Purification and Characterization of Native Conventional Kinesin, HSET, and CENP-E from Mitotic HeLa Cells

Abstract

We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at ∼30 μm/ min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at ∼75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of ∼5 μm/min, and microtubules glide for an average of 3 μm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.