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 (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). 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 kcat. Active-site measurements using [alpha-32P]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 microM tubulin; Km,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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gilbert
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SP
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Johnson
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D016547:Kinesins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; C036852:magnesium GTP; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D016547:Kinesins