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.

Production of prone‐to‐aggregate proteins

“…We employ this strategy in most of the projects that involve protein production in bacteria, with high success rates in dozens of cases. A few successful examples can be found in the following references [25][26][27][57][58][59].…”

“…It can easily be adjusted for many types of proteins, and when combined with the subsequent procedures for isolation and concentration, it proved to be extremely efficient for the vast majority of IDR, IDP, and other types of proteins prone to aggregate that were tested at our facility over the years [25][26][27][57][58][59].…”

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

“…The prevalence of this byproduct was dramatically reduced when ligation-deselenization reactions were performed as a one-pot protocol in the presence of exogenous MPAA. Under these conditions, the rate of deselenization also dramatically decreased, perhaps because of the ability of aryl thiols such as MPAA to act as competitive radical scavengers [126,127,144]. Nonetheless, native peptide products, including those bearing unprotected, non-ligation site Cys residues, were isolated in good yields following the one-pot protocol for ligation-deselenization at β-selenophenylalanine [149].…”

“…Interestingly, for the construction of the target protein, the authors also reported the use of a novel thiol additive, trifluoroethanethiol (TFET) 124, which facilitates the facile application of a one-pot ligation-desulfurization protocol [124]. Previous attempts to streamline the ligation-desulfurization strategy into a one-pot process have been hampered by the use of aryl thiol ligation additives which, because of their radical quenching ability [125], complicate the radical desulfurization process [126][127][128]. Given the importance of aryl thiol additives in modulating thioester reactivity and promoting rapid ligation reactions [15,16], a number of approaches have aimed to facilitate the post-ligation removal of aryl thiols.…”

Postligation-Desulfurization: A General Approach for Chemical Protein Synthesis

“…To realize the one-pot ligation and desulfurization, the additional aryl thiol must be removed from the reaction system before proceeding with desulfurization [86,88,97]. Interestingly, the Brik group examined several thiol additives and found that MESNa not only afforded the ligation at a fast rate but also allowed the rapid and efficient desulfurization, which promoted the one-pot ligation-desulfurization method with good efficiency [98]. Having used MESNa promoted NCL to obtain HIV-1 Rev protein, a 13 kDa protein plays an important role in the HIV replication cycle.…”