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. 2002 Nov 25;99(25):16058–16063. doi: 10.1073/pnas.252409199

Fig 1.

Fig 1.

Purification of heterodimeric kinesin. (a) Coexpression vector for generating heterodimeric kinesin. The two heavy chains in a kinesin heterodimer contain distinct motor domains attached to two different carrier sequences. The DNA fragments encoding the heavy chains A and B are placed side by side in a pGEX-2T-based vector. (b) The strategy for purifying heterodimeric kinesin. Three kinds of dimeric kinesins (A-B heterodimer, A-A homodimer, and B-B homodimer) form spontaneously via the coiled-coil regions. The heterodimeric kinesin with two different tags is separated from the homodimeric kinesins with two identical tags by the two-step affinity purification. (c) The SDS/PAGE patterns during the purification of WT/WT-His, after purification with glutathione Sepharose 4B/glutathione (lane 1), digestion by thrombin (lane 2), purification with Ni-NTA agarose/imidazole (lane 3), and removal of residual GST and GST-tagged protein with glutathione beads (lane 4). Lane 5 shows the WT dimer prepared by the same method but without digestion of GST (GST-WT/WT-His). The positions of K432 with a GST-tag and with a His-tag are indicated by an asterisk and double asterisks, respectively, and the cleaved GST is indicated by an arrowhead.