title: One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J.H. Dunant 3, Varese, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Diego
authors.initials: D
authors.lastname: Albani
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; C497604:SNCA protein, human; D051844:alpha-Synuclein; D054322:tat Gene Products, Human Immunodeficiency Virus
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8968-73
references.pmid: 12084935
references.citation: Biotechnol Appl Biochem. 2002 Aug;36(1):33-40
references.pmid: 12149120
references.citation: J Neurosci. 2012 Oct 17;32(42):14465-77
references.pmid: 23077033
references.citation: Nature. 1997 Aug 28;388(6645):839-40
references.pmid: 9278044
references.citation: J Biol Chem. 2001 Apr 6;276(14):10737-44
references.pmid: 11152691
references.citation: J Biol Chem. 1999 Apr 2;274(14):9843-6
references.pmid: 10092675
references.citation: J Mol Biol. 2002 Oct 4;322(5):1089-102
references.pmid: 12367530
references.citation: Nat Med. 1998 Dec;4(12):1449-52
references.pmid: 9846587
references.citation: J Biol Chem. 2005 Jun 17;280(24):22572-81
references.pmid: 15817448
references.citation: Protein Expr Purif. 2005 Jul;42(1):173-7
references.pmid: 15939304
references.citation: FEBS Lett. 2006 Mar 20;580(7):1775-9
references.pmid: 16513114
references.citation: Methods. 2001 Jul;24(3):247-56
references.pmid: 11403574
references.citation: Cell. 1988 Dec 23;55(6):1189-93
references.pmid: 2849510
references.citation: J Biol Chem. 1999 Mar 19;274(12):7619-22
references.pmid: 10075647
references.citation: Autophagy. 2012 Oct;8(10):1494-509
references.pmid: 22914317
references.citation: J Biol Chem. 2001 Nov 23;276(47):44284-96
references.pmid: 11553618
references.citation: BMC Biotechnol. 2010 Apr 21;10:33
references.pmid: 20409334
references.citation: Biopolymers. 2001;62(1):15-21
references.pmid: 11135188
references.citation: Restor Neurol Neurosci. 1995 Jan 1;8(1):11-2
references.pmid: 21551797
references.citation: Adv Drug Deliv Rev. 2005 Feb 28;57(4):597-608
references.pmid: 15722166
references.citation: J Neurochem. 2010 Oct;115(1):188-99
references.pmid: 20649845
references.citation: J Bacteriol. 2007 Apr;189(7):2777-86
references.pmid: 17277073
references.citation: Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):664-8
references.pmid: 8290579
references.citation: J Neurosci. 2007 Mar 21;27(12):3338-46
references.pmid: 17376994
references.citation: Br J Pharmacol. 2005 Aug;145(8):1093-102
references.pmid: 15937518
references.citation: Cell. 1988 Dec 23;55(6):1179-88
references.pmid: 2849509
references.citation: Protein Expr Purif. 2006 Jul;48(1):112-7
references.pmid: 16564705
references.citation: Neuroscience. 2011 Nov 10;195:128-37
references.pmid: 21906659
references.citation: Exp Neurol. 2006 Jun;199(2):249-56
references.pmid: 16310772
references.citation: FASEB J. 2004 Nov;18(14):1713-5
references.pmid: 15345691
references.citation: Protein Expr Purif. 2008 Oct;61(2):131-7
references.pmid: 18586517
references.citation: Protein Sci. 2002 Feb;11(2):313-21
references.pmid: 11790841
references.citation: Clin Biochem. 1997 Aug;30(6):455-63
references.pmid: 9316739
references.citation: Protein Eng. 1993 Jun;6(4):383-90
references.pmid: 8332596
references.citation: Trends Pharmacol Sci. 2000 Feb;21(2):45-8
references.pmid: 10664605
references.citation: Biochemistry. 1996 Oct 29;35(43):13709-15
references.pmid: 8901511
references.citation: FEBS Lett. 1994 May 23;345(1):27-32
references.pmid: 8194594
references.citation: Methods Mol Med. 2005;112:81-90
references.pmid: 16010012