Title of article :
Cloning, Expression, and In Vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
Author/Authors :
Sekhavati، Mohammad Hadi نويسنده Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran , , Tahmoorespur، Mojtaba نويسنده Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran , , Ghaedi، Kamran نويسنده , , Dormiani، Kianoush نويسنده Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran , , Nassiri، Mohammad Reza نويسنده Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran , , Khazaie، Yahya نويسنده , , Foruzanfar، Mahboubeh نويسنده Department of Molecular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran , , Hosseini، Morteza نويسنده Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran , , NASR ESFAHANI، MOHAMMAD HOSSEIN نويسنده ,
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.
