Cloning, expression, purification and characterization of the alternate splice Src variants for drug discovery

Protein tyrosine kinases (PTKs) are key members of intra- and extra-cellular signaling pathways. Aberrant signaling pathways are responsible for many human diseases, making these enzymes targets for drug development programs. The difficulty in PCR-amplification of Src due to the high G-C content was overcome using a commercial “G-C melt” reagent. The N06 Src was cloned along with the N12 and N23 neuronal variants. Neuronal variants of Src occur due to splicing within the N-loop of the SH3 domain. These variants have greater catalytic activity. Affinity purification methodologies were established that takes advantage of binding sites within the SH1 and SH2 domains. The purified enzyme is stable, without loss of activity for >1 year when frozen and more than 1 week at 4°C. A 96-well solution phase assay was developed and validated that overcomes many of the false positives and negatives generated by other assays. Studies of the catalytic mechanism have indicated that a second metal ion is essential for catalysis. Some transition metals can be substituted for the second metal ion and maintain activity while others act as dead-end inhibitors with binding constants in the sub-micromolar range. The precise role of this second metal ion is being studied.