Cloning, Expression, and Characterization of GFP:XKCM1 Deletion Constructs

Abstract

Kinesins are a type of motor protein that use the energy of ATP hydrolysis to move along microtubules. Kinesin Catastrophe Modulator One (XKCM1) of Xenopus laevis was recently identified as a kinesin that is essential for the establishment and maintenance of mitotic spindles by affecting the microtubule dynamics in Xenopus egg extracts. In the present research, three different deletion constructs of XKCM1 fused with Green Flourescent Protein (GFP) were cloned, expressed, and characterized in order to determine which part of the XKCM1 structure was necessary for proper destabilization of microtubules. These deletion constructs have previously been studied z'n vivo without GFP, and the GFP:XKCM1 (187-592) construct acted as the minimal domain—that is, the shortest construct that still retained the destabilization ability of the full length protein. I hypothesized that the deletion construct GFP:XKCM1 (187-592) would again act as the minimal domain. The results of my microtubule destabilization assays did not support this hypothesis.