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Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli.

Tavallaei O, Bandehpour M, Nafissi-Varcheh N, Kazemi B - Iran J Pharm Res (2015)

Bottom Line: However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream.Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay.The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

No MeSH data available.


Related in: MedlinePlus

Identification of periplasmic TRAIL protein by Western Blot. TRAIL was resolved by SDS-PAGE and then transferred to nitrocellulose membrane. After incubation with anti-TRAIL antibody and then secondary antibody, the membrane was developed with the BCIP/NBT substrate system. Lane 1: protein marker. Lane 2: periplasmic TRAIL.
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Figure 6: Identification of periplasmic TRAIL protein by Western Blot. TRAIL was resolved by SDS-PAGE and then transferred to nitrocellulose membrane. After incubation with anti-TRAIL antibody and then secondary antibody, the membrane was developed with the BCIP/NBT substrate system. Lane 1: protein marker. Lane 2: periplasmic TRAIL.

Mentions: In order to confirm the identity of recombinant TRAIL immunologically, western blot analysis was performed using anti-TRAIL antibody. The results showed that periplasmic fraction reacted with anti-TRAIL antibody and the size of the protein was approximately 21 kD as expected for recombinant TRAIL (Figure 6).


Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli.

Tavallaei O, Bandehpour M, Nafissi-Varcheh N, Kazemi B - Iran J Pharm Res (2015)

Identification of periplasmic TRAIL protein by Western Blot. TRAIL was resolved by SDS-PAGE and then transferred to nitrocellulose membrane. After incubation with anti-TRAIL antibody and then secondary antibody, the membrane was developed with the BCIP/NBT substrate system. Lane 1: protein marker. Lane 2: periplasmic TRAIL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403080&req=5

Figure 6: Identification of periplasmic TRAIL protein by Western Blot. TRAIL was resolved by SDS-PAGE and then transferred to nitrocellulose membrane. After incubation with anti-TRAIL antibody and then secondary antibody, the membrane was developed with the BCIP/NBT substrate system. Lane 1: protein marker. Lane 2: periplasmic TRAIL.
Mentions: In order to confirm the identity of recombinant TRAIL immunologically, western blot analysis was performed using anti-TRAIL antibody. The results showed that periplasmic fraction reacted with anti-TRAIL antibody and the size of the protein was approximately 21 kD as expected for recombinant TRAIL (Figure 6).

Bottom Line: However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream.Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay.The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

No MeSH data available.


Related in: MedlinePlus