한국화학공학회 1980 추계총회초록집 Vol.- No.-

Monoclonal Antibody Refolding and Assembly: Protein Disulfide Isomerase Reaction Kinetics

박선호,Dewey D. Y. Ryu 한국생물공학회 2003 Biotechnology and Bioprocess Engineering Vol.8 No.2

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (MAb) refolding and assembly which accompanies disulfide bond formation. The MAb in vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.

Yarrowia lipolytica의 Multicopy Integration Vector 개발

김정윤,우문희,Dewey D.Y. Ryu 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.5

Multicopy integration vector는 복제수가 많고 non-selective한 환경조건 하에서도 매우 안정되게 유지가 되기 때문에 heterologous 유전자를 발현시키는데 매우 유용한 벡터 시스템이다. Yarrowia lipolytica의 multicopy integration vector를 개발하기 위하여 Y. lipolytica로부터 P-type rDNA를 클로닝하였다. 이 클론된 rDNA의 HindIII-BglII 절편과 promoter 지역을 포함하고 있지 않은 URA3 유전자를 pGEM1 plasmid에 삽입하여 제조한 벡터를 pMIYL-1과 pMIYL-2로 명명하였다. RDNA 절편은 벡터와 chromosomal DNA 사이에 homologous recombination을 유도하기 위한 것이며, promoterless URA3는 불완전한 표지 유전자로서 multicopy integration을 유발시키기 위한 것이다. PMIYL-1은 rDNA의 HindIII-BglII 절편내에 유일한 제한효소 자리로서 KpnI을 가지고 있고, pMIYL-2는 KpnI과 EcoRI을 가지고 있다. 이 벡터들을 Y. lipolytica에 도입한 후에 형질 전환체를 선별하여 copy 수와 안정성을 검사한 결과, 벡터의 copy 수는 5개 이하로 존재하고 non-selective 배지에서도 매우 안정하게 유지가 됨을 알 수 있었다. Multicopy integration vector is a very useful vector system in that they can be integrated into chromosomal DNA in several copies and stably maintained under non-selective conditions. To develop a multicopy integration vector system in the yeast Yarrowia lipolytica, P-type ribosomal DNA was cloned from Y. lipolytica. A HindIII-BglII fragment of the cloned rDNA and a promoterless URA3 gene were inserted into pGEM1, generating multicopy integration vectors, pMIYL-1 and pMIYL-2. The rDNA fragment is for targeted homologous recombination between the vector and the chromosomal DNA of Y. lipolytica, and the promoterless URA3 gene is a defective selection marker for inducing multicopy integration. pMIYL-1 and pMIYL-2 have an unique restriction enzyme site, KpnI, and two unique restriction enzyme sites, KpnI and EcoRI, repectively, which can be used for targeting of the vectors into the rDNA of Y. lipolytica chromosomal DNA. After transformation of the vectors into Y. lipolytica, copy number and stability were analyzed by Southern hybridization. The vectors were found to be present in less than 5 copies per cell and were stably maintained during growth in non-selective media.

Monoclonal Antibody Refolding and Assembly: Protein Disulfide Isomerase Reaction Kinetics

Park, Sun-Ho,Ryu, Dewey D.Y. The Korean Society for Biotechnology and Bioengine 2003 Biotechnology and Bioprocess Engineering Vol.8 No.2

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (Mab) refolding and assembly which accompanies disulfide bend formation. The MAb in vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb in-termediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant fur a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.

Review Articles-Biomolecular Engineering and Drug Development

Nam, Doo-Hyun,Ryu, Dewey D. Y. 영남대학교 약품개발연구소 1999 영남대학교 약품개발연구소 연구업적집 Vol.9 No.-

Biomolecular engineering is a technology to create novel structures of high-value bio-molecules for use in medicine and industry, through the directed alteration of proteins and/or biologically active molecules in living cells to produce a novel biometabolites as well as engineered protein itself. For the development of new drugs by biomolecular en-gineering, desired biomolecules have to be rationally designed based on their structure-stability/structure-activity relationship, and then screened through well-established mutation and selection program. Over the past decade, there has been significant pro-gress in mutation and selection methodology; DNA shuffling technology mimicking natural evolution for artificial DNA recombination and phage-displayed combinatorial peptide library for rapid selection of proteins expressed from mutated genes. Bioinfor-matic tools including functional genomics and proteomics have been also developed for the ready access to the information related to the protein-function and genome-Protein,leading to the design and identification of new drug targets. Throughout the use of an enormous amount of bioinformatic databases, many protein/peptide drugs and biome-tabolite molecules have been designed The candidates of new drugs are monoclonal an-tibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two human-ized monoclonal antibodies approved by FDA became the first tine of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, far the treatment of breast cancer and pediatric respiratory syncytial viral infection, respectively. Many more newly engineered biomolecules are under developing for medicinal application. Some clinical trials fer therapeutic applications are now in progress, and very positive results are already anticipated.

Study of 3-Ketosteroid Dehydrogenase System Using Whole-cell-enzyme from Arthrobacter simplex

Eun Chung Park,Dewey Doo-Young Ryu 대한약학회 1977 약학회지 Vol.21 No.3

A new assay method for delta-l-dehydrogenated-90-ketocorticosteroid in the presence of proteinous material or whole-cell-enzyme and 3-ketocorticosteroid has been developed. This method makes use of the linear relationship between the ratio of absorbances at 265nm and at 242nm and the fractional concentration of delta-1-3-ketosteroid. Theoretical values were calculated based on the absorbances of proteinous material at fixed concentrations of the 3-ketosteroid and delta-l-dehydrogenated-90-ketosteroid. The values obtained experimentally showed good agreement with the values theoretically predicted. The new assay method developed for the steroid mixture containing proteinous material is of some practical importance. The use of such assay method enables one to determine the enzyme activity and the rate of enzyme reaction or conversion rather quickly, easily and accurately. By the use of this assay method, the reaction kinetics of whole-cell-enzyme has also been studied. It was found that it followed the simple Michaelis-Menten type enzyme kinetics. Also the reversibility of this reaction with actively metabolizing cell was examined. It was found that delta-l-dehydrogenated-3-ketosteroid could not be hydrogenated reversibly to 3-ketosteroid by this enzyme system.

Productivity Improvement of a Recombinant Fermentation through Decrease of Growth Rate Differentials

KIM, JEONG-YOON,RYU, DEWEY D.Y. 충남대학교 생물공학연구소 1998 생물공학연구지 Vol.6 No.-

The growth rate differential caused by the overexpression of a cloned gene accelerates takeover of bioreactor by plasmid-free cell population and results in productivity decrease. In an attempt to improve productivity of a recombinant fermentation by decreasing growth rate differentials, the par locus from pSC101 was inserted into a multicopy plasmid, pPLc-RP 4.5, giving rise to pPLP that is the same plasmid as pPLc-RP4.5 except for the presence of the par locus. It was found that pPLP content in the pPLP-bearing strain was decreased by a about 30% as compared to that in the pPLc-RP4.5-bearing strain although the presence of the par locus significantly increased plasmid stability. To see the effects of the par locus on specific growth rates, specific growth rates of the pPLc-RP4.5-bearing strain, pPLP-bearing strain and host strain were measured using a two-stage contiunous culture system. It was observed that the specific growth rates of the pPLc-RP4.5-bearing strain were lower than those of the host strain but the specific growth rates of the pPLP-bearing strain were similar to those of the host strain at the same growth conditions. These results suggest that the par locus decreased the growth rate differentials caused by the overproduction of β-galactosidase in the pPLP-bearing strain. In batch cultures, even though the pPLP-bearing strain and the pPLc-RP4.5-bearing strain gave similar specific productivities, the pPLP-bearing strain showed a significant increase in cell density as compared to the pPLc-RP4.5-bearing strain. As a result, the pPLP-bearing cells produced more β-galactosidase per volume per time than the pPLc-RP4.5-bearing cells.