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

Preparative Biochemistry & Biotechnology
Babak SaffariJamshid Davoodi

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 conveni...Continue Reading

References

Dec 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·K UédaT Saitoh
Aug 28, 1997·Nature·M G SpillantiniM Goedert
May 30, 1998·Proceedings of the National Academy of Sciences of the United States of America·M G SpillantiniM Goedert
Mar 27, 1999·The Journal of Biological Chemistry·L NarhiM Citron
Jan 22, 2001·The Journal of Biological Chemistry·V N UverskyA L Fink
Oct 26, 2001·Protein Expression and Purification·G De WildeG Haegeman
Jun 27, 2002·Proceedings of the National Academy of Sciences of the United States of America·Michael K LeeDon L Price
Sep 10, 2002·Journal of Molecular Biology·Wolfgang HoyerVinod Subramaniam
Dec 21, 2002·Methods in Molecular Biology·Edward R LaVallieJohn M McCoy
Feb 15, 2003·The Journal of Biological Chemistry·Sreeganga ChandraThomas C Südhof
May 29, 2004·Protein Engineering, Design & Selection : PEDS·Takeshi TennoHidekazu Hiroaki
Dec 21, 2004·Journal of Biotechnology·Hans Peter Sørensen, Kim Kusk Mortensen
Jun 9, 2005·Protein Expression and Purification·Chunjuan HuangChih-chen Wang
Oct 20, 2005·Journal of Bioscience and Bioengineering·Surinder Mohan Singh, Amulya Kumar Panda
Dec 14, 2005·Journal of Molecular Biology·Brian C McNultyGary J Pielak
Mar 4, 2006·FEBS Letters·Masami MasudaMasato Hasegawa
Mar 23, 2007·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Magali PeriquetMel B Feany
Apr 23, 2008·Current Protocols in Protein Science·J McCoy, E La Ville
Nov 7, 2008·Methods in Molecular Biology·Joseph E TropeaDavid S Waugh
Feb 6, 2009·Molecular Neurodegeneration·Mark R Cookson
Jun 23, 2009·Current Protein & Peptide Science·Vladimir N Uversky, David Eliezer
Jul 7, 2009·Biotechnology and Applied Biochemistry·Yifeng Li
Jul 22, 2009·Protein Science : a Publication of the Protein Society·Isaac T YonemotoJeffery W Kelly
Dec 2, 2010·Proteins·Christopher O Barnes, Gary J Pielak
Dec 18, 2010·Methods : a Companion to Methods in Enzymology·Lise GiehmDaniel E Otzen
Mar 8, 2011·Biochimica Et Biophysica Acta·S Fabio FalsoneKlaus Zangger
Apr 5, 2011·Protein Science : a Publication of the Protein Society·Minglei ZhaoDavid Eisenberg
Apr 26, 2011·Methods in Molecular Biology·Johan van MeerlooJacqueline Cloos
Oct 27, 2012·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Jacqueline BurréThomas C Südhof
Feb 13, 2013·Journal of the American Chemical Society·Alexander S MaltsevAd Bax

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Methods Mentioned

BETA
transmission
circular dichroism
PCR
electrophoresis
Assay
size-exclusion chromatography
gel-filtration
ion-exchange
thermal treatment
size exclusion chromatography

Software Mentioned

ImageJ
GraphPad PRISM

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