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

(Pages: 264-269)
Mohammad Hadi Sekhavati, M.Sc., 1Mojtaba Tahmoorespur, Ph.D., 1Kamran Ghaedi, Ph.D., 2,3,*Kianoush Dormiani, Pharm.D., 2,4Mohammad Reza Nassiri, Ph.D., 1Yahya Khazaie, Pharm.D., 2Mahboubeh Foruzanfar, B.Sc., 2Morteza Hosseini, D.V.M., 5Mohammad Hossein Nasr Esfahani, Ph.D., 2,5,*
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
3. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran
4. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
5. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
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
3. Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran
4. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
5. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
* Corresponding Address: P.O.Box: 8165131378 Department of Molecular Biotechnology at Cell Science Research Center Royan Institute for Biotechnology ACECR IsfahanIran Emails:kamranghaedi@royaninstitute.org mh_nasr@royaninstitute.org
Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sekhavati Mohammad Hadi, Tahmoorespur Mojtaba, Ghaedi Kamran, Dormiani Kianoush, Nassiri Mohammad Reza, Khazaie Yahya, Foruzanfar Mahboubeh, Hosseini Morteza, Nasr Esfahani Mohammad Hossein. Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli. Cell J. 2013; 14(4): 264-269.

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.