Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum

Abstract

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable.

Keywords: Agrobacterium tumefaciens; Molecular Farming; Nicotiana tabacum; Recombinant protein; TRAIL.

Figure 1

Schematic diagram of soluble human TRAIL cloning into intermediate 35S CaMV vector and pGreen-0179 plant expression vector. Panel A) represents cloning of TRAIL into BamHI and SacI regions of 35S-CaMV plasmid to obtain 35S promoter-TR inserted-CaMV polyA (1200 bp) fragment. Panel B) represents cloning of 35S promoter-TR inserted-CaMV polyA fragment into EcoRV region of the pGreen-0179 T-DNA to obtain final pGreen-TRAIL vector. LB and RB (Left border/right border) stands for left border and right border respectively.

Figure 2

Confirmation of soluble human TRAIL PCR products using electrophoresis on 2% Agarose gel. Panel (A) represents obtaining “a 620 bp fragment” from extracellular domain of TRAIL through specific RT-PCR. Panel (B) represents obtaining a 504 bp band encoding soluble human TRAIL through nested PCR.

Figure 3

Confirmation of soluble human TRAIL cloning into 35S-TR plasmid using electrophoresis on 2% Agarose gel. Panel (A) represents obtaining a 1209 bp “35S promoter-TR inserted-CaMV polyA fragment” via PCR. Panel (B) demonstrate separation of cloned 1200 bp TRAIL expressional region from 35S-TR plasmid through EcoRV digestion.

Figure 4

Confirmation of “35S promoter-TR inserted-CaMV polyA fragment” cloning into pGreen-0179 plasmid using electrophoresis on 2% Agarose gel. Panel (A) represents separation of the pGreen-0179 backbone (2495 bp) from the T-DNA region of the empty (2648 bp) and TRAIL contained (3860 bp) pGreen-0179 vector via BglII enzyme digestion: The empty pGreen-0179 (lane 1), DNA Ladder (lane 2), pGreen-TRAIL (lane 3). Panel (B) represents TRAIL specific PCR using F3R5 primers on TRAIL cloned pGreen-0179 vectors (lane 2) and DNA ladder (lane 1).

Figure 5

SDS-PAGE and western blot analyses of ShTRAIL obtained from transformed N. tabacum callus cells (A) silver staining of the transformed (TR) and untransformed (UN) N. tabacum total protein extract on 15% PAGE. The arrows indicate bands of monomer and dimer forms of TRAIL (B) Obtaining TRAIL specific monomer (22 kDa) and dimer (44 kDa) bands through western blot analysis of selected recombinant N. tabacum callus cells.

Figure 6

Estimation of TRAIL production by Semi-quantitative western blot analysis. Panel (A) represents western blot analysis of 200 μg total protein extract of transformed N. tabacum cells (1,2,3) beside 1 μg of recombinant standard TRAIL (TR). Panel (B) represent corresponding area of developed bands through ImageJ software analysis.

Figure 7

Confirmation of TRAIL Purification through silver staining method. Unstained Fermentase protein ladder #SM0431 (lane M), crude protein extract (lane 1), purified monomer and dimer forms of TRAIL (lane 2) are represented.