Hamid Reza Heidari
1,2, Mojgan Bandehpour
3,4, Hossein Vahidi
1, Jaleh Barar
5 
, Bahram Kazemi
3,4*, Hossein Naderi-Manesh
6*1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Student᾽s Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
6 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Introduction: In order to employ Nicotiana tabacum
cells as a profitable natural bioreactor for production of
bio-functional “Soluble human TRAIL” (ShTRAIL), endoplasmic reticulum
(ER) targeted expression and innovative extraction procedures were
exploited.
Methods: At first, the ShTRAIL encoding gene
was sub-cloned into designed H2 helper vector to equip it with potent
TMV omega leader sequences, ER sorting signal peptide, poly-histidine
tag and ER retention signal peptide (KDEL). Then, the ER targeted
ShTRAIL cassette was sequentially sub-cloned into “CaMV-35S” helper and
“pGreen-0179” final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum.
Next, the ShTRAIL protein was extracted through both phosphate and
innovative ascorbate extraction buffers. Subsequently, oligomerization
state of the ShTRAIL was evaluated through cross-linking assay and
western blot analysis. Then, semi-quantitative western blot analysis was
performed to estimate the ShTRAIL production. Finally, biological
activity of the ShTRAIL was evaluated through MTT assay.
Results: The phosphate buffer extracted
ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with
ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy
increased the ShTRAIL’s production level up to about 20 μg/g of fresh
weight of N. tabacum. MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line.
Conclusion: Endoplasmic reticulum expression
and reductive ascorbate buffer extraction procedure can be employed to
enhance the stability and overall production level of bio-functional
recombinant ShTRAIL from transgenic N. tabacum cells.