Purification of the Adenomatous Polyposis Coli (APC) Gene Product

Abstract

Mutation of the adenomatous polyposis coli (APC) gene is responsible for familial adenomatous polyposis and is an etiologic factor for digestive tract malignancies. Although the APC gene product (APC) is believed to play a role in growth suppression of colonocytes, the underlying mechanism is not clear. However, recent evidence does suggest that APC is a microtubule-associated protein (MAP), and like other MAPs, it can be phosphorylated, as we have shown. To facilitate studies of APC function, we purified the APC protein. To purify the full-length APC protein, HCT116 human colon cancer cells were lysed and the particulate fraction from the lysate was extracted with ammonium sulfate followed by Sepharose 4B and DEAE-Sephacel column fractionation and then by sucrose zonal density gradient centrifugation. The final purified APC fraction was determined to be about 1000-fold enriched in APC. The availability of purified APC will be valuable in investigating possible growth-suppressing mechanisms of APC including specific sites of APC phosphorylation and APC's interaction with other cellular proteins.