Browsing by Author "Ozcan, Dilek"

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Pokeweed (Phytolacca Americana L.) Antiviral Protein Inhibits Zucchini Yellow Mosaic Virus Infection in a Dose-Dependent Manner in Squash Plants
(Tubitak Scientific & Technological Research Council Turkey, 2017) Sipahioglu, Hikmet Murat; Kaya, Ilhan; Usta, Mustafa; Unal, Murat; Ozcan, Dilek; Ozer, Meryem; Pallas, Vicente
Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 mu g mL(-1) concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed.
Simultaneous Production of Alpha and Beta Amylase Enzymes Using Separate Gene Bearing Recombinant Vectors in the Same Escherichia Coli Cells
(Tubitak Scientific & Technological Research Council Turkey, 2020) Ozcan, Dilek; Sipahioglu, Hikmet Murat
The present study describes the simultaneous expression of thermostable industrial alpha (alpha) and beta (beta) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for alpha amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for beta amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ alpha Amy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/beta Amy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified alpha and beta amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified alpha and beta amylase were calculated as 4.59 mu g/mL and 3.17 mu g/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable alpha and beta amylases at the same E coli cells containing separate engineered plasmid vectors.