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Heterologous expression of biologically active Mambalgin-1 peptide as a new potential anticancer, using a PVX-based viral vector in Nicotiana benthamiana.

ABSTRACT: Mambalgin-1 is a peptide that acts as a potent analgesic through inhibiting acid-sensing ion channels (ASIC) in nerve cells. Research has shown that ASIC channels are involved in the proliferation and growth of cancer cells; therefore, Mambalgin-1 can be a potential anti-cancer by inhibiting these channels. In the present study, the Nicotiana benthamiana codon optimized Mambalgin-1 gene was synthesized and cloned in PVX (potato virus X) viral vector. The two cultures of Agrobacterium containing Mambalgin-1 and P19 silencing suppressor genes were co-agroinfiltrated into N. benthamiana leaves. Five days post infiltration, the production of recombinant Mambalgin-1 was determined by western blotting. For biological activity, MTT (3(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide) was analyzed for the cytotoxicity recombinant Mambalgin-1 from the transformed plants on nervous (SH-SY5Y) and breast (MCF7) cancer cells. The results showed that the plants expressing open reading frame of Mambalgin-1 showed recombinant 7.4 kDa proteins in the entire plant extract. In the MTT test, it was found that Mambalgin-1 had cytotoxic effects on SH-SY5Y cancer cells, yet no effects on MCF7 cancer cells were observed. According to the results, the expression of the biologically active recombinant Mambalgin-1 in the transformed plant leaves was confirmed and Mambalgin-1 can also have anti-cancer (inhibition of ASIC channels) potential along with its already known analgesic effect.

SUBMITTER: Khezri G

PROVIDER: S-EPMC7323601 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Heterologous expression of biologically active Mambalgin-1 peptide as a new potential anticancer, using a PVX-based viral vector in Nicotiana benthamiana.

Khezri Ghaffar G Baghban Kohneh Rouz Bahram B Ofoghi Hamideh H Davarpanah Seyed Javad SJ

Plant cell, tissue and organ culture 20200629 2

Mambalgin-1 is a peptide that acts as a potent analgesic through inhibiting acid-sensing ion channels (ASIC) in nerve cells. Research has shown that ASIC channels are involved in the proliferation and growth of cancer cells; therefore, Mambalgin-1 can be a potential anti-cancer by inhibiting these channels. In the present study, the Nicotiana benthamiana codon optimized Mambalgin-1 gene was synthesized and cloned in PVX (potato virus X) viral vector. The two cultures of Agrobacterium</ ...[more]

PMID: 32836586

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Project description:Oleanolic acid is a pentacyclic triterpenoid found in numerous plant species and is a precursor to several bioactive triterpenoids with commercial potential. However, oleanolic acid accumulates at low levels in plants, and its chemical synthesis is challenging. Here, we established a method for producing oleanolic acid in substantial quantities via heterologous expression of pathway enzymes in Nicotiana benthamiana. The “Tsukuba system” is one of the most efficient agroinfiltration-based transient protein expression systems using the vector pBYR2HS, which contains geminiviral replication machinery and a double terminator for boosting expression. Additionally, the pBYR2HS vector contains an expression cassette for the gene-silencing suppressor p19 protein from tomato bushy stunt virus, which can also contribute to enhancing the expression of target proteins. In this study, we evaluated the applicability of this system to heterologous triterpenoid production in N. benthamiana. Medicago truncatula cytochrome P450 monooxygenase (CYP) 716A12 is the first enzyme to be functionally characterized as β-amyrin C-28 oxidase producing oleanolic acid. A mutant CYP716A12 (D122Q) with improved catalytic activity engineered in our previous study was co-expressed with other enzymes in N. benthamiana leaves. Using pBYR2HS, oleanolic acid yield was increased 13.1-fold compared with that using the conventional binary vector, indicating the advantage of the Tsukuba system. We also demonstrated the efficacy of co-expressing a mutant Arabidopsis thaliana HMGR1 catalytic domain, additional NADPH-cytochrome P450 reductase (CPR) transferring electrons to heterologous CYPs, and application of ascorbic acid for preventing leaf necrosis after agroinfiltration, to improve product yield. As a result, the product yields of both simple (β-amyrin) and oxidized (oleanolic acid and maslinic acid) triterpenoids were significantly improved compared with the previously reported yield in heterologous triterpenoid production in N. benthamiana leaves.

Dataset Information

Heterologous expression of biologically active Mambalgin-1 peptide as a new potential anticancer, using a PVX-based viral vector in Nicotiana benthamiana.

Publications

Heterologous expression of biologically active Mambalgin-1 peptide as a new potential anticancer, using a PVX-based viral vector in <i>Nicotiana benthamiana</i>.

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