- Email: [email protected]
Novel separation and detection techniques for protein analysis
S18
Abstracts / Journal of Biotechnology 136S (2008) S16–S21
KN-044
KN-051
Novel concept of Escherichia coli minimum genome cell factory
Novel separation and detection techniques for protein analysis
Hideharu Anazawa 1,∗ , Hideo Mori 2
Yukui Zhang
1
Kyowa Hakko Kogyo Co., Ltd., Science & Technology Department, Tokyo, Japan 2 Bio Frontier Labs., Tokyo, Japan
National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Acedemy of Sciences, Dalian, China
E-mail address: [email protected] (H. Anazawa).
The proteome study is of great significance to discover the truth of life. Human proteome is much more complex than genome, due to wide dynamic distribution range, large number and dynamic change with time and space. Therefore, the development of high efficient separation and high sensitive detection techniques for proteome study has been regarded as one of bottleneck problems to accelerate the proteomics. In our recent work, various novel liquid phase separation techniques for proteome study have been developed. For sample preparation, universal methods have been proposed to improve the detection sensitivity of low abundance proteins by dialysis and multi-chamber isoelectric focusing. In addition, several new kinds of materials, such as protein-imprinted polymers and monolithic Zr4+ -IMAC materials have been synthesized and applied for the selective enrichment of target proteins or phosphorated peptides. In addition, several kinds of new 2D-liquid phase separation platforms, such as 2D-monolithic immobilized pH gradient CIEF-CZE, 2D-CE on microchip (Cong et al., 2008) and 2D-WAX/WCX-on column digestion-RPLC (Ma et al., 2008), have been established to improve the separation capacity. Furthermore, in situ fluorescence labeling technique, on-line desalting device, and AP treatment method have been proposed to achieve high sensitive detection of proteins or peptides by fluorescence detector and ESI-MS/MS. Based on the above-mentioned techniques, two integrated liquid phase separation platforms, based respectively on multidimensional HPLC and CE, have been established, and successfully applied into the analysis of proteins extracted from cells and tissues.
Production of commodity chemicals by biochemical process at low cost is generally considered to be difficult. Genetic recombination technology has made it easy to enhance and improve terminal biosynthetic pathways. However, the whole picture of metabolisms and regulations in the host cell is still in a black box. Though Escherichia coli K-12 is one of the major microorganisms industrially and scientifically used, 40% of its genes are still assigned unknown function. We expected that the microorganism without function-unknown genes would be simple and useful for process development since we could avoid unexpected regulations. In the process to make defined chromosome, it is expected that unnecessary and negative regulators would be eliminated and result in activated metabolism. We call the host cell with a defined chromosome and activated metabolism as “Minimum-Genome Factory (MGF)”. We have developed an efficient technique to manipulate chromosome. On the other hand, we have been analyzing all genes of K-12 from the viewpoints of growth, essentiality and metabolism. These results have been used for designing defined chromosome and MGF. Non-conserved genes in mosaic-distributed regions are thought to be less important and candidates for deletion to MGF. We have deleted 53 regions, 1.03 Mbp, 22% of genome size was deleted without any negative growth characters. Surprisingly this strain, MGF-01, show improved growth characters and the productivities with introduced recombinant production plasmids have increased comparing to wild-type strain. 4320 single gene knock-out mutants assayed for selection of candidate genes for elimination using the GN2-MicroPlate (Biolog Inc.), which permits assay of 95 carbonsource utilizations simultaneously. Our analysis would be useful not only for functional genomics but also detecting important genes for metabolism and cell proliferation. Acknowledgement This work was supported by Ministry of Economy, Trade & Industry (METI), and New Energy and Industrial Technology Development Organization (NEDO) in Japan.
E-mail address: [email protected].
References Cong, Y., Zhang, L., Tao, D., Liang, Y., Zhang, W., Zhang, Y., 2008. Miniaturized twodimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins. J. Sep. Sci. 31 (3), 588–594. Ma, J., Liang, Z., Qiao, X., Deng, Q., Tao, D., Zhang, L., Zhang, Y., 2008. Organic–inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 80 (8), 2949–2956.
doi:10.1016/j.jbiotec.2008.07.027
References
IL-003
Hara, K., et al., 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. J. Biomol. Screen. 11 (3), 310–317. Hashimoto, M., et al., 2005. Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol. Microbiol. 55 (1), 137–149. Hibi, M., et al., 2007. Improvement of NADPH-dependent bioconversion by transcriptome-based molecular breeding. Appl. Environ. Microbiol. 73 (23), 7657–7663. Ito, M., et al., 2005. Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays. Metab. Eng. 7 (4), 318–327. Mizoguchi, K., et al., 2008. Superpositioning of deletions promotes growth of Escherichia coli with a reduced genome. DNA Res. (in revised).
From systems biology to biosystems engineering for industrial biotechnology
doi:10.1016/j.jbiotec.2008.07.026
An-Ping Zeng Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, D-21071 Hamburg, Germany E-mail address: [email protected]. The rapid development in genome sequencing makes it now possible to sequence almost any organisms of industrial interest in a very short time. Furthermore, technological and instrumental advances in functional genomics allow faster and more accurate quantitative measurements at different molecular levels (gene, mRNA, protein and metabolite) and thus the reconstruction and analysis of genome-scale metabolic and regulatory networks. These devel-
Abstracts / Journal of Biotechnology 136S (2008) S16–S21
KN-044
KN-051
Novel concept of Escherichia coli minimum genome cell factory
Novel separation and detection techniques for protein analysis
Hideharu Anazawa 1,∗ , Hideo Mori 2
Yukui Zhang
1
Kyowa Hakko Kogyo Co., Ltd., Science & Technology Department, Tokyo, Japan 2 Bio Frontier Labs., Tokyo, Japan
National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Acedemy of Sciences, Dalian, China
E-mail address: [email protected] (H. Anazawa).
The proteome study is of great significance to discover the truth of life. Human proteome is much more complex than genome, due to wide dynamic distribution range, large number and dynamic change with time and space. Therefore, the development of high efficient separation and high sensitive detection techniques for proteome study has been regarded as one of bottleneck problems to accelerate the proteomics. In our recent work, various novel liquid phase separation techniques for proteome study have been developed. For sample preparation, universal methods have been proposed to improve the detection sensitivity of low abundance proteins by dialysis and multi-chamber isoelectric focusing. In addition, several new kinds of materials, such as protein-imprinted polymers and monolithic Zr4+ -IMAC materials have been synthesized and applied for the selective enrichment of target proteins or phosphorated peptides. In addition, several kinds of new 2D-liquid phase separation platforms, such as 2D-monolithic immobilized pH gradient CIEF-CZE, 2D-CE on microchip (Cong et al., 2008) and 2D-WAX/WCX-on column digestion-RPLC (Ma et al., 2008), have been established to improve the separation capacity. Furthermore, in situ fluorescence labeling technique, on-line desalting device, and AP treatment method have been proposed to achieve high sensitive detection of proteins or peptides by fluorescence detector and ESI-MS/MS. Based on the above-mentioned techniques, two integrated liquid phase separation platforms, based respectively on multidimensional HPLC and CE, have been established, and successfully applied into the analysis of proteins extracted from cells and tissues.
Production of commodity chemicals by biochemical process at low cost is generally considered to be difficult. Genetic recombination technology has made it easy to enhance and improve terminal biosynthetic pathways. However, the whole picture of metabolisms and regulations in the host cell is still in a black box. Though Escherichia coli K-12 is one of the major microorganisms industrially and scientifically used, 40% of its genes are still assigned unknown function. We expected that the microorganism without function-unknown genes would be simple and useful for process development since we could avoid unexpected regulations. In the process to make defined chromosome, it is expected that unnecessary and negative regulators would be eliminated and result in activated metabolism. We call the host cell with a defined chromosome and activated metabolism as “Minimum-Genome Factory (MGF)”. We have developed an efficient technique to manipulate chromosome. On the other hand, we have been analyzing all genes of K-12 from the viewpoints of growth, essentiality and metabolism. These results have been used for designing defined chromosome and MGF. Non-conserved genes in mosaic-distributed regions are thought to be less important and candidates for deletion to MGF. We have deleted 53 regions, 1.03 Mbp, 22% of genome size was deleted without any negative growth characters. Surprisingly this strain, MGF-01, show improved growth characters and the productivities with introduced recombinant production plasmids have increased comparing to wild-type strain. 4320 single gene knock-out mutants assayed for selection of candidate genes for elimination using the GN2-MicroPlate (Biolog Inc.), which permits assay of 95 carbonsource utilizations simultaneously. Our analysis would be useful not only for functional genomics but also detecting important genes for metabolism and cell proliferation. Acknowledgement This work was supported by Ministry of Economy, Trade & Industry (METI), and New Energy and Industrial Technology Development Organization (NEDO) in Japan.
E-mail address: [email protected].
References Cong, Y., Zhang, L., Tao, D., Liang, Y., Zhang, W., Zhang, Y., 2008. Miniaturized twodimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins. J. Sep. Sci. 31 (3), 588–594. Ma, J., Liang, Z., Qiao, X., Deng, Q., Tao, D., Zhang, L., Zhang, Y., 2008. Organic–inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal. Chem. 80 (8), 2949–2956.
doi:10.1016/j.jbiotec.2008.07.027
References
IL-003
Hara, K., et al., 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. J. Biomol. Screen. 11 (3), 310–317. Hashimoto, M., et al., 2005. Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol. Microbiol. 55 (1), 137–149. Hibi, M., et al., 2007. Improvement of NADPH-dependent bioconversion by transcriptome-based molecular breeding. Appl. Environ. Microbiol. 73 (23), 7657–7663. Ito, M., et al., 2005. Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays. Metab. Eng. 7 (4), 318–327. Mizoguchi, K., et al., 2008. Superpositioning of deletions promotes growth of Escherichia coli with a reduced genome. DNA Res. (in revised).
From systems biology to biosystems engineering for industrial biotechnology
doi:10.1016/j.jbiotec.2008.07.026
An-Ping Zeng Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, D-21071 Hamburg, Germany E-mail address: [email protected]. The rapid development in genome sequencing makes it now possible to sequence almost any organisms of industrial interest in a very short time. Furthermore, technological and instrumental advances in functional genomics allow faster and more accurate quantitative measurements at different molecular levels (gene, mRNA, protein and metabolite) and thus the reconstruction and analysis of genome-scale metabolic and regulatory networks. These devel-