PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8-15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous expression and purification has slowed progress in the AR-PD research field. To pave the way for direct structural study of this important protein, in this study, we developed an efficient expression system of recombinant PINKI kinase domain (rPINK1) using Pichia pastoris (P. pastoris). Our results showed that rPINK1 is best expressed in P. pastoris at 25 degrees C induction. Additionally, we determined that the optimal induction time was 72 h and the optimal induction methanol concentration was 1% for the expression of rPINK1 in P. pastoris. Subsequent purification by Ni affinity chromatography (Ni-NTA) and cation-exchange chromatography (Mono S) produced the protein with purity higher than 95%. The pure rPINK1 was active to phosphorylate ubiquitin in a substrate phosphorylation assay. Overall, these studies provide the first effective method for heterologous expression and purification of the rPINK1 with a high purity. These findings can help contribute to further researches on the interactions study and biochemical characterization of PINKI. (C) 2016 Elsevier Inc. All rights reserved.