EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE DROSOPHILA KINESIN MOTOR DOMAIN PRODUCED IN ESCHERICHIA-COLI

The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (k(cat) = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (k(cat) = 10 s-1; K0.5,MT = 0.9 muM tubulin; K(m,ATP) = 31 muM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase k(cat). Active-site measurements using [alpha-P-32]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 muM tubulin; K(m,GTP) = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules. Furthermore, we found that truncation of the kinesin heavy chain to approximately 400 amino acids was necessary to purify the milligram amounts of active motor domain necessary for further mechanistic and structural studies to establish the basis for force production.