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Articles

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

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  • Spillantini, M. G.; Crowther, R. A.; Jakes, R.; Hasegawa, M.; Goedert, M. Alpha-Synuclein in Filamentous Inclusions of Lewy Bodies from Parkinson’s Disease and Dementia with Lewy Bodies. Proc. Natl. Acad. Sci. U S A. 1998, 95, 6469–6473. DOI: 10.1073/pnas.95.11.6469.
  • Spillantini, M. G.; Schmidt, M. L.; Lee, V. M.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Alpha-Synuclein in Lewy Bodies. Nature 1997, 388, 839–840. DOI: 10.1038/42166.
  • Cookson, M. R. Alpha-Synuclein and Neuronal Cell Death. Mol. Neurodegener. 2009, 4, 9. DOI: 10.1186/1750-1326-4-9.
  • Bendor, J. T.; Logan, T. P.; Edwards, R. H. The Function of Alpha-Synuclein. Neuron 2013, 79, 1044–1066. DOI: 10.1016/j.neuron.2013.09.004.
  • Rodriguez, J. A.; Ivanova, M. I.; Sawaya, M. R.; Cascio, D.; Reyes, F. E.; Shi, D.; Sangwan, S.; Guenther, E. L.; Johnson, L. M.; Zhang, M.; et al. Structure of the Toxic Core of Alpha-Synuclein from Invisible Crystals. Nature 2015, 525, 486–490. DOI: 10.1038/nature15368.
  • Lee, M. K.; Stirling, W.; Xu, Y.; Xu, X.; Qui, D.; Mandir, A. S.; Dawson, T. M.; Copeland, N. G.; Jenkins, N. A.; Price, D. L. Human Alpha-Synuclein-Harboring Familial Parkinson’s Disease-Linked Ala-53 –> Thr Mutation Causes Neurodegenerative Disease with Alpha-Synuclein Aggregation in Transgenic Mice. Proc. Natl. Acad. Sci. U S A. 2002, 99, 8968–8973. DOI: 10.1073/pnas.132197599.
  • Periquet, M.; Fulga, T.; Myllykangas, L.; Schlossmacher, M. G.; Feany, M. B. Aggregated Alpha-Synuclein Mediates Dopaminergic Neurotoxicity in Vivo. J. Neurosci. 2007, 27, 3338–3346. DOI: 10.1523/JNEUROSCI.0285-07.2007.
  • Chen, R. Bacterial Expression Systems for Recombinant Protein Production: E. coli and Beyond. Biotechnol. Adv. 2012, 30, 1102–1107. DOI: 10.1016/j.biotechadv.2011.09.013.
  • Yonemoto, I. T.; Wood, M. R.; Balch, W. E.; Kelly, J. W. A General Strategy for the Bacterial Expression of Amyloidogenic Peptides Using BCL-XL-1/2 Fusions. Protein Sci. 2009, 18, 1978–1986. DOI: 10.1002/pro.211.
  • Sharpe, S.; Yau, W. M.; Tycko, R. Expression and Purification of a Recombinant Peptide from the Alzheimer’s Beta-Amyloid Protein for Solid-State NMR. Protein Expr. Purif. 2005, 42, 200–210. DOI: 10.1016/j.pep.2005.03.005.
  • Sorensen, H. P.; Mortensen, K. K. Advanced Genetic Strategies for Recombinant Protein Expression in Escherichia coli. J. Biotechnol. 2005, 115, 113–128. DOI: 10.1016/j.jbiotec.2004.08.004.
  • LaVallie, E. R.; DiBlasio-Smith, E. A.; Collins-Racie, L. A.; Lu, Z.; McCoy, J. M. Thioredoxin and Related Proteins as Multifunctional Fusion Tags for Soluble Expression in E. coli. Methods Mol. Biol. 2003, 205, 119–140. DOI: 10.1385/1-59259-301-1:119.
  • Lunn, C. A.; Kathju, S.; Wallace, B. J.; Kushner, S. R.; Pigiet, V. Amplification and Purification of Plasmid-Encoded Thioredoxin from Escherichia coli K12. J. Biol. Chem. 1984, 259, 10469–10474.
  • Lunn, C. A.; Pigiet, V. P. Localization of Thioredoxin from Escherichia coli in an Osmotically Sensitive Compartment. J. Biol. Chem. 1982, 257, 11424–11430.
  • Tropea, J. E.; Cherry, S.; Waugh, D. S. Expression and Purification of Soluble His(6)-Tagged TEV Protease. Methods Mol. Biol. 2009, 498, 297–307. DOI: 10.1007/978-1-59745-196-3_19.
  • Shevchik, V. E.; Condemine, G.; Robert-Baudouy, J. Characterization of DsbC, a Periplasmic Protein of Erwinia chrysanthemi and Escherichia coli with Disulfide Isomerase Activity. Embo J. 1994, 13, 2007–2012. DOI: 10.1002/j.1460-2075.1994.tb06470.x.
  • de Marco, A. Two-Step Metal Affinity Purification of Double-Tagged (NusA-His6) Fusion Proteins. Nat. Protoc. 2006, 1, 1538–1543. DOI: 10.1038/nprot.2006.289.
  • Van Meerloo, J.; Kaspers, G. J.; Cloos, J. Cell Sensitivity Assays: The MTT Assay. In Cancer Cell Culture. Springer, Berlin, Germany, 2011; pp. 237–245.
  • Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. DOI: 10.1016/0003-2697(76)90527-3.
  • Choi, J. Y.; Sung, Y. M.; Park, H. J.; Hur, E. H.; Lee, S. J.; Hahn, C.; Min, B. R.; Kim, I. K.; Kang, S.; Rhim, H. Rapid Purification and Analysis of Alpha-Synuclein Proteins: C-Terminal Truncation Promotes the Conversion of Alpha-Synuclein into a Protease-Sensitive Form in Escherichia coli. Biotechnol. Appl. Biochem. 2002, 36, 33–40. DOI: 10.1042/BA20020004.
  • Hu, H. Y.; Li, Q.; Cheng, H. C.; Du, H. N. Beta-Sheet Structure Formation of Proteins in Solid State as Revealed by Circular Dichroism Spectroscopy. Biopolymers 2001, 62, 15–21. DOI: 10.1002/1097-0282(2001)62:1<15::AID-BIP30>3.0.CO;2-J.
  • Fu, X. Y.; Tong, W. Y.; Wei, D. Z. Extracellular Production of Human Parathyroid Hormone as a Thioredoxin Fusion Form in Escherichia coli by Chemical Permeabilization Combined with Heat Treatment. Biotechnol. Prog. 2005, 21, 1429–1435. DOI: 10.1021/bp050137z.
  • Caldinelli, L.; Albani, D.; Pollegioni, L. One Single Method to Produce Native and Tat-Fused Recombinant Human Alpha-Synuclein in Escherichia coli. BMC Biotechnol. 2013, 13, 32. DOI: 10.1186/1472-6750-13-32.
  • Huang, C.; Ren, G.; Zhou, H.; Wang, C. C. A New Method for Purification of Recombinant Human Alpha-Synuclein in Escherichia coli. Protein Expr. Purif. 2005, 42, 173–177. DOI: 10.1016/j.pep.2005.02.014.
  • McNulty, B. C.; Young, G. B.; Pielak, G. J. Macromolecular Crowding in the Escherichia coli Periplasm Maintains Alpha-Synuclein Disorder. J. Mol. Biol. 2006, 355, 893–897. DOI: 10.1016/j.jmb.2005.11.033.
  • Waudby, C. A.; Camilloni, C.; Fitzpatrick, A. W.; Cabrita, L. D.; Dobson, C. M.; Vendruscolo, M.; Christodoulou, J. In-Cell NMR Characterization of the Secondary Structure Populations of a Disordered Conformation of Alpha-Synuclein Within E. coli Cells. PLOS One 2013, 8, e72286. DOI: 10.1371/journal.pone.0072286.
  • Barnes, C. O.; Pielak, G. J. In-Cell Protein NMR and Protein Leakage. Proteins 2011, 79, 347–351. DOI: 10.1002/prot.22906.
  • Slade, K. M.; Baker, R.; Chua, M.; Thompson, N. L.; Pielak, G. J. Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion in Escherichia coli. Biochemistry 2009, 48, 5083–5089. DOI: 10.1021/bi9004107.
  • Oliveira, C.; Domingues, L. Guidelines to Reach High-Quality Purified Recombinant Proteins. Appl. Microbiol. Biotechnol. 2018, 102, 81–92. DOI: 10.1007/s00253-017-8623-8.
  • Singh, S. M.; Panda, A. K. Solubilization and Refolding of Bacterial Inclusion Body Proteins. J. Biosci. Bioeng. 2005, 99, 303–310. DOI: 10.1263/jbb.99.303.
  • Uversky, V. N.; Eliezer, D. Biophysics of Parkinson’s Disease: Structure and Aggregation of Alpha-Synuclein. CPPS. 2009, 10, 483–499. DOI: 10.2174/138920309789351921.
  • Correddu, D.; Montano Lopez, J. J.; Vadakkedath, P. G.; Lai, A.; Pernes, J. I.; Watson, P. R.; Leung, I. K. H. An Improved Method for the Heterologous Production of Soluble Human Ribosomal Proteins in Escherichia coli. Sci. Rep. 2019, 9, 8884. DOI: 10.1038/s41598-019-45323-8.
  • LaVallie, E. R.; DiBlasio, E. A.; Kovacic, S.; Grant, K. L.; Schendel, P. F.; McCoy, J. M. A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm. Nat. Biotechnol. 1993, 11, 187–193. DOI: 10.1038/nbt0293-187.
  • Giasson, B. I.; Uryu, K.; Trojanowski, J. Q.; Lee, V. M. Mutant and Wild Type Human Alpha-Synucleins Assemble into Elongated Filaments with Distinct Morphologies in Vitro. J. Biol. Chem. 1999, 274, 7619–7622. DOI: 10.1074/jbc.274.12.7619.
  • Kloepper, K. D.; Woods, W. S.; Winter, K. A.; George, J. M.; Rienstra, C. M. Preparation of Alpha-Synuclein Fibrils for Solid-State NMR: Expression, Purification, and Incubation of Wild-Type and Mutant Forms. Protein Expr. Purif. 2006, 48, 112–117. DOI: 10.1016/j.pep.2006.02.009.
  • Masuda, M.; Dohmae, N.; Nonaka, T.; Oikawa, T.; Hisanaga, S.; Goedert, M.; Hasegawa, M. Cysteine Misincorporation in Bacterially Expressed Human Alpha-Synuclein. FEBS Lett. 2006, 580, 1775–1779. DOI: 10.1016/j.febslet.2006.02.032.
  • Narhi, L.; Wood, S. J.; Steavenson, S.; Jiang, Y.; Wu, G. M.; Anafi, D.; Kaufman, S. A.; Martin, F.; Sitney, K.; Denis, P.; et al. Both Familial Parkinson’s Disease Mutations Accelerate Alpha-Synuclein Aggregation. J. Biol. Chem. 1999, 274, 9843–9846. DOI: 10.1074/jbc.274.14.9843.
  • Ueda, K.; Fukushima, H.; Masliah, E.; Xia, Y.; Iwai, A.; Yoshimoto, M.; Otero, D. A.; Kondo, J.; Ihara, Y.; Saitoh, T. Molecular Cloning of cDNA Encoding an Unrecognized Component of Amyloid in Alzheimer Disease. PNAS. 1993, 90, 11282–11286. DOI: 10.1073/pnas.90.23.11282.
  • Uversky, V. N.; Li, J.; Fink, A. L. Evidence for a Partially Folded Intermediate in Alpha-Synuclein Fibril Formation. J. Biol. Chem. 2001, 276, 10737–10744. DOI: 10.1074/jbc.M010907200.
  • Weinreb, P. H.; Zhen, W.; Poon, A. W.; Conway, K. A.; Lansbury, P. T. Jr. NACP, a Protein Implicated in Alzheimer’s Disease and Learning, is Natively Unfolded. Biochemistry 1996, 35, 13709–13715. DOI: 10.1021/bi961799n.
  • Coelho-Cerqueira, E.; Carmo-Gonçalves, P.; Pinheiro, A. S.; Cortines, J.; Follmer, C. α-Synuclein as an Intrinsically Disordered Monomer-Fact or Artefact? FEBS J. 2013, 280, 4915–4927. DOI: 10.1111/febs.12471.
  • Hoyer, W.; Antony, T.; Cherny, D.; Heim, G.; Jovin, T. M.; Subramaniam, V. Dependence of Alpha-Synuclein Aggregate Morphology on Solution Conditions. J. Mol. Biol. 2002, 322, 383–393. DOI: 10.1016/S0022-2836(02)00775-1.
  • Hwang, P. M.; Pan, J. S.; Sykes, B. D. Targeted Expression, Purification, and Cleavage of Fusion Proteins from Inclusion Bodies in Escherichia coli. FEBS Lett. 2014, 588, 247–252. DOI: 10.1016/j.febslet.2013.09.028.
  • Ponzini, E.; De Palma, A.; Cerboni, L.; Natalello, A.; Rossi, R.; Moons, R.; Konijnenberg, A.; Narkiewicz, J.; Legname, G.; Sobott, F.; et al. Methionine Oxidation in α-Synuclein Inhibits Its Propensity for Ordered Secondary Structure. J. Biol. Chem. 2019, 294, 5657–5665. DOI: 10.1074/jbc.RA118.001907.
  • Zhou, W.; Long, C.; Reaney, S. H.; Di Monte, D. A.; Fink, A. L.; Uversky, V. N. Methionine Oxidation Stabilizes Non-Toxic Oligomers of Alpha-Synuclein Through Strengthening the Auto-Inhibitory Intra-Molecular Long-Range Interactions. Biochim. Biophys. Acta 2010, 1802, 322–330. DOI: 10.1016/j.bbadis.2009.12.004.
  • Maltsev, A. S.; Chen, J.; Levine, R. L.; Bax, A. Site-Specific Interaction Between Alpha-Synuclein and Membranes Probed by NMR-Observed Methionine Oxidation Rates. J. Am. Chem. Soc. 2013, 135, 2943–2946. DOI: 10.1021/ja312415q.
  • Falsone, S. F.; Leitinger, G.; Karner, A.; Kungl, A. J.; Kosol, S.; Cappai, R.; Zangger, K. The Neurotransmitter Serotonin Interrupts Alpha-Synuclein Amyloid Maturation. Biochim. Biophys. Acta 2011, 1814, 553–561. DOI: 10.1016/j.bbapap.2011.02.008.
  • Volles, M. J.; Lansbury, P. T. Jr. Relationships Between the Sequence of Alpha-Synuclein and Its Membrane Affinity, Fibrillization Propensity, and Yeast Toxicity. J. Mol. Biol. 2007, 366, 1510–1522. DOI: 10.1016/j.jmb.2006.12.044.
  • Burre, J.; Sharma, M.; Sudhof, T. C. Systematic Mutagenesis of Alpha-Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities. J. Neurosci. 2012, 32, 15227–15242. DOI: 10.1523/JNEUROSCI.3545-12.2012.
  • Diao, J.; Burre, J.; Vivona, S.; Cipriano, D. J.; Sharma, M.; Kyoung, M.; Sudhof, T. C.; Brunger, A. T. Native Alpha-Synuclein Induces Clustering of Synaptic-Vesicle Mimics via Binding to Phospholipids and Synaptobrevin-2/VAMP2. Elife 2013, 2, e00592. DOI: 10.7554/eLife.00592.
  • Zhao, M.; Cascio, D.; Sawaya, M. R.; Eisenberg, D. Structures of Segments of Alpha-Synuclein Fused to Maltose-Binding Protein Suggest Intermediate States during Amyloid Formation. Protein Sci. 2011, 20, 996–1004. DOI: 10.1002/pro.630.
  • Giehm, L.; Lorenzen, N.; Otzen, D. E. Assays for Alpha-Synuclein Aggregation. Methods 2011, 53, 295–305. DOI: 10.1016/j.ymeth.2010.12.008.
  • Li, Y. Carrier Proteins for Fusion Expression of Antimicrobial Peptides in Escherichia coli. Biotechnol. Appl. Biochem. 2009, 54, 1–9. DOI: 10.1042/BA20090087.
  • McCoy, J.; La Ville, E. Expression and Purification of Thioredoxin Fusion Proteins. Curr. Protoc. Protein Sci. 2001, Chapter 6, Unit 6.7.
  • Venkataraman, G.; Goswami, M.; Tuteja, N.; Reddy, M. K.; Sopory, S. K. Isolation and Characterization of a Phospholipase C Delta Isoform from Pea That is Regulated by Light in a Tissue Specific Manner. Mol. Genet. Genomics 2004, 270, 378–386. DOI: 10.1007/s00438-003-0925-0.
  • De Wilde, G.; Mertens, N.; Boone, E.; De Vreese, B.; Van Beeumen, J.; Fiers, W.; Haegeman, G. Expression in Escherichia coli of the Death Domain of the Human p55 Tumor Necrosis Factor Receptor. Protein Expr. Purif. 2001, 23, 226–232. DOI: 10.1006/prep.2001.1499.
  • Guo, Q. R.; Wei, D. Z.; Tong, W. Y. Partial Purification of Human Parathyroid Hormone 1-84 as a Thioredoxin Fusion Form in Recombinant Escherichia coli by Thermoosmotic Shock. Protein Expr. Purif. 2006, 49, 32–38. DOI: 10.1016/j.pep.2006.03.004.
  • Tenno, T.; Goda, N.; Tateishi, Y.; Tochio, H.; Mishima, M.; Hayashi, H.; Shirakawa, M.; Hiroaki, H. High-Throughput Construction Method for Expression Vector of Peptides for NMR Study Suited for Isotopic Labeling. Protein Eng. Des. Sel. 2004, 17, 305–314. DOI: 10.1093/protein/gzh044.
  • Ajouz, B.; Berrier, C.; Garrigues, A.; Besnard, M.; Ghazi, A. Release of Thioredoxin via the Mechanosensitive Channel MscL During Osmotic Downshock of Escherichia coli Cells. J. Biol. Chem. 1998, 273, 26670–26674. DOI: 10.1074/jbc.273.41.26670.
  • Berrier, C.; Garrigues, A.; Richarme, G.; Ghazi, A. Elongation Factor Tu and DnaK Are Transferred from the Cytoplasm to the Periplasm of Escherichia coli During Osmotic Downshock Presumably via the Mechanosensitive Channel MscL. J. Bacteriol. 2000, 182, 248–251. DOI: 10.1128/JB.182.1.248-251.2000.
  • el Yaagoubi, A.; Kohiyama, M.; Richarme, G. Localization of DnaK (Chaperone 70) from Escherichia coli in an Osmotic-Shock-Sensitive Compartment of the Cytoplasm. J. Bacteriol. 1994, 176, 7074–7078. DOI: 10.1128/JB.176.22.7074-7078.1994.
  • Ewis, H. E.; Lu, C. D. Osmotic Shock: A Mechanosensitive Channel Blocker Can Prevent Release of Cytoplasmic but Not Periplasmic Proteins. FEMS Microbiol. Lett. 2005, 253, 295–301. DOI: 10.1016/j.femsle.2005.09.046.
  • Vazquez-Laslop, N.; Lee, H.; Hu, R.; Neyfakh, A. A. Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli. J. Bacteriol. 2001, 183, 2399–2404. DOI: 10.1128/JB.183.8.2399-2404.2001.
  • Hoarau, M.; Malbert, Y.; Irague, R.; Hureau, C.; Faller, P.; Gras, E.; André, I.; Remaud-Siméon, M.; Remaud-Simeon, M. A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-Beta Peptides. PLOS One 2016, 11, e0161209. DOI: 10.1371/journal.pone.0161209.
  • Meisl, G.; Yang, X.; Frohm, B.; Knowles, T. P.; Linse, S. Quantitative Analysis of Intrinsic and Extrinsic Factors in the Aggregation Mechanism of Alzheimer-Associated Abeta-Peptide. Sci. Rep. 2016, 6, 18728. DOI: 10.1038/srep18728.
  • Powers, A. E.; Patel, D. S. Expression and Purification of Untagged α-Synuclein. In Alpha-Synuclein. Springer, 2019; pp. 261–269. DOI: 10.1007/978-1-4939-9124-2_2.
  • Volpicelli-Daley, L. A.; Luk, K. C.; Lee, V. M. Addition of Exogenous α-Synuclein Preformed Fibrils to Primary Neuronal Cultures to Seed Recruitment of Endogenous α-Synuclein to Lewy Body and Lewy Neurite–like Aggregates. Nat. Protoc. 2014, 9, 2135–2146. DOI: 10.1038/nprot.2014.143.
  • Chandra, S.; Chen, X.; Rizo, J.; Jahn, R.; Sudhof, T. C. A Broken Alpha -Helix in Folded alpha -Synuclein. J. Biol. Chem. 2003, 278, 15313–15318. DOI: 10.1074/jbc.M213128200.
  • Zhao, J.; Liang, Q.; Sun, Q.; Chen, C.; Xu, L.; Ding, Y.; Zhou, P. (−)-Epigallocatechin-3-Gallate (EGCG) Inhibits Fibrillation, Disaggregates Amyloid Fibrils of α-Synuclein, and Protects PC12 Cells against α-Synuclein-Induced Toxicity. RSC Adv. 2017, 7, 32508–32517. DOI: 10.1039/C7RA03752J.
  • Burre, J.; Sharma, M.; Sudhof, T. C. Definition of a Molecular Pathway Mediating Alpha-Synuclein Neurotoxicity. J. Neurosci. 2015, 35, 5221–5232. DOI: 10.1523/JNEUROSCI.4650-14.2015.
  • Burre, J.; Sharma, M.; Sudhof, T. C. alpha-Synuclein Assembles into Higher-Order Multimers upon Membrane Binding to Promote SNARE Complex Formation. Proc. Natl. Acad. Sci. USA. 2014, 111, E4274–4283. DOI: 10.1073/pnas.1416598111.
  • Xicoy, H.; Wieringa, B.; Martens, G. J. The SH-SY5Y Cell Line in Parkinson’s Disease Research: A Systematic Review. Mol. Neurodegener. 2017, 12, 10. DOI: 10.1186/s13024-017-0149-0.

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