Ps-4: Cloning and Expression of PhiC31 Integrase cDNA in Escherichia coli and Test of its In vitro Activity Using an Intra-Molecular Assay Vector (Pages: 0-0)

Sekhavati MH *, Tahmoorespur M , Ghaedi K , Khazaie Y , Nassiri MR , Dormiani K , Forouzanfar M ,


Objective: Gene therapy and creation of transgenic animals are two main areas of research which is growing too fast recently. In these fields, there is a serious need for integration into safe locations viewing not to destroy the other genes or important sites in the target genome. Applying site specific integrase such as phage phiC31 is one of the best tools for this approach. Phage phiC31 performs recombination between some specific attachment sites on the phage DNA and its bacterial host genome, known as attP and attB, respectively. There are lots of reports which state that there are some site-specific regions in the mammalian genomes similar to attP. These sites are known as pseudo attP. There is possibility of producing transgenic animals and performing safer gene therapy studies by using phiC31 integrase to integrate the gene of interest in to pseudo attP. The objective of the present study is to clone the phiC31 integrase cDNA in a bacterial expression vector and to express it in E.coli. After purification of integrase protein, we tried to test its in vitro activity by a molecular assay vector. This molecular assay vector contains both attP and attB sites in correct orientations. There is a specific fragment between these two sites in this vector. If phiC31 integrase acts properly, it can do a recombination in this molecular assay vector leading to delete the specific DNA fragment located between attP and attB in the vector. This deletion can be determined easily by PCR. Materials and Methods: cDNA of phiC31 was amplified and cloned in an bacterial expression vector. The construct was expressed in E.coli. BL21 (DE3). Produced integrase was purified under non-denaturing condition using Ni2+ agarose columns. Its concentration was determined by Bradford assay and the related size was confirmed by SDS-PAGE analysis. The purified protein was concentrated and stored at -70◦C. The molecular assay plasmid was incubated with 1µg purified integrase in a reaction buffer for 1 hour at 37°C. A portion of this reaction was transferred to a PCR reaction mixture as template in order to investigate the desired deletion showing proper activity of the produced integrase. Results: SDS-PAGE analysis of the purified phiC31 integrase illustrates the expected size of the protein (65 kDa). Moreover, amplification of a 401-bp band confirmed the correct functionality of purified phiC31 integrase. Conclusion: The results of this study indicated that the purified integrase has a great potential of in vitro site-specific integration. This protein could be useful as a means of genetic engineering in some studies such as protein microinjection in oocyte in the aim of creation of transgenic animal or gene therapy.