Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated
Protocol

J 2024

Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol

THAKKAR, Harsh; Sayan CHATTERJEE; Purvi SAXENA; Rameswari EERLA; Sachin WAGH et al.

Základní údaje

Originální název

Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol

Autoři

THAKKAR, Harsh; Sayan CHATTERJEE; Purvi SAXENA; Rameswari EERLA; Sachin WAGH; Amit Suresh KHAIRNAR a Ravi P SHAH

Vydání

Journal of Proteome Research, Washington, American Chemical Society, 2024, 1535-3893

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 3.600

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14110/24:00139081

Organizační jednotka

Lékařská fakulta

Klíčová slova anglicky

Parkinson's disease; recombinant alpha-synuclein; protein purification; Gage R&R; analyticalcharacterization; PCA

Štítky

14110132, 14110515, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 28. 3. 2025 08:34, Mgr. Tereza Miškechová

Anotace

V originále

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.

Citovat

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, 2024, roč. 23, č. 1, s. 16-24. ISSN 1535-3893. Dostupné z: https://doi.org/10.1021/acs.jproteome.3c00190.

@article{2484645,
   author = {Thakkar, Harsh and Chatterjee, Sayan and Saxena, Purvi and Eerla, Rameswari and Wagh, Sachin and Khairnar, Amit Suresh and Shah, Ravi P},
   article_location = {Washington},
   article_number = {1},
   doi = {https://doi.org/10.1021/acs.jproteome.3c00190},
   keywords = {Parkinson's disease; recombinant alpha-synuclein; protein purification; Gage R&R; analyticalcharacterization; PCA},
   language = {eng},
   issn = {1535-3893},
   journal = {Journal of Proteome Research},
   title = {Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol},
   url = {https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00190},
   volume = {23},
   year = {2024}
}

TY  - JOUR
ID  - 2484645
AU  - Thakkar, Harsh - Chatterjee, Sayan - Saxena, Purvi - Eerla, Rameswari - Wagh, Sachin - Khairnar, Amit Suresh - Shah, Ravi P
PY  - 2024
TI  - Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol
JF  - Journal of Proteome Research
VL  - 23
IS  - 1
SP  - 16-24
EP  - 16-24
PB  - American Chemical Society
SN  - 15353893
KW  - Parkinson's disease
KW  - recombinant alpha-synuclein
KW  - protein purification
KW  - Gage R&R
KW  - analyticalcharacterization
KW  - PCA
UR  - https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00190
N2  - 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.
ER  -

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\&{}amp;R Validated Protocol. \textit{Journal of Proteome Research}. Washington: American Chemical Society, 2024, roč.~23, č.~1, s.~16-24. ISSN~1535-3893. Dostupné z: https://doi.org/10.1021/acs.jproteome.3c00190.

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