In Vitro Synthesis and Design of Kinesin Biomolecular Motors by Cell-Free Protein Synthesis

Kinesin is a biomolecular motor that generates forceand motilityalong microtubule cytoskeletons in cells. Owing to their ability tomanipulate cellular nanoscale components, microtubule/kinesin systemsshow great promise as actuators of nanodevices. However, classical in vivo protein production has some limitations for thedesign and production of kinesins. Designing and producing kinesinsis laborious, and conventional protein production requires specificfacilities to create and contain recombinant organisms. Here, we demonstratedthe in vitro synthesis and editing of functionalkinesins using a wheat germ cell-free protein synthesis system. Thesynthesized kinesins propelled microtubules on a kinesin-coated substrateand showed a higher binding affinity with microtubules than E. coli-produced kinesins. We also successfully incorporatedaffinity tags into the kinesins by extending the original sequenceof the DNA template by PCR. Our method will accelerate the study ofbiomolecular motor systems and encourage their wider use in variousnanotechnology applications.