Kinesin Chimera Protein Fused with Calmodulin as a Molecular Shuttle

Recently attention is focused on the application of molecular shuttles based on the motor protein kinesin and microtubule to drug delivery system (DDS) and lab-on-a-chip. In vivo, kinesin carries cargoes of biomolecular e.g., organelle which attach to the tail domain of kinesin. However, the molecular mechanism of the attaching and detaching of cargo is still obscure. Therefore, artificial binding systems have to be introduced on the molecular shuttle. Previously biotin-avidin and antigen-antibody reaction system have been used to attach kinesin to target cargoes. Although the systems are highly specific and tight, these are flawed as irreversible binding. In this study, we employed reversible cargo loading system using calmodulin (CaM) and M13 peptide for the molecular shuttle. We have designed kinesin K560 chimera protein fused with CaM at the C-terminal tail region of kinesin (K560-CaM). K560-CaM was successfully expressed by E. coli. expression system and purified. And M13 peptide fused with yellow fluorescent protein (M13-YFP) was also prepared as a target cargo. The ATPase activity and the microtubules gliding activity of K560-CaM were almost in the normal range of the kinesin wild type. The Ca2+ dependent reversible binding of K560-CaM and M13-YFP was observed with HPLC using size-exclusion column.