title: Expression and purification of the kinase domain of PINK1 in Pichia pastoris.
abstract: 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 PINK1 kinase domain (rPINK1) using Pichia pastoris (P. pastoris). Our results showed that rPINK1 is best expressed in P. pastoris at 25 °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 PINK1.
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Daichao
authors.initials: D
authors.lastname: Wu
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Lingzhi
authors.initials: L
authors.lastname: Qu
authors.affiliation: Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Longying
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Guo
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China.
authors.forename: Zhuchu
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China; Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA. Electronic address: linchen@usc.edu.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.affiliation: Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, XiangYa Hospital, Central South University, Changsha, Hunan 410008, China; Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA. Electronic address: yonghenc@163.com.
authors.forename: Yongheng
authors.initials: Y
authors.lastname: Chen
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D015870:Gene Expression; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000099202:PTEN-Induced Putative Kinase
chemical_list: D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D000099202:PTEN-Induced Putative Kinase
keywords: Heterologous expression; PINK1; Parkinson's disease; Pichia pastoris; Ubiquitin