Expressed Protein Selenoester Ligation
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ABSTRACT:
SUBMITTER: Kulkarni S
PROVIDER: S-EPMC10947028 | biostudies-literature | 2022 Mar
REPOSITORIES: biostudies-literature
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Project description:Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.
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Project description:Proteins with a functionalized C-terminus such as a C-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its N-side amide for undergoing nucleophilic acyl substitution with amines including a number of l- and d-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this activated cysteine-directed protein ligation (ACPL) approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a C-terminal posttranslational modification, RNase H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.
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Project description:Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.
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Project description:The development of a rapid and chemoselective selenocystine-selenoester peptide ligation that operates at nanomolar reactant concentrations has been developed by utilising PNA templation. Kinetic analysis of the templated peptide ligation revealed that the selenocystine-selenoester reaction was 10 times faster than traditional native chemical ligation at cysteine and to our knowledge is the fastest templated ligation reaction reported to date. The efficiency and operational simplicity of this technology is highlighted through the formation of hairpin molecular architectures and in a novel paper-based lateral flow assay for the rapid and sequence specific detection of oligonucleotides, including miRNA in cell lysates.
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Project description:Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.
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Project description:A sizeable fraction of the selenoproteome encodes oxidoreductases possessing a thioredoxin fold, a structural motif that is shared among a diverse group of enzymes. In these oxidoreductases, the active site is comprised of a cysteine and a selenocysteine separated by one to two amino acids. In a subset of these selenoproteins, such as human SELENOH, SELENOM, SELENOT, SELENOV, SELENOW, and SELENOF, this redox motif is positioned immediately after the first β-sheet in a short loop, and is essential for interactions with its substrate or partners. Here, we describe the preparation of a representative member of this group, SELENOM, by selenocysteine-driven expressed protein ligation. The preparation employs a peptide bond formation between two protein fragments expressed recombinantly in E. coli. This method can be employed to prepare other selenoproteins.
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Project description:Employing selenocysteine-containing protein fragments to form the amide bond between respective protein fragments significantly extends the current capabilities of the widely used protein engineering method, expressed protein ligation. Selenocysteine-mediated ligation is noteworthy for its high yield and efficiency. However, it has so far been restricted to solid-phase synthesized seleno-peptides and thus constrained by where the selenocysteine can be positioned. Here we employ heterologously expressed seleno-fragments to overcome the placement and size restrictions in selenocysteine-mediated chemical ligation. Following ligation, the selenocysteine can be deselenized into an alanine or serine, resulting in nonselenoproteins. This greatly extends the flexibility in selecting the conjugation site in expressed protein ligations with no influence on native cysteines. Furthermore, the selenocysteine can be used to selectively introduce site-specific protein modifications. Therefore, selenocysteine-mediated expressed protein ligation simplifies incorporation of post-translational modifications into the protein scaffold.
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