Creation of Ultra Super Fusion Protein for Antibody Binding and Purification

2014 Fiscal Year Final Research Report

• Project/Area Number: 25630370
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biofunction/Bioprocess
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: UEDA Hiroshi 東京工業大学, 資源化学研究所, 教授 (60232758)
• Co-Investigator(Renkei-kenkyūsha): MATSUYAMA Yuki (OHMURO Yuki) 学術振興会RPD/神戸大学, 大学院工学研究科, 特命助教 (30571088)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: antibody / purification / affinity / avidity / immunoassay

Outline of Final Research Achievements

Two antibody binding domains from Protein A D (PA) and Protein G C1 (PG) were fused with a linker peptide with various lengths (DDAKK)n, n=0,2,4,6, to create a novel antibody binding protein PAxnPG. When their affinity to Fab and Fc of antibody was evaluated with a biosensor based on biolayer interferometry, the highest binding constant was obtained with n=4 for Fab, and n=6 for Fc, probably reflecting their length between two binding sites. Moreover, the obtained binding constants are several tens-fold higher than those with PA/PG alone. The utility of PAxPG as an antibody detection reagent was also suggested by immunoblot analysis using a fusion protein with chimeric human alkaline phosphatase (AP) with higher catalytic activity and stability than the parent APs from intestinal and placental origins.

Free Research Field

タンパク質工学