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.
Highlights
Synucleinopathies are a group of neurodegenerative diseases, characterized by the abnormal accumulation of the protein alpha-synuclein. aSyn is an intrinsically disordered protein that can adopt different aggregation states, some of which may be associated with disease
It is the main component of Lewy bodies and Lewy neurites, pathological hallmarks of Parkinsons disease (PD) and related synucleinopathies. aSyn is considered to be intrinsically disordered in its native state [4,10,11,12]
Monomeric recombinant aSyn is incubated in a well-defined manner to generate aggregated species consisting primarily of aSyn amyloid fibrils
Summary
ASyn is defined as a natively unfolded protein, it is of high importance to keep in mind that it exists as a dynamic ensemble of conformations Some of these conformations are more aggregation prone than others. These conformations are affected by the surrounding environment which can affect the equilibrium among this dynamic ensemble of conformations [50] It is still largely unknown how the different purification methods, and how variations within a certain method can affect the final equilibrium of the different conformations. A recently published systematic comparison of aSyn isolated via boiling, acid precipitation, ammonium sulfate precipitation, or periplasmic lysis revealed that the aggregation propensity is affected by the isolation method [51]. Variations in Purification Protocols Can Change the Outcome of Downstream Applications
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