One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

Background: Human alpha-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that alpha-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of alpha-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate alpha-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-alpha-synuclein. Results: A reliable protocol was designed to efficiently express and purify two different forms of human alpha-synuclein. The synthetic cDNAs encoding for the native alpha-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (>= 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant alpha-synuclein protein forms could be purified in a single step to >= 95% purity. Both alpha-synuclein recombinant proteins form fibrils and the TAT-alpha-synuclein is also cytotoxic in the micromolar concentration range. Conclusions: To further characterize the molecular mechanisms of alpha-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular alpha-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant alpha-synuclein than previously described procedures.