title: Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran ; Student s Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Heidari
authors.affiliation: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran ; Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Vahidi
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Jaleh
authors.initials: J
authors.lastname: Barar
authors.affiliation: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran ; Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
authors.affiliation: Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Naderi-Manesh
keywords: Ascorbate extraction buffer; Endoplasmic expression; Molecular farming; Nicotiana tabacum; TRAIL