title: Chemical synthesis and expression of the HIV-1 Rev protein.
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.
authors.affiliation: Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: Peter
authors.initials: P
authors.lastname: Siman
authors.forename: Ofrah
authors.initials: O
authors.lastname: Blatt
authors.forename: Tal
authors.initials: T
authors.lastname: Moyal
authors.forename: Tsafi
authors.initials: T
authors.lastname: Danieli
authors.forename: Mario
authors.initials: M
authors.lastname: Lebendiker
authors.forename: Hilal A
authors.initials: HA
authors.lastname: Lashuel
authors.forename: Assaf
authors.initials: A
authors.lastname: Friedler
authors.forename: Ashraf
authors.initials: A
authors.lastname: Brik
mesh_terms: D015394:Molecular Structure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1