Rapid purification and analysis of alpha-synuclein proteins: C-terminal truncation promotes the conversion of alpha-synuclein into a protease-sensitive form in Escherichia coli.
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 alpha-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 alpha-synuclein proteins in suitable forms for biochemical studies of their functions. The wild-type alpha-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 alpha-synuclein. With the exception of the alpha-synuclein protein that was truncated at amino acid residue 95, all domain constructs of alpha-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 alpha-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 alpha-synuclein regulates the pathogenesis of PD.
Choi
Ju-Youn
JY
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.
Sung
Young-Mo
YM
Park
Hyo-Jin
HJ
Hur
Eun-Hye
EH
Lee
Sun-Joo
SJ
Hahn
Chul
C
Min
Byung-Re
BR
Kim
In-Kyung
IK
Kang
Seongman
S
Rhim
Hyangshuk
H
D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051843:Synucleins; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D051843:Synucleins; D051844:alpha-Synuclein; D005982:Glutathione Transferase