A Facile Method to Produce N-Terminally Truncated α-Synuclein

Abstract

α-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinson’s disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of α-synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients’ brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of α-synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of α-synuclein play an important role in the formation of the amyloids.

The N-terminal Region of α-Synuclein Controls Amyloid Nucleation at the Condensate Interface

“…Thus, increasing N-terminal truncation appears to reduce the structural stability of -syn fibrils. This result is consistent with existing evidence that truncation of the first 6 N-terminal residues reduces fibril stability against mechanical agitation and protease activity 44 . As the amyloids formed during our assay experience significant mechanical agitation due to shaking, our data indicate that residues 5 and 6 may facilitate the formation of amyloids with enhanced resistance to mechanical stress-induced fragmentation.…”

“…Protein expression and purification. FL -syn (residues 1-140) was expressed and purified as detailed previously 44 . Briefly, pT7-7 -syn FL plasmid (a gift from Hilal Lashuel, Addgene 66 ) was transformed into BL21-Gold (DE3) competent Escherichia coli (E. coli) cells (Agilent Technologies) according to the manufacturer's instructions and previous methodology 44 .…”

“…FL α-syn (residues 1-140) was expressed and purified as detailed previously 44 . Briefly, pT7-7 α-syn FL plasmid (a gift from Hilal Lashuel, Addgene 66 ) was transformed into BL21-Gold (DE3) competent Escherichia coli ( E. coli ) cells (Agilent Technologies) according to the manufacturer’s instructions and previous methodology 44 . Transformed cells were grown (37 °C, 200 rpm shaking) in LB media containing ampicillin (100 µg/ml) to an OD 600 of ∼ 0.7 before FL α-syn expression induced with 1 mM IPTG overnight (28 °C, 200 rpm shaking).…”

“…To generate 11-140 and 19-140 α-syn, deletion polymerase chain reaction (PCR) was performed on the pT7-7 Int7-140αSyn plasmid, which encodes a protein sequence where an intein is fused to the N-terminus of residues 7-140 of α-syn 44 , eliminating the need for a non-native starting Met in the protein sequences. PCR was carried out using the Q5 high-fidelity DNA polymerase (New England Biolabs), the 5’ phosphorylated primer pairs and their corresponding annealing temperatures (T a ), listed in Table 1.…”

“…The intein-α-syn fusion proteins corresponding to 11-140 and 19-140 α-syn were then expressed and the cell lysate harvested as described for FL α-syn. Subsequently, the fusion proteins were immobilized on a chitin resin before the target α-syn variant was cleaved, eluted and further purified by size-exclusion chromatography as previously described 44 .…”