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Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−206−Synthesiology - English edition Vol.5 No.3 (2012) soluble enzymes. Therefore, the membrane binding moiety of the candidate genes was omitted and the area supposed to be the enzyme activity domain was incorporated into the gateway vector tagged with FLAG, and transfected into the human embryonic kidney blast cells (HEK293T cell). The obtained recombinant proteins secreted into the medium were partially purified from the supernatant. We concentrated on comprehensive, easy, and fast detection of activities in recombinant enzymes derived from many candidate glycogenes. We purchased nine human-derived substrates labeled with radioisotope, and added into the culture media. Moreover, we added monosaccharides and oligosaccharides, as well as a mixture of glycolipid and a mixture of glycoproteins obtained from cultured cells as the acceptor substrates. The HEK293T cell was used for recombinant expression of glycosyltransferases, because it was already known that human derived glycosyltransferases are quite unstable and fragile proteins, and impossible to be expressed in active forms by Escherichia coli or yeast. The HEK293T cell is derived from humans and its glycoproteins are highly glycosylated. It is estimated that many glycosyltransferases are expressed endogenously in the HEK293T cell, which leads to a prediction that there are machineries required for expression of ectogenic human recombinant glycosyltransferases with their own activity. Even under the current knowledge, the human derived HEK293T cell is the most suitable for expression of human recombinant glycosyltransferases. The newly developed glycosyltransferase genes in this project were applied for the substance patents and most of them were published in major journals.[1]-[29] However, in the future, the mass production of glycans will be required. For the mass production, an inexpensive bulk production method is necessary. Production by vertebrate-derived cultured cells costs much and is not suitable for mass production. From this point, Chiba and his group of RCMG are establishing a mass production system of human-derived glycosyltransferases in yeast.[30]These accomplishments are accumulated in the glycogene databases for open access at Japan Consortium for Glycobiology and Glycotechnology Database (http://jcggdb.jp/). This database is being expanded to contain not only the information about glycogenes, but the wider range of contents to form an advanced database by Shikanai and other members of the RCMG. Elemental technology 3: Quantitative assay method for 186 kinds of glycogene expressionsGlycosyltransferases, which synthesize the main structure of N-glycans, are expressed in every cell. The expression level of glycosyltransferases is large and not affected by the conditions of cells. The expression level of other glycogenes is low; especially those synthesizing the terminal moiety of glycans are very slight compared with other genes. They are impossible to be detected by ordinary DNA chips, and even if it could be detected, their modifications cannot be measured correctly. We developed a technology to measure the expression levels of all 186 glycogenes accurately in a comprehensive, high throughput manner. The quantitative real-time PCR (qPCR) for comprehensive glycogene expression analysis is a matured experimental technique, which is the most reliable biological analysis method in terms of detection sensitivity and measurement accuracy. Glycogenes are experimentally known as low in the expression levels in most of the cases, thus qPCR was considered to be suitable for the development of the expression analysis system.[31] Specifically, customized qPCR arrays for the 186 glycogenes encoding the glycosyltransferases and modification enzymes were established by the members of RCMG with Sawaki as the core member. The plasmid DNA pool of the glycogene clone library used as the calibrator enabled the system to indicate the amount of transcription products of all 186 glycogenes based on their copy number at a one-time measurement. Classification of cells in terms of the expression profiling of glycogenes is well correlated with the cell differentiation or canceration, and the expression of glycogenes is known to be proportional to the expression of glycans. Elemental technology 4: Establishment of in vitro synthesis method of glycans and glycopeptides by recombinant glycosyltransferaseAs the recombinant glycosyltransferases are derived from humans, most of them can be purified as soluble recombinant enzymes without losing activity by means of human-derived HEK293T cells. Based on this principle, we established an in vitro synthesis method of glycans and glycopeptides. As an exception, the glycosyltransferases attributed to the main structure of N-glycans cannot be recombined as they are synthesized on the rough endoplasmic reticulum (ER) and they pass through the lipid membrane several times. As for the main structure of N-glycans, commercially available purified natural forms were used as the starting materials. As for the O-glycans, representative peptides possessing O-glycans, such as mucin were used as the starting materials and glycans were elongated by adding glycosyltransferases sequentially.[30][32][33] Synthesis methods were established for two objectives by Ito et al. of RCMG. The first objective is the mass production of one kind of glycans in the largest quantity. The conditions for enzyme reaction were set at a certain level, and the largest amount of enzymes was reacted for the longest time possible. The obtained products were separated and refined by liquid chromatography. The second objective was the simultaneous synthesis of multiple glycan structures at small quantities in one tube. Reaction of each enzyme was terminated by heat when the production reached about 50 % saturation. Then the next enzyme was added to the tube and similarly the reaction was terminated at the 50 % reaction point. Thearetically, 2n

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