HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

AbstractBACKGROUND:
In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.
RESULTS:
The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.
CONCLUSION:
We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.
AuthorsRaffaele Lombardi, Patrizia Circelli, Maria Elena Villani, Giampaolo Buriani, Luca Nardi, Valentina Coppola, Linda Bianco, Eugenio Benvenuto, Marcello Donini, Carla Marusic
JournalBMC biotechnology (BMC Biotechnol) Vol. 9 Pg. 96 (Nov 20 2009) ISSN: 1472-6750 [Electronic] England
PMID19930574 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • CBX5 protein, human
  • RNA, Small Interfering
  • Viral Core Proteins
  • nef Gene Products, Human Immunodeficiency Virus
  • nef protein, Human immunodeficiency virus 1
  • Chromobox Protein Homolog 5
Topics
  • Chromatography, Affinity
  • Chromobox Protein Homolog 5
  • Gene Expression Regulation, Plant
  • Genetic Engineering (methods)
  • Mass Spectrometry
  • RNA Interference
  • RNA, Small Interfering (genetics)
  • Tobacco (genetics, virology)
  • Tombusvirus (genetics)
  • Viral Core Proteins (genetics)
  • nef Gene Products, Human Immunodeficiency Virus (biosynthesis, genetics, isolation & purification)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: