Kyushu University develops a simple method for kinesin biomolecular motor synthesis without using living organisms

Kinesin biomolecular motors are proteins several tens of nanometers long that bind to microtubule cytoskeletons and move along them at a speed of approximately one micrometer per second. At first glance, this may seem like a leisurely movement. However, if a kinesin molecule were the same size as a human, it would be faster than a Shinkansen bullet train. Additionally, because kinesin converts the chemical energy of ATP into motion with high efficiency, its use as a power source for microfluidic devices and as a rotary motor is also being researched.

Until now, kinesin has been produced using genetically modified organisms such as Escherichia coli. However, the use of genetically modified organisms requires government approval, and special equipment and highly refined skills are needed for the extraction and purification of kinesin. This has limited the use of kinesin by researchers and engineers in a wide range of fields.

The research group led by Assistant Professor Daisuke Inoue of the Faculty of Design at Kyushu University, Graduate Student Keisuke Ohashi of the Research Faculty of Agriculture at Hokkaido University Associate Professor Taichi Takasuka of the Graduate School of Agriculture at Hokkaido University and Professor Akira Kakugo of the Graduate School of Science at Kyoto University has succeeded in synthesizing kinesin in vitro, using a wheat-germ-extract cell-free protein synthesis system that does not require genetically modified organisms such as E. coli. The better access to kinesins will expand its range of applications, and the results were published in ACS Synthetic Biology.

In a cell-free protein synthesis system, proteins can be easily synthesized in vitro by mixing the DNA that encodes their genetic information into the necessary reagents. Kinesins can even be produced at home, because the protein can be synthesized without violating the Cartagena Act, which regulates genetic modification technology. Additionally, in vitro synthesis does not require much purification work, unlike production using genetically modified E. coli.

To evaluate the performance of the synthesized kinesins, a sliding motion test was performed in which microtubules were allowed to move on a kinesin-immobilized glass substrate. Microtubule movement on the synthesized kinesins was also observed, and the movement rate was not significantly different from that on E. coli-produced kinesins. However, the number of microtubules adsorbed to the synthetic kinesin substrate was higher than that adsorbed to the E. coli-produced kinesins, and the proportion of microtubules that did not move was lower on synthetic kinesins. This may be due to the difference in accuracy between the primitive E. coli protein synthesis process and the eukaryotic synthesis system used in this study.

The researchers also succeeded in easily modifying the structure and function of kinesins using the cell-free protein synthesis system. Normally, to modify the structure of a protein, the DNA sequence containing the original genetic information must first be edited and then a circular DNA called a plasmid is inserted. To eliminate this complicated process, PCR (a DNA amplification technique) was used to incorporate additional genetic information into the kinesin DNA template. The linear DNA template produced by this technique can be used directly for kinesin synthesis.

Additionally, a new structure that binds to a specific antibody was incorporated into kinesin. Using fluorescent proteins for visualization of the kinesins, the amount bound to the antibody-immobilized glass substrate was evaluated. The number of kinesins bound to the substrate increased in the presence of the antibody, indicating that additional structures can be easily introduced into these biomolecular motors through the use of the cell-free protein synthesis system.

The new kinesin synthesis method is so simple that even third-year undergraduate students of the Faculty of Design of Kyushu University, who had never held a micropipette before, were able to synthesize and move the proteins successfully. This is an important result from the perspective of do-it-yourself biotechnology, an activity aimed at democratizing the biotechnology field. With the minimum necessary equipment and reagents, kinesin research can now be performed outside of research institutes such as universities, and the range of applications may expand in the future.