Molecular Tension Probe for In Vitro Bioassays

Cell-free bioassays (CFBs) provide their own distinctive merits over cell-based bioassays (CBBs) including (i) rapid and on-site applicability, (ii) long-term utility, and (iii) bioanalytical versatility. The authors previously introduced a unique bioluminescent imaging probe for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. In this chapter, we exemplify that a full-length artificial luciferase is sandwiched between FRB (FKBP-rapamycin-binding domain of FKBP12-rapamycin-associated protein) and FKBP (FK506-binding protein) via minimal flexible linkers, named FRB-A23-FKBP. The rapamycin-activated PPIs between FRB and FKBP append molecular tension to the sandwiched luciferase, enhancing the enzymatic activity in a quantitative manner. The fusion protein, FRB-A23-FKBP, is three-step column-purified and the bioanalytical utility is characterized in various CFB conditions. This chapter guides the detailed protocols from the purification to the practical bioassays of FRB-A23-FKBP.