Details

Autor(en) / Beteiligte

• Janarthanam, Chelva
• Clabaugh, Griffin
• Wang, Zerui
• Melvin, Bradley R
• Scheibe, Ileia
• Jin, Huajun
• Anantharam, Vellareddy
• Urbauer, Ramona J B
• Urbauer, Jeffrey L
• Ma, Jiyan
• Kanthasamy, Arthi
• Huang, Xuemei
• Donadio, Vincenzo
• Zou, Wenquan
• Kanthasamy, Anumantha G

Titel

High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility

Ist Teil von

• International journal of molecular sciences, 2024-06, Vol.25 (11), p.5988

Ort / Verlag

Switzerland: MDPI AG

Erscheinungsjahr

2024

Quelle

MEDLINE

Beschreibungen/Notizen

• Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 , lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.