2022
DOI: 10.3390/biom12020324
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Production of Recombinant Alpha-Synuclein: Still No Standardized Protocol in Sight

Abstract: Synucleinopathies are a group of neurodegenerative diseases, characterized by the abnormal accumulation of the protein alpha-synuclein (aSyn). aSyn is an intrinsically disordered protein that can adopt different aggregation states, some of which may be associated with disease. Therefore, understanding the transitions between such aggregation states may be essential for deciphering the molecular underpinnings underlying synucleinopathies. Recombinant aSyn is routinely produced and purified from E. coli in many … Show more

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Cited by 8 publications
(10 citation statements)
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“…To produce labeled protein, we twitched the media composition, wherein 14 N ammonium chloride was replaced by 15 N ammonium chloride as a source of nitrogen. The protocol followed for the expression of the labeled protein was the same as mentioned in the previous section.…”
Section: Expression Of Labeled α-Syn Using Metabolic Labelingmentioning
confidence: 99%
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“…To produce labeled protein, we twitched the media composition, wherein 14 N ammonium chloride was replaced by 15 N ammonium chloride as a source of nitrogen. The protocol followed for the expression of the labeled protein was the same as mentioned in the previous section.…”
Section: Expression Of Labeled α-Syn Using Metabolic Labelingmentioning
confidence: 99%
“…Hence, the same protocol can be used for producing labeled α-Syn, which can be used for absolute quantification. Metabolic labeling utilizes the ability of the bacteria to take up 15 N from the culture media as a source of nitrogen. During the translation process,…”
Section: Expression and Purification Of α-Synucleinmentioning
confidence: 99%
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“…Currently, recombinant αSyn is produced and purified from E. coli in many research laboratories. 30 The strategies reported comprise the whole cell extract obtained by sonication, with subsequent protein precipitation by ammonium sulfate or acidic pH to eliminate most of the cytoplasm proteins followed by dialysis and different types of chromatography, resulting in up to 96% of purity. 31 Other methods include fusion to a protein partner to improve purification such as the glutathione S-transferase (GST) system, which is used for purification using a glutathione-Sepharose 4B column.…”
Section: Purification and Confirmation Of Rltb-synmentioning
confidence: 99%