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 laboratories, and in vitro preparations of aSyn aggregated species became central for modeling neurodegeneration in cell and animal models. Thus, reproducibility and reliability of such studies largely depends on the purity and homogeneity of aSyn preparations across batches and between laboratories. A variety of different methods are in use to produce and purify aSyn, which we review in this commentary. We also show how extraction buffer composition can affect aSyn aggregation, emphasizing the importance of standardizing protocols to ensure reproducibility between different laboratories and studies, which are essential for advancing the field.

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

“…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.…”

“…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,…”

“…Tiago Fleming Outeiro and the group have published an article summarizing the unsolved challenges in producing consistent α-Syn. 15 The majority of the protocols lack reproducibility and robustness; a compilation of methodologies used to date, along with their limitation, has been given in Table 1. For establishing isolation protocol, a few critical points must be considered, for example, selecting an expression system resulting in higher yields, extraction, and purification protocols, which will impact the form, amount, and purity of the protein obtained.…”

“…In both cases, variations in produced r α-Syn have been observed and pointed out in a few research studies. Tiago Fleming Outeiro and the group have published an article summarizing the unsolved challenges in producing consistent α-Syn . The majority of the protocols lack reproducibility and robustness; a compilation of methodologies used to date, along with their limitation, has been given in Table .…”

Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies

“…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.…”