Volume 9, Issue 1 (2018)
JMBS 2018, 9(1): 145-152 |
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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
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
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 ,
Abstract: (6925 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.
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.
Keywords: Parkinson Disease, Alpha- Synuclein, Cloning, Polymerase Chain Reaction, Genetic Engineering
Article Type: Original Manuscript |
Subject:
Agricultural Biotechnology
Received: 2017/08/12 | Accepted: 2017/10/22 | Published: 2018/03/20
Received: 2017/08/12 | Accepted: 2017/10/22 | Published: 2018/03/20
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