Chemical Synthesis and Expression of the HIV-1 Rev Protein
Autor: | Tsafi Danieli, Peter Siman, Hilal A. Lashuel, Assaf Friedler, Tal Moyal, Mario Lebendiker, Ashraf Brik, Ofrah Blatt |
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Rok vydání: | 2011 |
Předmět: |
solid-phase synthesis
Size-exclusion chromatography Peptide 010402 general chemistry 01 natural sciences Biochemistry Chemical synthesis Solid-phase synthesis Virus Type-Iii Thioesters Cysteine protein expression Vilsmeier-Haack reaction Molecular Biology Racemization chemistry.chemical_classification Molecular Structure 010405 organic chemistry Chemistry Organic Chemistry Glycopeptides desulfurization rev Gene Products Human Immunodeficiency Virus Binding Native chemical ligation Combinatorial chemistry Recombinant Proteins 3. Good health 0104 chemical sciences Efficient Yield (chemistry) Sugar-Assisted Ligation Molecular Medicine Chemical ligation Kinetically Controlled Ligation HIV-1 Rev protein |
Zdroj: | ChemBioChem |
ISSN: | 1439-4227 |
Popis: | 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. |
Databáze: | OpenAIRE |
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