study of low-immunogenic monomeric streptavidin

2014 Fiscal Year Final Research Report

• Project/Area Number: 25640088
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Tumor therapeutics
• Research Institution: The University of Tokyo
• Principal Investigator: DOI Hirofumi 東京大学, 先端科学技術研究センター, 特任教授 (80403335)
• Co-Investigator(Kenkyū-buntansha): SUGIYAMA Akira 東京大学, アイソトープ総合センター, 助教 (40562715)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: テトラマー / モノマー / 巻き戻し / ビオチン結合 / 分子動力学 / シミュレ＝ション / 塩橋

Outline of Final Research Achievements

Based on the low immunogenicity streptavidin LISA314 we tried to make a monomer of LISA314. Hydrophobic amino acids in the tetramer interfaces, or in the dimer interfaces were replaced by Thr or Ser soluble amino acids. Also, 120Trp was replaced to Gly. Genes introducing these mutations (with His tag) was artificially synthesized, and then LISA314 monomers were expressed in E. coli and purified. The binding affinity of the monomer with biotin was measured using a Biacore system, but binding affinity was lost.
In order to explore the cause, we carried out molecular dynamics simulations in the monomer state. The simulation suggested that the interaction between two amino acids that form a salt bridge is important for biotin binding.

Free Research Field

タンパク質工学