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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

OE-PCR protocol for the insertion of the OmpA signal sequence into the TRAIL cDNA. Reaction 1: hybridization of 3´ region of primers F1 with terminal regions of TRAIL cDNA and synthesis a part of the OmpA signal sequence. Reaction 2: using the product of the reaction 1 as a template by primer F2 and completion of whole OmpA signal sequence.
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Figure 1: OE-PCR protocol for the insertion of the OmpA signal sequence into the TRAIL cDNA. Reaction 1: hybridization of 3´ region of primers F1 with terminal regions of TRAIL cDNA and synthesis a part of the OmpA signal sequence. Reaction 2: using the product of the reaction 1 as a template by primer F2 and completion of whole OmpA signal sequence.

Mentions: Overlapping Extension Polymerase Chain Reaction (OE-PCR) method was used in order to insert OmpA signal sequence into the 5´ region of human TRAIL cDNA, which had been previously prepared in our laboratory (20). For this purpose, DNA encoding OmpA signal sequence was added at 5' end of TRAIL cDNA through two cycles of PCR using pfu DNA polymerase (Fermentas, Lithuania) (Figure 1). In the first run of PCR, a primer pair was partially annealed to TRAIL cDNA with overlapping end region. During PCR reaction, the overhang region of the forward primer, which is a part of OmpA signal sequence, was inserted at 5' end of DNA template. On the other hand, XhoI restriction site was introduced at 3' end of DNA template. The first PCR product was used as a template for the second run and the second forward primer was partially annealed with 5' end region of this template resulting in the synthesis of full-length OmpA signal sequence and NdeI restriction site at 5' end of TRAIL cDNA.


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)

OE-PCR protocol for the insertion of the OmpA signal sequence into the TRAIL cDNA. Reaction 1: hybridization of 3´ region of primers F1 with terminal regions of TRAIL cDNA and synthesis a part of the OmpA signal sequence. Reaction 2: using the product of the reaction 1 as a template by primer F2 and completion of whole OmpA signal sequence.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: OE-PCR protocol for the insertion of the OmpA signal sequence into the TRAIL cDNA. Reaction 1: hybridization of 3´ region of primers F1 with terminal regions of TRAIL cDNA and synthesis a part of the OmpA signal sequence. Reaction 2: using the product of the reaction 1 as a template by primer F2 and completion of whole OmpA signal sequence.
Mentions: Overlapping Extension Polymerase Chain Reaction (OE-PCR) method was used in order to insert OmpA signal sequence into the 5´ region of human TRAIL cDNA, which had been previously prepared in our laboratory (20). For this purpose, DNA encoding OmpA signal sequence was added at 5' end of TRAIL cDNA through two cycles of PCR using pfu DNA polymerase (Fermentas, Lithuania) (Figure 1). In the first run of PCR, a primer pair was partially annealed to TRAIL cDNA with overlapping end region. During PCR reaction, the overhang region of the forward primer, which is a part of OmpA signal sequence, was inserted at 5' end of DNA template. On the other hand, XhoI restriction site was introduced at 3' end of DNA template. The first PCR product was used as a template for the second run and the second forward primer was partially annealed with 5' end region of this template resulting in the synthesis of full-length OmpA signal sequence and NdeI restriction site at 5' end of TRAIL cDNA.

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