کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020157 1542313 2016 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression and purification of the kinase domain of PINK1 in Pichia pastoris
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression and purification of the kinase domain of PINK1 in Pichia pastoris
چکیده انگلیسی


• The kinase domain of PINK1 was successfully expressed in Pichia pastoris.
• The induction conditions of recombinant PINK1 transformant were optimized.
• Recombinant PINK1 was obtained after two steps purification.
• The purity of purified recombinant PINK1 was higher than 95%.
• Recombinant PINK1 was able to phosphorylate ubiquitin.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 128, December 2016, Pages 67–72
نویسندگان
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