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http://dx.doi.org/10.5352/JLS.2005.15.6.916

The Expression Patterns of Human Parkin in E. codi and Mammalian Cells  

Nam Min-Kyung (Department of Biomedical Sciences, Research Institute of Molecular Genetics, the Catholic University of Korea)
Park Hye-Min (School of Life Science and Biotechnology, Korea University)
Choi Ju-Youn (Research Insitute of Molecular Genetics, the Catholic University of Korea)
Park Hyo-Jin (Research Institute of Molecular Genetics, The Catholic University of Korea, School of Life Science and Biotechnology, Korea University)
Chung Kwang Chul (Department of Biology, College of Sciences, Yonsei University)
Kang Seong man (School of Life Science and Biotechnology, Korea University)
Rhim Hyangshuk (Department of Biomedical Sciences, Research Institute of Molecular Genetics, the Catholic University of Korea)
Publication Information
Journal of Life Science / v.15, no.6, 2005 , pp. 916-922 More about this Journal
Abstract
Parkin, known as an E3 ubiquitin ligase, has essential role in protein quality control, and its severe dysfunction leads to neurodegenerative disorders. Human Parkin was excessively degraded when expressed in Escherichia coli under the conventional induction condition ($37^{\circ}C$ culture condition with 0.5 mM IPTG). To optimize the induction and culture conditions for recombinant human Parkin and develop a rapid method for the Parkin purification, we expressed Parkin by using PCEX system at the different culture temperatures and IPTC concentrations. The intact Parkin protein was purified to approximately $90\%$ purity with suitable amounts of protein under the optimal culture condition ($25^{\circ}C$E with 0.01 mM IPTG). Additionally, we constructed various parkin plasmids with different tagging systems and investigated their expression patterns in HEK293 cells. We found that the proteolytically sensitive site is localized within a ubiquitin-like domain of Parkin. This study developes a method for generating useful reagents to investigate biochemical properties of Parkin.
Keywords
Parkin; Protein quality control; PEST sequence;
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