Chemical Synthesis and Expression of the HIV-1 Rev Protein

Peter Siman, Ofrah Blatt, Tal Moyal, Tsafi Danieli, Mario Lebendiker, Hilal A. Lashuel, Assaf Friedler, Ashraf Brik

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

The HIV-1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV-1 lifecycle, thus making Rev an attractive target for the design of anti-HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self-assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one-pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein. Winning HIV-1 Rev: The protein HIV-1 Rev plays an important role in the HIV lifecycle; however, its high tendency to aggregate has hindered several studies that aimed at deciphering better its structure and function. Two highly reproducible methods to generate this protein in large quantities, based on chemical synthesis and recombinant expression, are presented.

Original languageEnglish
Pages (from-to)1097-1104
Number of pages8
JournalChemBioChem
Volume12
Issue number7
DOIs
Publication statusPublished - 2 May 2011
Externally publishedYes

Fingerprint

Desulfurization
Purification
Ligation
HIV-1
rev Gene Products
Anti-HIV Agents
Proteins
Recombinant proteins
Recombinant Proteins
Solubility
Gel Chromatography
Distillation columns
Cations
Self assembly
HIV
Ion exchange
Agglomeration
Messenger RNA
Gels
Human Immunodeficiency Virus-1 rev protein

Keywords

  • Desulfurization
  • HIV-1 Rev protein
  • Protein expression
  • Solid-phase synthesis
  • Vilsmeier-Haack reaction

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology

Cite this

Siman, P., Blatt, O., Moyal, T., Danieli, T., Lebendiker, M., Lashuel, H. A., ... Brik, A. (2011). Chemical Synthesis and Expression of the HIV-1 Rev Protein. ChemBioChem, 12(7), 1097-1104. https://doi.org/10.1002/cbic.201100033

Chemical Synthesis and Expression of the HIV-1 Rev Protein. / Siman, Peter; Blatt, Ofrah; Moyal, Tal; Danieli, Tsafi; Lebendiker, Mario; Lashuel, Hilal A.; Friedler, Assaf; Brik, Ashraf.

In: ChemBioChem, Vol. 12, No. 7, 02.05.2011, p. 1097-1104.

Research output: Contribution to journalArticle

Siman, P, Blatt, O, Moyal, T, Danieli, T, Lebendiker, M, Lashuel, HA, Friedler, A & Brik, A 2011, 'Chemical Synthesis and Expression of the HIV-1 Rev Protein', ChemBioChem, vol. 12, no. 7, pp. 1097-1104. https://doi.org/10.1002/cbic.201100033
Siman P, Blatt O, Moyal T, Danieli T, Lebendiker M, Lashuel HA et al. Chemical Synthesis and Expression of the HIV-1 Rev Protein. ChemBioChem. 2011 May 2;12(7):1097-1104. https://doi.org/10.1002/cbic.201100033
Siman, Peter ; Blatt, Ofrah ; Moyal, Tal ; Danieli, Tsafi ; Lebendiker, Mario ; Lashuel, Hilal A. ; Friedler, Assaf ; Brik, Ashraf. / Chemical Synthesis and Expression of the HIV-1 Rev Protein. In: ChemBioChem. 2011 ; Vol. 12, No. 7. pp. 1097-1104.
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