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KMID : 0613820050150060916
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Journal of Life Science
2005 Volume.15 No. 6 p.916 ~ p.922
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The Expression Patterns of Human Parkin in E. coli and Mammalian Cells
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Nam Min-Kyoung
Park Hye-Min
Choi Ju-Youn
Park Hyo-Jin
Chung Kwang-Chul
Kang Seong-Man
Rhim Hyang-Shuk
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Abstract
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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℃ 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 pGEX system at the different culture temperatures and IPTG concentrations. The intact Parkin protein was purified to approximately 90% purity with suitable amounts of protein under the optimal culture condition (25℃ 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.
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