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Biotechnology and Applied Biochemistry (2002) 36, (33–40) (Printed in Great Britain)

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Rapid purification and analysis of a-synuclein proteins: C-terminal truncation promotes the conversion of a-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‡ and Hyangshuk Rhim*¹

*Research Institute of Molecular Genetics, Catholic Research Institutes of Medical Science, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-701, South Korea, †Department of Biology, Sangmyung University, Seoul 110-743, South Korea, ‡Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea, and §Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, South Korea

Key words: Lewy body, non-amyloid-b component (NAC), neuronal cytoplasmic inclusion, Parkinson's disease, pGEX expression system.

Abbreviations used: AD, Alzheimer's disease; NAC, non-amyloid-b component of AD amyloid plaques; PD, Parkinson's disease; FPD, familial PD; GST, glutathione S-transferase; IPTG, isopropyl b-D-thiogalactoside; LB, Lewy body; BD, DNA-binding domain.

¹To whom correspondence should be addressed (e-mail hrhim@catholic.ac.kr).

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 a-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 a-synuclein proteins in suitable forms for biochemical studies of their functions. The wild-type a-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 a-synuclein. With the exception of the a-synuclein protein that was truncated at amino acid residue 95, all domain constructs of a-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 a-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 a-synuclein regulates the pathogenesis of PD.

Received 17 January 2002/12 April 2002; accepted 16 May 2002

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