46] Purification of functional Sec13p–Sec31p complex, a subunit of COPII coat

Abstract

This chapter describes the purification of the Sec13p-Sec31p complex that employs a hexahistidine (His 6 )-tagged version of Sec31p. This procedure requires two purification steps and yields approximately 3 mg of pure Sec13p-Sec31p from a 15-liter culture of yeast cells. To determine the activity of purified Secl3p-Sec31p and monitor the yield through each purification step, a cell-free assay that depends on this protein complex is employed. The assay is based on an in vitro budding reaction that measures the release of radiolabeled yeast secretory protein, 35 S-labeled glyco-pro-α-factor, into endoplasmic reticulum-derived COPII vesicles. Using this assay, the specific activity of Secl3p-Sec31p complex is determined at each stage of the purification. In this preparation, a 287-fold purification is obtained and 67% of the activity contained in the starting cytosol is recovered. This compares favorably with a previous method that used a Secl3- dihydrofolate reductase fusion protein where a 3.5% recovery is reported.