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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14110%2F24%3A00139081" target="_blank" >RIV/00216224:14110/24:00139081 - isvavai.cz</a>

Výsledek na webu

<a href="https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00190" target="_blank" >https://pubs.acs.org/doi/10.1021/acs.jproteome.3c00190</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acs.jproteome.3c00190" target="_blank" >10.1021/acs.jproteome.3c00190</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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&amp;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&amp;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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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&amp;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&amp;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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Proteome Research

ISSN

1535-3893

e-ISSN

1535-3907

Svazek periodika

23

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

16-24

Kód UT WoS článku

001137570700001

EID výsledku v databázi Scopus

2-s2.0-85179151181