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.
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.
Bioimpacts. 2014;4(3):123-32. doi: 10.15171/bi.2014.005. Epub 2014 May 17.
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.
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.
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.
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.
为了利用烟草细胞作为生产生物功能“可溶性人 TRAIL”（ShTRAIL）的有利的天然生物反应器，利用了内质网（ER）靶向表达和创新的提取程序。
首先，将 ShTRAIL 编码基因亚克隆到设计的 H2 辅助载体中，为其配备强大的 TMV omega 启动子序列、ER 分拣信号肽、多组氨酸标签和 ER 保留信号肽（KDEL）。然后，将 ER 靶向 ShTRAIL 盒依次亚克隆到“CaMV-35S”辅助和“pGreen-0179”最终表达载体中。之后，采用农杆菌介导的转化方法将 ShTRAIL 在烟草细胞的 ER 中表达。接下来，通过磷酸盐和创新抗坏血酸提取缓冲液提取 ShTRAIL 蛋白。随后，通过交联实验和 Western blot 分析评估 ShTRAIL 的寡聚状态。然后，进行半定量 Western blot 分析以估计 ShTRAIL 的产量。最后，通过 MTT 测定评估 ShTRAIL 的生物活性。
磷酸盐缓冲液提取的 ShTRAIL 以暗淡的形式产生，而抗坏血酸缓冲液提取的 ShTRAIL 则产生三聚体形式。ER 靶向 ShTRAIL 策略将 ShTRAIL 的产量提高到约 20μg/g 新鲜烟草细胞。MTT 测定表明，抗坏血酸缓冲液提取的 ShTRAIL 可以抑制 A549 细胞系的增殖。
内质网表达和还原型抗坏血酸缓冲液提取程序可用于提高来自转基因烟草细胞的生物功能重组 ShTRAIL 的稳定性和整体产量水平。
