Establishment of post-translational modification system in the cell free protein system

• Project/Area Number: 25850071
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Applied biochemistry
• Research Institution: Toyama Prefectural University (2014-2016); Ehime University (2013)
• Principal Investigator: Izumi Atsushi 富山県立大学, 工学部, 研究員 (80637562)
• Research Collaborator: YUZURU TOZAWA (90363267)
• Project Period (FY): 2013-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2014: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 膜タンパク質 / フェロキラターゼ / 脂質二重膜 / 無細胞タンパク質合成系 / 脂質膜 / 無細胞系 / 翻訳後修飾

Outline of Final Research Achievements

We introduced a simple method to synthesize and purify a membrane-associated protein in the wheat germ cell free system as a model production system. We used ferrochelatase from Saccharomyces cerevisiae, catalyzing the insertion of ferrous ion into the tetrapyrrole center of protoporphyrin IX, as a model protein. The luminol-horse radish peroxidase assay indicated that the active ferrochelatase was only synthesized in the presence of assolectin liposomes. The Accudenz density gradient centrifugation experiment indicated that ferrochelatase clearly localized at the liposome fraction and the complex of the synthesized protein with liposomes can be simply separated from soluble protein fraction by a single step centrifugation.