Open Access Research article

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

Laura Caldinelli12, Diego Albani3 and Loredano Pollegioni12*

Author Affiliations

1 Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, via J.H. Dunant 3, Varese, Italy

2 The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano, ICRM – CNR Milano, and Università degli Studi dell’Insubria, via Mancinelli 7, Milano, Italy

3 Dipartimento di Neuroscienze, Unità di Genetica delle Malattie Neurodegenerative, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, via La Masa 19, Milano, Italy

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BMC Biotechnology 2013, 13:32  doi:10.1186/1472-6750-13-32

Published: 4 April 2013



Human α-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 α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein.


A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-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 α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range.


To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-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 α-synuclein than previously described procedures.

α-Synuclein; TAT-fusion protein; Recombinant proteins; Parkinson’s disease; Oxidative stress; Protein aggregation