Expression and purification of recombinant human Parkin and pSer65 Parkin v1

Mutations in PARK2 encoding Parkin are causal for early-onset Parkinson’s disease. Parkin is a ubiquitin E3 ligase and is activated by the PINK1 kinase to stimulate ubiquitin-dependent removal of damaged mitochondria via autophagy (mitophagy). The ability to generate recombinant Parkin in its inactive unmodified conformation and activated phosphorylated conformation represent important tools for studying the its regulation by PINK1-dependent phosphorylation as well as understanding how disease mutations impact on its function. This protocol describes the expression and purification of full-length human untagged Parkin along with how to make the phosphorylated protein. In cells the removal of malfunctioning organelles is important for cellular survival. Damaged mitochondria are removed via mitophagy upon activation of the PTEN induced kinase 1 (PINK1) / Parkin pathway. In healthy mitochondria PINK1 is cleaved by PARL and released into the cytoplasm where it is degraded by the N-end rule. In damaged mitochondria depolarisation of the mitochondrial membrane stabilises PINK1, causing it to accumulate on the outer mitochondrial membrane. Stabilised PINK1 phosphorylates Ser65 on both ubiquitin and the ubiquitin like (Ubl) domain of Parkin, this activated Parkin. Active Parkin ubiquitinates outer mitochondrial membrane proteins, leading to ubiquitin chains containing pSer65 ubiquitin. The formation of these chains on the mitochondria identifies it for removal by mitophagy. Loss of activity in either Parkin or PINK1 have been observed in cases of early-onset Parkinson’s disease. Parkin is a RING-in-between-RING (RBR) E3 ubiquitin ligase containing a ubiquitin like (UBL) domain, three RING domains (referred to as RING0, RING1, and RING2 domains), and an in between ring (IBR) domain. Unmodified Parkin exists in an autoinhibited conformation, and phosphorylation of Ser65 on the Ubl domain along with binding to pSer65 ubiquitin causes a significant conformational change that activates Parkin. Previous studies have shown that a small purification tag on the N-terminus of Parkin can lead to constitutive activation, making the production of untagged Parkin an important tool in the study of its regulation and ubiquitylation activity. We describe a protocol for the generation of untagged recombinant Parkin using a His-SUMO tagged fusion construct. In addition we describe the additional steps to produce phospho-Parkin. The yield of Parkin produced varies between 2 and 6 mg, and this is 1/3 lower for pSer65 Parkin. For pSer65 Parkin the stoichiometry of phosphorylation can vary from 60 to 80 %.