Abstract
HsEg5 is a homotetrameric BimC/kinesin‐5 family member that plays a vital role in the mitotic spindle. This crucial role has identified HsEg5 as a promising target for cancer chemotheraphy.The aim of a project is to clarify the role of the sequence regions in kinetic mechanism of HsEg5 and to determine the various conformational states in which this protein occurs while performing its biological function. Intein‐mediated protein ligation (IPL) was used as a tool for segmental labeling of proteins of interest. Based on various criteria including the presence of cysteine required for peptide bound formation (native or artificially introduced), the breaking points for IPL were designed, so that the separately expressed fragments of protein could be joined together. The rebuilt protein has been obtained in a very high yield compared to values reported in the literature. The resulting IPL product reconstructed from fragments exhibits catalytic activity of native enzyme, however, due to the amino acid exchange (substitution for cysteine), the activity differs from that of wild type enzyme. The segmentally labeled protein is then subjected to analysis by Fourier‐Transform Infrared Spectroscopy (FTIR).
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