Fig. 6. Purification and analysis of His-tagged CfNek from Crithidia. (A) Purification of His-CfNek over a Ni-column. 10% polyacrylamide gel stained with silver of the thrombin eluate from wild-type (WT) or CfNek-expressing cells. Note that a contaminating triple band of about 98 kDa is present in both eluates while only the CfNek eluate contains the expected 56 kDa polypeptide. (B) 5 µl aliquots of the thrombin eluates from A were assayed for tubulin polyglutamylation activity. Only the CfNek eluate displays significant tubulin polyglutamylation activity. (C) 5 µl aliquots of the thrombin eluates from A were used in kinase assays with ß-casein as substrate. The kinase reactions were run on an SDS-gel and analyzed by autoradiography. Again, only the CfNek eluate catalyses the incorporation of radioactve phosphate into ß-casein. (D) 5 µl aliquots of the CfNek eluate from A were used in different glutamylation assays with the indicated components. Glutamylation activity is only observed with CfNek, tubulin and ATP in the reaction mixture.

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