Informace o publikaci
Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol
| Autoři | |
|---|---|
| Rok publikování | 2023 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Journal of Proteome Research |
| Citace | THAKKAR, Harsh, Sayan CHATTERJEE, Purvi SAXENA, Rameswari EERLA, Sachin WAGH, Amit Suresh KHAIRNAR a Ravi P SHAH. Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol. Journal of Proteome Research. Washington: American Chemical Society, 2023, roč. 23, č. 1, s. 16-24. ISSN 1535-3893. Dostupné z: https://dx.doi.org/10.1021/acs.jproteome.3c00190. |
| www | https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00190 |
| Doi | http://dx.doi.org/10.1021/acs.jproteome.3c00190 |
| Klíčová slova | Parkinson's disease; recombinant alpha-synuclein; protein purification; Gage R&R; analyticalcharacterization; PCA |
| Popis | alpha-Synuclein (alpha-Syn) misfolding and its presence in Lewy bodies are observed in almost all Parkinson's disease (PD) patients. Basic biomedical research would benefit from a quick, low-cost approach to purifying alpha-Syn and developing in vitro and in vivo models for PD. Several research groups utilize PFF-based models, yet the production of alpha-Syn PFFs is inconsistent, resulting in nonconclusive findings. Some research laboratories prepare recombinant alpha-Syn (r alpha-Syn) by molecular cloning to overexpress alpha-Syn with various purifying techniques. Laboratory-to-laboratory protocols cause considerable variability and sometimes contradictory findings. PD researchers spend more on protein than solving alpha-Syn's riddles. This article uncovered a novel method for expressing and purifying r alpha-Syn validated through gage reproducibility and repeatability (Gage R&R). For the production of r alpha-Syn, we have employed the ability of a high-cell-density-based expression system to overexpress protein in BL21(DE3). A simple, high-throughput, nonchromatographical purification protocol has been devised to facilitate research with higher reproducibility, which was validated through Gage R&R. A crossover experimental design was utilized, and the purified protein was characterized using orthogonal high-end analytical methods, which displayed higher similarity between the isolated r alpha-Syn. Batch-to-batch variability was the least for produced protein and hence can be utilized for exploring the iceberg of PD. |