A lanthipeptide library used to identify a protein–protein interaction inhibitor

Abstract

We describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmidencoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, a critical protein–protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay for the identification of lanthipeptides with new biological activities.

In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues

“…Such combinations would further expand the molecular complexity of peptides produced by the artificial in vitro biosynthesis system strategy . A significant advantage of bioengineering with RiPP‐modifying enzymes is the potential applicability for the construction of combinatorial peptide libraries and screening by display technologies . Therefore, the reprogrammed FIT‐PatD system, demonstrating the combination of reprogrammed translation and post‐translational modification, can pave the way for the development of new bioactive peptides with diverse non‐proteinogenic structures.…”

Chemoenzymatic Posttranslational Modification Reactions for the Synthesis of Ψ[CH2NH]‐Containing Peptides

“…Thew ide substrate scope,s ite selectivity,a nd waterfriendly mild reaction conditions of our methodology have offered template-dependent synthesis of diverse Y[CH 2 NH]containing peptides by means of an in vitro translation system. This method can be applied not only for the synthesis of rationally designed peptidomimetic compounds [28] but also to the construction of peptidomimetic libraries with randomized peptide sequences, [29] which can be screened by display technologies [30][31][32][33][34] to develop de novo bioactive molecules bearing the reduced amide bonds.I nc onclusion, the posttranslational modifications reported here provide an ew tool to produce aw ide array of peptidomimetics containing the Y[CH 2 NH] bond.…”

A cell-free platform based on nisin biosynthesis for discovering novel lanthipeptides and guiding their overproductionin vivo

“…N-Ethylmaleimide (NEM) alkylation assay of cyclization NEM alkylation assays were performed according to a previously reported method (7). Briefly, an aliquot of the protease-digested peptide or full-length peptide solution was diluted into a two-fold volume of NEM alkylation buffer containing 500 mM HEPES, 3 mM NEM, 0.3 mM TCEP (pH 6.5).…”

“…Construction of a mutant lanthipeptides library and performing high-throughput screening is another useful method for discovering novel lanthipeptides. Further, method have been developed that allow for the high-throughput screening of new bioactive (anti-viral) lanthipeptides in vivo (7), and ingenious high-throughput screening of anti-microbials lanthipeptides by expression of inactive mLanAs in living cells prior to removal of the leader peptide in vitro (including in dead cells) to form active lanthipeptides (8,9). However, the in vivo systems pose unavoidable problems, such as intracellular toxicity; the formation of inclusion bodies, which is problematic for subsequent purification; lack of special tRNA Glu for heterologous dehydrase (dehydration in lanthipeptide PTM process is catalyzed in a tRNA Glu -dependent manner).…”