Home Bratislava Medical Journal 2019 Bratislava Medical Journal Vol.120, No.2, p.106–112, 2019

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Published Monthly, in English
Founded: 1919
ISSN 0006-9248
(E)ISSN 1336-0345
 

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Bratislava Medical Journal Vol.120, No.2, p.106–112, 2019

Title: Generation of a fluorescent oncoprotein in soluble form and its delivery into mammalian cells
Author: F. Motevalli, A. Khodaei, M. Anvari, A. Bolhassani

Abstract: BACKGROUND: Protein fusion technology was widely used to improve expression, purification and solubility of the recombinant proteins expressed in E. coli for in vitro/in vivo delivery.
METHODS: We developed a method for successful expression of soluble +36GFP-A2-E7 protein in E. coli and its effective delivery into mammalian cells. At first, the plasmid harboring +36GFP-A2-E7 was transformed into E. coli Rosetta competent cells. Then, the recombinant protein fused to histidine tag was expressed and purified using affinity chromatography. Different conditions such as inducer dose, time and temperature of induction, pH and urea concentration were evaluated. Finally, the delivery of the recombinant protein was detected in HEK-293T cells using fluorescent microscopy and flow cytometry.
RESULTS: Our data showed that the expressed protein formed inclusion bodies at 37 °C and 3 h post-induction. The soluble protein was generated using 0.5 mM IPTG and growth at 16 °C for 20 h, and purified by low concentrations of urea and 200 mM imidazole. The soluble fraction of +36GFP-A2-E7 protein could significantly represent higher fluorescent property and stronger delivery into mammalian cells compared to the insoluble form.
CONCLUSION: Generally, soluble form of fusion protein retained its biological activity and could directly penetrate into the cells without the fusion tags (Tab. 1, Fig. 7, Ref. 23).

Keywords: HPV, E7, +36 GFP, E. coli expression system, transfection
Published online: 17-Feb-2019
Year: 2019, Volume: 120, Issue: 2 Page From: 106, Page To: 112
doi:10.4149/BLL_2019_017


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