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Cloning, Expression, and in Vitro Functional Activity Assay of Phic31 Integrase Cdna in Escherichia Coli



Sekhavati MH1 ; Tahmoorespur M1 ; Ghaedi K2, 3 ; Dormiani K2, 4 ; Nassiri MR1 ; Khazaie Y2 ; Foruzanfar M2 ; Hosseini M5 ; Hossein M2, 5 ; Esfahani N2, 5
Authors
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Authors Affiliations
  1. 1. Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran
  2. 2. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, P.O.Box: 8165131378, Iran
  3. 3. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran
  4. 4. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
  5. 5. Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

Source: Cell Journal Published:2013

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.
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