Mater. Res. Soc. Symp. Proc. Vol. 1316 © 2011 Materials Research Society DOI: 10.1557/opl.2011. Construction of Molecular Shuttles Based on Kinesin Motor Proteins and Microtubules Daniel Oliveira 1 , Kim Domyoung 2 , Mitsuo Umetsu 1,2 , Tadafumi Adschiri 1 and Winfried Teizer 1,3 1 World Premier International - Advanced Institute for Materials Research, Tohoku University, 2- 1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan; 2 Dept. of Biomolecular Engineering, Tohoku University, 6-6-11 Aramaki, Aoba-ku, Sendai 980-8579, Japan; 3 Dept. of Physics and Astronomy, Texas A&M University, College Station, TX 77843-4242, USA ABSTRACT The intracellular cargo delivery performed by kinesin motor proteins can be biomimetically employed to engineer tailor-made artificial nanotransport systems. Kinesin (expressed on an Escherichia coli system) and microtubules (obtained from the polymerization of tubulin proteins) were prepared and characterized. We report recent results and explore the aim of the construction of biomotor-based NanoElectroMechanical Systems (NEMS) and their potential applications, e.g. as drug delivery systems. INTRODUCTION Molecular shuttles, also referred to as nanotransporters, are among the best known naturally occurring biological nanomachines, performing a variety of tasks such as vesicles transport and mitosis. In this system, a motor protein (e.g., kinesin) steps along a cytoplasmic system of fibers (microtubules) employing energy harnessed by the hydrolysis of adenosine-5'- triphosphate (ATP) 1 . Recent efforts to engineer tailor-made artificial nanotransport systems in order to carry out directional transport of nanoobjects in a cell-free environment are thus hardly surprising 2 . In a typical design, ATP-fueled kinesin motor proteins are immobilized on a glass surface while microtubules are propelled over the motors 3,4 . Alternatively, molecular shuttles can be assembled mimicking the natural cell’s intracellular transport mechanism where the kinesin protein moves over microtubule tracks. Therefore, it is conceivable to direct both concepts to the development of NEMS capable of nanoscale transport of highly functional hybrid nanomaterials. In this report, we demonstrate the preparation and characterization of the two major components of such molecular shuttles, namely, kinesin and microtubule. In addition, the strategies and directions aimed at assembling synthetic nanotransport systems are addressed. EXPERIMENTAL DETAILS Protein preparations The Drosophila melanogaster DNA fragment encoding the full-length kinesin heavy- chain motor domain with the sequences of poly-histidine tag (HHHHHH) and biotin acceptor peptide (AviTag; GGLNDIFEAQKIEWH) in this order at the C-terminus was inserted in the 519