An efficient method for recombinant production of human alpha synuclein in Escherichia coli using thioredoxin as a fusion partner

Abstract

Herein, we describe a simple and efficient approach to produce recombinant human α-synuclein (hAS) with high purity from Escherichia coli (E. coli). The cDNA for hAS was inserted into plasmid pET32a and expressed in E. coli BL21 (DE3) with an N-terminal tag containing E. coli thioredoxin (trx), followed by a histidine hexapeptide, and a tobacco etch virus (TEV) protease cleavage site (trx-6His-TEV). The fusion protein, trx-hAS, was initially released by osmotic shock treatment from the host cells and subsequently purified using a nickel affinity chromatography. A TEV protease cleavage step was performed to liberate the target protein, hAS, from the fusion partner, trx. Finally, an additional nickel affinity chromatography was performed to further purify the digested product. The yield of this method is ∼25 mg of tag-less protein (with ∼99% purity) per liter of culture volume. Reverse phase HPLC (RP-HPLC) and electrospray ionization (ESI) mass spectrometry confirmed the purity and authenticity of the purified protein. Thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy demonstrated that the purified proteins form fibrils. Our protocol not only provides a convenient procedure for preparing highly pure hAS, but also requires very little specialized laboratory techniques.