Development of Thermal Refolding and application for antibody production

• Project/Area Number: 15K14695
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Applied microbiology
• Research Institution: Okayama University
• Principal Investigator: Takashi Tamura 岡山大学, 環境生命科学研究科, 教授 (40253009)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2017: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: disulfide oxidoreductase / Thermus thermophilus / redox tuning / GFP / antibody / redox motif / ジスルフイド架橋 / 高度好熱菌 / 緑色蛍光タンパク質 / リフォールディング / グルタチオン / ジスルフィド異性化酵素 / 変性タンパク質 / 酸化還元電位 / ジスルフィド架橋

Outline of Final Research Achievements

In this research, a novel technique was developed which enabled the refolding of proteins at high temperatures with catalysis by thermostable protein disulfide isomerase. For this purpose a novel reporter protein sfGFP-2SS was invented, which emits green fluorescence depending on restoration of correct S-S bonds to be introduced. The fluorescence can be once vanished by erroneous S-S crosslink upon oxidation under denaturation, but green fluorescence was restored by disulfide isomerase from Thermus thermophilus HB8 in the programmed thermal cycling with redox buffer GSH/GSSG. The invention of Protein Thermal Refolding technique, termed as PTR, was established as a protein version of PCR, leading denatured proteins with abbrenet disulfide bonds to the correct structure with high expectation for recombinant antibody production.

Research Products

• [Journal Article] Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis (2017)
  • Author(s): Sato D, Shiba T, Yunoto S, Furutani K, Fukumoto M, Kudou D, Tamura T, Inagaki K, Harada S.
  • Journal Title: Protein Science Volume: 26 Issue: 6 Pages: 152-158
  • DOI: 10.1002/pro.3158
  • NAID: 130007628790
  • Peer Reviewed / Open Access
• [Journal Article] The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights (2017)
  • Author(s): Sato D, Shiba T, Mizuno S, Kawamura A, Hanada S, Yamada T, Shinozaki M, Yanagitani M, Tamura T, Inagaki K, Harada S
  • Journal Title: Acta Cryst., F Volume: 73 Issue: 3 Pages: 152-158
  • DOI: 10.1107/s2053230x17002011
  • Peer Reviewed / Open Access
• [Journal Article] A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8 (2016)
  • Author(s): Kamada, S., Okugochi, T., Asano, K., Tobe, R., Mihara, H., Nemoto, M., Inagaki, K., and Tamura, T.
  • Journal Title: Biosci. Biotechnol. Biochem. Volume: 80 Issue: 10 Pages: 1970-1972
  • DOI: 10.1080/09168451.2016.1200458
  • Peer Reviewed
• [Journal Article] Identification and Characterization of the zosA Gene Involved in Copper Uptake in Bacillus subtilis 168 (2016)
  • Author(s): Fukuhara, T., Kobayashi, K., Kanayama, Y., Enomoto, S., Kondo, T., Tsunekawa, N., Nemoto, M., Ogasawara, N., Inagaki, K., and Tamura, T.
  • Journal Title: Biosci. Biotechnol. Biochem Volume: 80 Issue: 3 Pages: 600-609
  • DOI: 10.1080/09168451.2015.1107462
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060. (2016)
  • Author(s): Fukuda, Y., Abe, A., Tamura, T., Kishimoto, T., Sogabe, A., Akanuma, S., Yamagishi, A., Imada, K., and Inagaki, K
  • Journal Title: J. Biosci. Bioeng Volume: 121 Issue: 5 Pages: 497-502
  • DOI: 10.1016/j.jbiosc.2015.09.011
  • NAID: 40020838799
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico (2016)
  • Author(s): Takashi Tamura, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Toshiyuki Hirano, Fumitoshi Sato
  • Journal Title: Scientific Reports Volume: 6 Issue: 1
  • DOI: 10.1038/srep19742
  • NAID: 120005723278
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 量子化学計算が解き明かすピリドキサール酵素の反応機構 (2016)
  • Author(s): 田村 隆
  • Journal Title: ビタミン Volume: 90 Pages: 281-285
  • Peer Reviewed
• [Journal Article] Reactivity of sorbose dehydrogenase from Sinorhizobium sp. 97507 for 1,5-anhydro-D-glucitol. (2015)
  • Author(s): Araki T., Nakatsuka T., Kobayashi F., Watanabe-Ishimaru E., Sanada H., Tamura T., Inagaki K.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: Epub ahead of print Issue: 7 Pages: 1130-1132
  • DOI: 10.1080/09168451.2015.1012148
  • Peer Reviewed
• [Journal Article] Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, l-lysine α-oxidase from Trichoderma viride. (2015)
  • Author(s): Amano M, Mizuguchi H, Sano T, Kondo H, Shinyashiki K, Inagaki J, Tamura T, Kawaguchi T, Kusakabe H, Imada K, Inagaki K.
  • Journal Title: J Biochem. Volume: 157 Issue: 6 Pages: 1-11
  • DOI: 10.1093/jb/mvv012
  • NAID: 110010000888
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin (2015)
  • Author(s): Oshima, K., Hattori, M., Shimizu, H., Fukuda, K., Nemoto, M., Inagaki, K., and Tamura, T
  • Journal Title: Genome Announcements Volume: 3 Issue: 4
  • DOI: 10.1128/genomea.00715-15
  • NAID: 120005672474
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Molecular Cloning and Characterrization of L-Methionine γ-Lyase from Streptomyces avermitilis. (2015)
  • Author(s): Kudou, D., Yasuda, E., Hirai, Y., Tamura, T., and Inagaki, K.
  • Journal Title: Journal of Bioscience and Bioengineering Volume: Epub ahead of print Issue: 4 Pages: 380-383
  • DOI: 10.1016/j.jbiosc.2015.02.019
  • Peer Reviewed
• [Presentation] 高度好熱菌由来PDIのタンパク質の高温リフォールディング活性 (2017)
  • Author(s): 平島史崇，倉光成紀,根本理子，稲垣賢二，田村 隆
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] ジスルフィド架橋導入sfGFPを用いた蛍光回復過程の経時変化 (2016)
  • Author(s): 平島史崇，福山慎也，根本理子，稲垣賢二，田村 隆
  • Organizer: 日本分子生物学会
  • Place of Presentation: 横浜
• [Presentation] ジスルフィド架橋導入sfGFPを用いた高温リフォールディング評価法 (2015)
  • Author(s): 福山慎也，森野智美，根本理子，稲垣賢二，田村 隆
  • Organizer: 日本分子生物学会
  • Place of Presentation: 神戸
  • Year and Date: 2015-12-01
• [Remarks] 微生物遺伝子化学研究室web site
  • URL: http://www.agr.okayama-u.ac.jp/ApplEnz/