Doo-Hyun Nam

Recent progress in biomolecular engineering

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 disciplines, 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 Mabs, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, 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.

Role of corticosterone in ethyl carbamate-induced immunosuppression in female BALB:c mice

We have recently demonstrated that the antibody response to the T-cell-dependent antigen, sheep red blood cells (SRBCs), was suppressed by ethyl carbamate in female BALB/c mice. At the same doses, ethyl carbamate decreased in the numbers of splenic macrophages, B cells, total T cells, CD4(+) T cells and CD8(+) T cells. In addition, the serum level of corticosterone was increased dose-dependently. To investigate the possible role of corticosterone in ethyl carbamate-induced immunosuppression, the antibody response to SRBCs and the subpopulation changes of splenocytes and thymocytes were determined in naive, sham-operated and adrenalectomized (ADX) female BALB/c mice. When the mice were treated intraperitoneally with 400 mg/kg ethyl carbamate, the antibody response was significantly suppressed by ethyl carbamate in naive and sham-operated mice in accompanying the decrease in spleen and thymus weights and/or the increase in the level of serum corticosterone. Meanwhile, the antibody response was not suppressed by ethyl carbamate in the ADX mice. The splenic numbers of total cells, macrophages, B and T cells, and CD4(+) cells were decreased by ethyl carbamate in naive and sham-operated mice. Meanwhile, each cell number was comparable with control in the ADX mice. The flow cytometric analyses on thymocytes did not show obvious differences as seen in the spleen. Finally, when the ADX mice were treated intraperitoneally with 25 mg/kg corticosterone, the antibody response was significantly suppressed. Taken together, our present results suggested that corticosterone might be, at least partially, responsible for ethyl carbamate-induced immunosuppression in female BALB/c mice.

Biomolecular engineering : a new frontier in biotechnology

Abstract 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 patterns 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 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 ever 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 biomolecular engineering, the rate of finding new high-value peptides or proteins including antibodies, vaccines, enzymes, and therapeutic peptides will continue to be accelerated. The targets for rational design of biomolecules will be very broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge base on biomolecules of interest and their roles and functions 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 industrial 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 antibiotics that are active against resistant strains will be also produced by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here and a great deal of benefits can be derived from this field of scientific research for many years to come if we are willing to put it to good use.

Utilization of DNA marker-assisted selection in Korean native animals

The recent progress of DNA technologies including DNA fingerprinting (DFP) and random amplified DNA polymorphism (RAPD) analysis make it possible to identify the specific genetic traits of animals and to analyze the genetic diversity and relatedness between or within species or populations. Using those techniques, some efforts to identify and develop the specific DNA markers based on DNA polymorphism, which are related with economic traits for Korean native animals, Hanwoo (Korean native cattle), Korean native pig and Korean native chicken, have been made in Korea for recent a few years. The developed specific DNA markers successfully characterize the Korean native animals as the unique Korean genetic sources, distinctively from other imported breeds. Some of these DNA markers have been related to some important economic traits for domestic animals, for example, growth rate and marbling for Hanwoo, growth rate and back fat thickness for native pig, and growth rate, egg weight and egg productivity for native chicken. This means that those markers can be used in important marker-assisted selection (MAS) of Korean native domestic animals and further contribute to genetically improve and breed them.

Cloning, Expression, and Biological Function of a dTDP-Deoxyglucose Epimerase (gerF) Gene from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules which has antimicrobial activities against Gram-positive bacteria. The deoxyhexose biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-155 genome has now been isolated. Four orf were identified and a putative orf, supposed to code for the dTDP-deoxyglucose epimerase gene, was designated as gerF. gerF was expressed in E. coli using recombinant expression vector pHJ3. The recombinant protein expressed in a soluble form. The enzyme was purified by Ni-affinity column using imidazole buffer as eluents. The molecular mass of the expressed protein correlated with the predicted mass (36 000 Da) deduced from the cloned gene sequence data. The purified enzyme produced maltol from dTDP-4-keto-6-deoxyglucose and it was confirmed that the expressed protein was dTDP-deoxyglucose epimerase catalyzing epimerization of C-3 and C-5 or C-3 of dTDP-4-keto-6-deoxyglucose.

Cloning and sequencing of a novel glutaryl acylase β-subunit gene ofPseudomonas cepacia BY21 from bioinformatics

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from variousPseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences ofPseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene fromP. cepacia BY21. The unknown β-subunit gene of glutaryl acylase from chromosomal DNA ofP. cepacia BY21 was cloned successfully by PCR. The β-subunit amino acids ofP. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those ofPseudomonas diminuta KAC-1 acylase except that Asn408 ofP. diminuta KAC-1 acylase was changed to Leu408.

Biomolecular engineering and drug development

Biomolecular engineering is a technology to create novel structures of high-value biomolecules 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 engineering, 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 progress 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. Bioinformatic 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 biometabolite molecules have been designed. The candidates of new drugs are monoclonal antibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two humanized monoclonal antibodies approved by FDA became the first line of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, for 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 for therapeutic applications are now in progress, and very positive results are already anticipated.

Effects of flupyrazofos on liver microsomal cytochrome P450 in the male Fischer 344 rat

1. The effects of flupyrazofos on liver microsomal cytochrome P450 were investigated in the male Fischer 344 rat.When rats were treated intraperitoneally with flupyrazofos for 3 consecutive days, the activities of ethoxyresorufin O-deethylase and testosterone 2 β-hydroxylase were significantly reduced, whereas the activities of pentoxyresorufin O-depentylase and testosterone 6 β- and 7 α-hydroxylases were induced in liver microsomes. 2. Within 24 h after treatment with 50 m kg−1 flupyrazofos, most enzyme activities were decreased, indicating the interaction of flupyrazofos with cytochrome P450. 3. In Western immunoblotting, cytochrome P4502B1/2 proteins were clearly induced by treatment with flupyrazofos, whereas P4501A1/2 and 2C6 proteins were reduced in liver microsomes. 4. The present results indicate that flupyrazofos modulates the expression of cytochrome P450 in rat.