KLP-3 is a C-terminal kinesin-like protein in nematode C.elegans , which shares extensive homology with yeast Kar3 and Drosophila ncd motor. These kinesins have previously been shown to mediate chromosomal movement and segregation during mitosis and meiosis. Overexpression of
klp-3 gene causes production of dead eggs and reduction in the number of males due to non-disjunction of the X-chromosome, suggesting, that this kinesin may be involved in chromosome movement and segregation and specially in the movement of the sex chromosome during meiosis. Antisense
klp-3 RNA expression causes many developmental abnormalities in the progeny, suggesting again a critical role for the
klp-3 function in chromosome segregation. Determination of the rate and direction of motility and steady state kinetic properties of KLP-3 may be important for elucidating its function in vivo . The
klp-3 full-length cDNA was subcloned in a bacterial expression vector and expressed as 6xHis-tag protein and a truncated version as GST-fusion protein in E.coli cells (BL-21 pLysS). The 6x His-tagged full-length protein obtained from the bacterial cell lysate was purified over Ni-NTA (Qiagen) affinity column and Mono-Q ion exchange column (Pharmacia). Microtubule binding assay was carried out with the full-length and the truncated GST-fusion protein of KLP-3. It was found that both the fragments bound specifically to the taxol-polymerised tubulin and released from MT at 10mM ATP but required additional 100mM KCl, which is consistent with the data of other C-terminal kinesin, ncd, in Drosophila . Work is in progress to determine the local conformation of ATP binding site, the MT-activated steady state ATPase activity and the motor properties of KLP-3.