Volume 9, Issue 1 (2018)                   JMBS 2018, 9(1): 145-152 | Back to browse issues page

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Sheykhi S, Amininasab M, Saffari B, Abdi S. DNA Cloning and Expression of alpha-Synuclein Protein in E. coli. JMBS 2018; 9 (1) :145-152
URL: http://biot.modares.ac.ir/article-22-12579-en.html
1- Cell & Molecular Biology Department, Faculty Biology, University of Tehran, Tehran, Iran
2- Cell & Molecular Biology Department, Faculty Biology, University of Tehran, Tehran, Iran, Cell & Molecular Biology Department, Faculty Biology, College of Science, University of Tehran, Enghelab Street, Tehran, Iran. Postal Code: 141556658 , amininasab@khayam.ut.ac.ir
Abstract:   (5690 Views)
Aims: Identifying the structure and function of alpha-Synuclein protein can lead to the development of appropriate treatments for Parkinson disease. The aim of the current study was to investigate DNA cloning and the expression of alpha-Synuclein protein in E. coli.
Materials and Methods: In this experimental study, the sequence of encoding alpha-Synuclein in pRK172 recombinant plasmid was amplified by Polymerase Chain Reaction (PCR), using best primers. The synthesized DNA was, then, digested by restriction enzymes and cloned into pET28a and recombinant plasmid was transferred into the expression strain of E. coli (BL21) by Calcium Chloride method. The expression of alpha-Synuclein gene was induced by Isopropyl-Beta-D-Thiogalactoside (IPTG) and the expression of alpha-Synuclein was investigated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) method. Sequencing was done, using the ClustalW algorithm by the BioEdit 5.0.9 program.
Findings: In products of DNA enzymatic digestive reactions and pET28a plasmid with restriction enzymes, the size of the fragments indicated the correctness of the enzymatic reactions. The synthesized DNA and pET28a plasmid were 407 and 5369 nucleotides, respectively. The translation of the sequence of the cloned fragment revealed a 100% similarity to the human alpha-Synuclein protein. In expressing the recombinant protein in comparison with negative control samples, adding IPTG increased the expression of alpha-Synuclein protein in all samples, especially 2 hours after induction. Most of alpha-Synuclein expressed from the pET28a-alpha-Synuclein plasmid accumulated in the bacteria as incorporated objects.
Conclusion: The alpha-Synuclein protein is cloned into the pET28a plasmid and formation of the objects incorporated by alpha-Synuclein is confirmed by the expression of the pET28a-alpha-Synuclein system and paves the way for producing this protein in high scale.
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Article Type: Original Manuscript | Subject: Agricultural Biotechnology
Received: 2017/08/12 | Accepted: 2017/10/22 | Published: 2018/03/20

1. Goldstein DS, Sewell L, Holmes C. Association of anosmia with autonomic failure in Parkinson disease. Neurology. 2010;74(3):245-51. [Link] [DOI:10.1212/WNL.0b013e3181ca014c]
2. Park A, Stacy M. Non-motor symptoms in Parkinson's disease. J Neurol. 2009;256(Suppl 3):293-8. [Link] [DOI:10.1007/s00415-009-5240-1]
3. Wullner U, Schmitz-Hubsch T, Antony G, Fimmers R, Spottke A, Oertel WH, et al. Autonomic dysfunction in 3414 Parkinson's disease patients enrolled in the German Network on Parkinson's disease (KNP e.V.): The effect of ageing. Eur J Neurol. 2007;14(12):1405-8. [Link] [DOI:10.1111/j.1468-1331.2007.01982.x]
4. Reichmann H. Clinical criteria for the diagnosis of Parkinson's disease. Neurodegener Dis. 2010;7(5):284-90. [Link] [DOI:10.1159/000314478]
5. Braak H, Bohl JR, Muller CM, Rub U, de Vos RA, Del Tredici K. Stanley Fahn Lecture 2005: The staging procedure for the inclusion body pathology associated with sporadic Parkinson's disease reconsidered. Mov Disord. 2006;21:2042-51. [Link] [DOI:10.1002/mds.21065]
6. Shults CW. Lewy bodies. Proc Natl Acad Sci U S A. 2006;103(6):1661-8. [Link] [DOI:10.1073/pnas.0509567103]
7. Spillantini MG, Crowther RA, 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(11):6469-73. [Link] [DOI:10.1073/pnas.95.11.6469]
8. Olanow CW, Tatton WG. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci. 1999;22:123-44. [Link] [DOI:10.1146/annurev.neuro.22.1.123]
9. Martin I, Dawson VL, Dawson TM. Recent advances in the genetics of Parkinson's disease. Annu Rev Genomics Hum Genet. 2011;12:301-25. [Link] [DOI:10.1146/annurev-genom-082410-101440]
10. Ozansoy M, Basak AN. The central theme of Parkinson's disease: Alpha-synuclein. Mol Neurobiol. 2013;47(2):460-5. [Link] [DOI:10.1007/s12035-012-8369-3]
11. Bruinsma IB, Bruggink KA, Kinast K, Versleijen AA, Segers-Nolten IM, Subramaniam V, et al. Inhibition of alpha-synuclein aggregation by small heat shock proteins. Proteins. 2011;79(10):2956-67. [Link] [DOI:10.1002/prot.23152]
12. Buchman VL, Hunter HJ, Pinon LG, Thompson J, Privalova EM, Ninkina NN, et al. Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J Neurosci. 1998;18(22):9335-41. [Link] [DOI:10.1523/JNEUROSCI.18-22-09335.1998]
13. Lavedan C, Leroy E, Dehejia A, Buchholtz S, Dutra A, Nussbaum RL, Polymeropoulos MH. Identification, localization and characterization of the human gamma-synuclein gene. Hum Genet. 1998;103(1):106-12. [Link] [DOI:10.1007/s004390050792]
14. Maroteaux L, Scheller RH. The rat brain synucleins; family of proteins transiently associated with neuronal membrane. Brain Res Mol Brain Res. 1991;11(3):335-43. [Link] [DOI:10.1016/0169-328X(91)90043-W]
15. Shibasaki Y, Baillie DA, St Clair D, Brookes AJ. High-resolution mapping of SNCA encoding alpha-synuclein, the non-A beta component of Alzheimer's disease amyloid precursor, to human chromosome 4q21.3-->q22 by fluorescence in situ hybridization. Cytogenet Cell Genet. 1995;71(1):54-5. [Link] [DOI:10.1159/000134061]
16. Biskup S, Gerlach M, Kupsch A, Reichmann H, Riederer P, Vieregge P, et al. Genes associated with Parkinson syndrome. J Neurol. 2008;255(Suppl 5):8-17. [Link] [DOI:10.1007/s00415-008-5005-2]
17. Loov C, Scherzer CR, Hyman BT, Breakefield XO, Ingelsson M. Alpha-synuclein in extracellular vesicles: Functional implications and diagnostic opportunities. Cell Mol Neurobiol. 2016;36(3):437-48. [Link] [DOI:10.1007/s10571-015-0317-0]
18. Fink AL. The aggregation and fibrillation of alpha-synuclein. Acc Chem Res. 2006;39(9):628-34. [Link] [DOI:10.1021/ar050073t]
19. Lee MK, Stirling W, Xu Y, Xu X, Qui D, Mandir AS, et al. 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(13):8968-73. [Link] [DOI:10.1073/pnas.132197599]
20. Periquet M, Fulga T, Myllykangas L, Schlossmacher MG, Feany MB. Aggregated alpha-synuclein mediates dopaminergic neurotoxicity in vivo. J Neurosci. 2007;27(12):3338-46. [Link] [DOI:10.1523/JNEUROSCI.0285-07.2007]
21. Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, et al. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci U S A. 1993;90(23):11282-6. [Link] [DOI:10.1073/pnas.90.23.11282]
22. Neve RL, Selkoe DJ, Kurnit DM, Kosik KS. A cDNA for a human microtubule associated protein 2 epitope in the Alzheimer neurofibrillary tangle. Brain Res. 1986;387(2):193-6. [Link] [DOI:10.1016/0169-328X(86)90011-2]
23. Jakes R, Spillantini MG, Goedert M. Identification of two distinct synucleins from human brain. FEBS Lett. 1994;345(1):27-32. [Link] [DOI:10.1016/0014-5793(94)00395-5]
24. Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, et al. Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation. J Biol Chem. 1999;274(14):9843-6. [Link] [DOI:10.1074/jbc.274.14.9843]
25. Choi JY, Sung YM, Park HJ, Hur EH, Lee SJ, Hahn C, et al. 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(Pt 1):33-40. [Link] [DOI:10.1042/BA20020004]
26. Bisaglia M, Trolio A, Tessari I, Bubacco L, Mammi S, Bergantino E. Cloning, expression, purification, and spectroscopic analysis of the fragment 57-102 of human alpha-synuclein. Protein Expr Purif. 2005;39(1):90-6. [Link] [DOI:10.1016/j.pep.2004.09.018]
27. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673-80. [Link] [DOI:10.1093/nar/22.22.4673]
28. Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-8. [Link]
29. Huang C, Ren G, Zhou H, Wang CC. A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr Purif. 2005;42(1):173-7. [Link] [DOI:10.1016/j.pep.2005.02.014]
30. Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT Jr. NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry. 1996;35(43):13709-15. [Link] [DOI:10.1021/bi961799n]
31. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J Biol Chem. 2001;276(14):10737-44. [Link] [DOI:10.1074/jbc.M010907200]
32. Hashimoto M, Hsu LJ, Sisk A, Xia Y, Takeda A, Sundsmo M, et al. Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease. Brain Res. 1998;799(2):301-6. [Link] [DOI:10.1016/S0006-8993(98)00514-9]
33. De Groot NS, Ventura S. Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett. 2006;580(27):6471-6. [Link] [DOI:10.1016/j.febslet.2006.10.071]
34. Iljina, M, Garcia GA, Horrocks MH, Tosatto, L, Choi, ML, Ganzinger KA, et al. Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading. Proc Natl Acad Sci U S A. 2016;113(9):E1206-15. [Link] [DOI:10.1073/pnas.1524128113]
35. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;(17)5:172. [Link] [DOI:10.3389/fmicb.2014.00172]

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