Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli

Document Type : Research Article

Authors

1 . Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran

2 . Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran

3 . Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;4. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Scien

4 . Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

5 5. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

Abstract

Objective
The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified. Conclusion: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.

Keywords

Cell Journal (Yakhteh)
Volume 14, Issue 4
February 2013
Pages 264-269

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