| 초록 |
The coiled coil (CC) motif, which is a protein helix structure, has a rich potential as a molecular tool because it has the characteristics of oligomerization specifically with the pair coil. Therefore, various methods are being studied for binding stability against unavoidable dissociation of CC. In this study, we produced and then co-expressed a cysteine-mutated coil motif capable of forming disulfide bonds to prevent dissociation of CC and to perform an efficient purification process. We fused different fluorescent proteins to the C-terminus of each complementary coil. After that, residues in each coil, were mutated to cysteine to form a disulfide bridge. Each constructed coil motif was efficiently co-expressed in the bacterial system. The dissociation constant (KD) value of the CC before cysteine mutation was measured by isothermal titration calorimetry, and non-dissociating behavior of the CC complex was analyzed by Native-PAGE, size exclusion chromatography, and fluorescence resonance energy transfer assay. As a result, the disulfide-bonded CC complex was found to be a stable and dilution-resistant heterodimer, holding a promise as a bispecific scaffold for drug delivery. |
