Development of Thermal Refolding and application for antibody production

2017 Fiscal Year Final Research Report

• 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
• Keywords: disulfide oxidoreductase / Thermus thermophilus / redox tuning / GFP / antibody / redox motif

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.

Free Research Field

応用微生物学