Abstract B121: Influence of affinity tags on the biochemical properties of recombinant human tyrosine kinase ALK.

Abstract

Anaplastic lymphoma kinase (ALK) is a human receptor tyrosine kinase which has important functions in the development and maintenance of the peripheral and central nervous system. It has also been shown to be one causative agent in the development of several human malignancies including neuroblastoma, anaplastic lymphoma and non small cell lung cancer (NSCLC). One mechanism of pathological activation of ALK occurs by chromosomal translocation of the ALK gene to EML4 giving rise to an abnormal fusion protein. In order to develop small molecule inhibitors targeting ALK, in-vitro kinase assays have been developed which use recombinant ALK fragments fused to affinity tags to facilitate purification. Here we present data which demonstrate that biochemical parameters of recombinant ALK fusion proteins, like substrate specificity, ATP Km, Vmax and effects of chemical compounds, are significantly influenced by the type of affinity tag used. That influence partially persists even after cleavage of the affinity tag during the purification process. Since affinity-tagged recombinant proteins are widely used in drug discovery, a careful evaluation of potential tag-related effects should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B121.