Cell Journal (Jan 2013)

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

  • Mohammad Hadi Sekhavati,
  • Mojtaba Tahmoorespur,
  • Kamran Ghaedi,
  • Kianoush Dormiani, Pharm,
  • Mohammad Reza Nassiri,
  • Yahya Khazaie,
  • Mahboubeh Foruzanfar,
  • Morteza Hosseini,
  • Mohammad Hossein Nasr Esfahani

Journal volume & issue
Vol. 14, no. 4
pp. 264 – 269

Abstract

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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 purifi ed 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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