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High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves.

ABSTRACT:

Key message

The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand. The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand.

SUBMITTER: Hoelscher M

PROVIDER: S-EPMC6061503 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana.

Project description:The exploration of emerging host organisms for the economic and efficient production of protein microbicides against HIV is urgently needed in resource-poor areas worldwide. In this study, the production of the novel HIV entry inhibitor candidate, griffithsin (GRFT), was investigated using Nicotiana benthamiana as the expression platform based on a non-viral vector. To increase the yield of recombinant GRFT, the RNA silencing defense mechanism of N. benthamiana was abolished by using three gene silencing suppressors. A transient expression system was used by transferring the GRFT gene, which encodes 122 amino acids, under the control of the enhanced CaMV 35S promoter. The presence of correctly assembled GRFT in transgenic leaves was confirmed using immunoglobulin-specific sandwich ELISA. The data demonstrated that the use of three gene silencing suppressors allowed the highest accumulation of GRFT, with a yield of 400??g?g-1 fresh weight, and this amount was reduced to 287??g?g-1 after purification, representing a recovery of 71.75%. The analysis also showed that the ability of GRFT expressed in N. benthamiana to bind to glycoprotein 120 is close to that of the GRFT protein purified from E. coli. Whole-cell assays using purified GRFT showed that our purified GRFT was potently active against HIV. This study provides the first high-level production of the HIV-1 entry inhibitor griffithsin with a non-viral expression system and illustrates the robustness of the co-agroinfiltration expression system improved through the use of three gene silencing suppressors.

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