Rapid purification and analysis of α-synuclein proteins: C-terminal truncation promotes the conversion of α-synuclein into a protease-sensitive form in Escherichia coli

Ju Youn Choi, Young Mo Sung, Hyo Jin Park, Eun Hye Hur, Sun Joo Lee, Chul Hahn, Byung Re Min, In Kyung Kim, Seongman Kang, Hyangshuk Rhim

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons and the formation of eosinophilic intracytoplasmic inclusion bodies known as Lewy bodies. Although α-synuclein is known to be a pivotal factor implicated in the pathogenesis of PD, its function remains to be elucidated. We used the pGEX expression system to develop a simple and rapid method for purifying α-synuclein proteins in suitable forms for biochemical studies of their functions. The wild-type α-synuclein protein was overexpressed in Escherichia coli and purified to approx. 80% purity with relatively high yields. We also used this expression system to investigate the expression pattern of the various domains of α-synuclein. With the exception of the α-synuclein protein that was truncated at amino acid residue 95, all domain constructs of α-synuclein were purified at similar levels with relatively high yields. Unexpectedly, removal of amino acid residues 96-140 in the C-terminal acidic region of α-synuclein promotes its conversion to a protease-sensitive form during expression and purification in E. coli. Our study suggests a method for generating useful reagents to investigate the molecular mechanism by which α-synuclein regulates the pathogenesis of PD.

Original languageEnglish
Pages (from-to)33-40
Number of pages8
JournalBiotechnology and Applied Biochemistry
Volume36
Issue number1
DOIs
Publication statusPublished - 2002

Keywords

  • Lewy body
  • Neuronal cytoplasmic inclusion
  • Non-amyloid-β component (NAC)
  • Parkinson's disease
  • pGEX expression system

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Molecular Medicine
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Drug Discovery
  • Process Chemistry and Technology

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