Recombinant production in baculovirus-infected insect cells and purification of the mammalian Sec13/Sec31 complex.

Membrane traffic along the eukaryotic secretory pathway starts with the selective packing of biosynthetic cargo into nascent vesicles that are forming on the endoplasmic reticulum (ER). This process is mediated by the coat protein complex II (COPII) machinery, which at the minimum, comprises the Sar1 GTPase and the cytosolic protein complexes Sec23/Sec24 (Sec23/24) and Sec13/Sec31 (Sec13/31). While the components of the basic COPII machinery are highly conserved from yeast to human, it is now clearly evident that the overall process is under tighter spatial and temporal regulation in higher eukaryotes. Here we describe recombinant production in baculovirus-infected insect cells and subsequent purification to homogeneity of the mammalian Sec13/31 complex for biochemical and biophysical characterization.