Using protein purification to isolate constructs of the Glo3 protein
Cell functioning relies on the package and delivery of macromolecular resources to various compartments via vesicle-dependent shuttling systems. The success of this trafficking scheme depends on the viability of various regulatory proteins such as ADP-ribosylation factors (Arfs). Arfs are a family of GTPases that, when activated by guanine nucleotide exchange factors (GEFs), associate with specific membranes and interact with other protein components to form coat complexes and promote vesicle formation. These proteins rely on GTPase-activating proteins (GAPs) to stimulate them and initiate GTP hydrolysis. Glo3 and Gcs1 are yeast ArfGAPs critical to retrograde transfer of materials from the Golgi to the ER via COPI coated vesicles. Inactivation of Glo3 leads to impairment of the cell, but breakdown of both Glo3 and Gcs1 leads to cell death. Although both proteins overlap in function, Glo3 is a component of COPI-coated vesicles and is thought to be more relevant to COPI trafficking. However, we don’t fully comprehend Glo3’s definitive roles in vesicle formation. We hope to study Glo3 and investigate which regions specifically interact with the β’-COPI subunit, and how specific interactions relate to coat assembly and disassembly. Furthermore, we hope to map Glo3 biochemically and understand the functions of its domains. In order to study Glo3 and identify the rules by which it functions, we have to successfully isolate and purify it. Using common expression lab protocols, we transform E.coli competent cells with Glo3 constructs, then express and harvest them for subsequent purification. We then use cell-disrupting techniques, followed by stages including centrifugation and affinity purification, to isolate the proteins and prepare them for use.