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:: Volume 17, Issue 6 (Iranian South Medical Journal 2015) ::
Iran South Med J 2015, 17(6): 1168-1175 Back to browse issues page
Cloning and expression of aequorin photoprotein using intein tag
Elah sadat Seyed Hosseini1, Mehdi Zeinoddini 2, Zeinoddini Fallah1, Alireza Saeedinia1
1- Department of Bioscience and Biotechnology, Mallek Ashtar University of Technology, Tehran, IRAN
2- Department of Bioscience and Biotechnology, Mallek Ashtar University of Technology, Tehran, IRAN , zeinoddini52@mut.ac.ir
Abstract:   (3584 Views)

Background: Intein (INT), is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn’t require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated.

Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by electrophoresis, western blotting and sequencing.

Results: The photoprotein aequorin was cloned into SapI/PstI restriction site of pTYB21 plasmid accurately and successfully. Aequorin- INT hybrid protein expression confirmed using traditional methods.

Conclusion: The photoprotein aequorin constract in fused with INT confirmed by molecular methods. Also rate of Aequorin- INT expression determined about %25 of cell total protein.

Keywords: Intein, Protein splicing, Cloning, Expression, Aequorin photoprotein
Full-Text [PDF 670 kb]   (1325 Downloads)    
Type of Study: Original | Subject: Biochemistry. Cell Biology and Genetics
Received: 2014/12/18 | Accepted: 2014/12/18 | Published: 2014/12/18
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Seyed Hosseini E S, Zeinoddini M, Fallah Z, Saeedinia A. Cloning and expression of aequorin photoprotein using intein tag. Iran South Med J. 2015; 17 (6) :1168-1175
URL: http://ismj.bpums.ac.ir/article-1-633-en.html


Volume 17, Issue 6 (Iranian South Medical Journal 2015) Back to browse issues page
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