Expression, purification and functional analysis of advenovirus type 5 e4 orf3 protein

Koyuncu, Emre
In this study, structural and functional aspects of adenovirus type 5 (Ad5) E4orf3 protein were analyzed by biophysical and biochemical methods. Ad5 is one of the mostly used gene therapy vectors to date. However, some of its proteins possess oncogenic potential and their presence comprises safety risks. E4orf3 is one of the oncoproteins of Ad5. It also takes important roles in viral infection, and is beneficial for therapy vectors. Therefore, understanding the functions of E4orf3 is very important for developing efficient and safe adenovirus vectors. Most of the present knowledge about the functions of E4orf3 comes from its mutational analysis. It has never been expressed or purified successfully due to its extreme insolubility. Therefore, this study focused on the optimization of expression of E4orf3 protein. As a result, full-length E4orf3 was obtained in soluble form as a Glutathione-S-transferase (GST) fusion protein and purified by GST affinity chromatography for the first time. Subsequently, the interaction of E4orf3 with four different proteins, DNA-PK, Aup1, E1B-55 kDa and E4orf6 was analyzed in detail by GST-pulldown technique. In these experiments, E4orf3 was shown to associate with Aup1, E1B-55 kDa and E4orf6 in vitro, and the C-terminal of E4orf3 was determined to be responsible for these interactions. Finally, basic structural information about E4orf3 protein was also obtained for the first time by the direct analysis of the fusion protein in glutathione beads with Fourier Transform Infrared (FTIR) spectroscopy. Since the purified E4orf3 protein could not be separated from the glutathione beads due to its hydrophobic regions, the secondary structures in this protein were determined after subtracting glutathione and H2O absorption bands, and the GST moiety.


Determination of Autophagy in the Caco-2 Spontaneously Differentiating Model of Intestinal Epithelial Cells
Tunçer, Sinem; Banerjee, Sreeparna (Springer New York, 2017)
The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the...
Expression Analysis of TaNAC69-1 and TtNAMB-2, Wheat NAC Family Transcription Factor Genes Under Abiotic Stress Conditions in Durum Wheat (Triticum turgidum)
Baloglu, Mehmet Cengiz; Oz, Mehmet Tufan; Öktem, Hüseyin Avni; Yücel, Ayşe Meral (2012-10-01)
NAC-type plant-specific transcription factor genes encode proteins that play important roles in abiotic stress responses, as well as regulation of plant development. In the current study, expression profiles of wheat NAC-type transcription factor genes, TaNAC69-1 and TtNAMB-2, were examined under drought, salt, cold, and heat stress conditions in wheat. Based on reverse transcription quantitative PCR results, TaNAC69-1 was strongly expressed under drought, salinity, and high-temperature stress conditions. C...
Alterations of ceramide/sphingosine 1-phosphate rheostat involved in the regulation of resistance to imatinib-induced apoptosis in K562 human chronic myeloid leukemia cells
Baran, Yusuf; Salas, Arelis; Senkal, Can E.; Gündüz, Ufuk; Bielawski, Jacek; Obeid, Lina M.; Ogretmen, Besim (2007-04-13)
In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed similar to 2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C-18-ceramide, which is generated ...
Synthesis and Stimuli-Responsive Properties of Metallo-Supramolecular Phosphazene Polymers Based on Terpyridine Metal Complexes
In this study, terpyridine functionalized phosphazene based metallo-supramolecular polymers were synthesized by three step reaction method. In the first step, 4 '-(4-aminophenyl)-2,2 ':6 ',2 ''-terpyridine was synthesized with p-nitro benzaldehyde and 2-acetylpyridine. Then, the monomer containing six terpyridine (TPY) units attached to the phosphazene was prepared from 4 '-(4-aminophenyl)-2,2 ':6 ',2 ''-terpyridine and hexachlorocyclotriphosphazene by a condensation reaction. Finally, metallo-supramolecula...
Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens
Arinc, E; Kocabıyık, Semra; Su, E (2001-02-01)
In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5 ' -dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3 ' was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positi...
Citation Formats
E. Koyuncu, “Expression, purification and functional analysis of advenovirus type 5 e4 orf3 protein,” M.S. - Master of Science, Middle East Technical University, 2004.