Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Selenocysteine-Mediated Expressed Protein Ligation of SELENOM.

ABSTRACT: 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 ?-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.

SUBMITTER: Liu J

PROVIDER: S-EPMC5821492 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Selenocysteine-Mediated Expressed Protein Ligation of SELENOM.

Liu Jun J Chen Qingqing Q Rozovsky Sharon S

Methods in molecular biology (Clifton, N.J.) 20180101

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 essentia ...[more]

PMID: 28917051

Similar Datasets

Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations.

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.

| S-EPMC5824972 | biostudies-literature

Expressed Protein Selenoester Ligation.

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.

| S-EPMC9314092 | biostudies-literature

Enzyme-catalyzed expressed protein ligation.

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.

| S-EPMC5088058 | biostudies-literature

Expressed Protein Ligation without Intein.

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.

| S-EPMC7249697 | biostudies-literature

Streamlined expressed protein ligation using split inteins.

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 ?-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 ?-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein ?-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein ?-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 ?-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.

| S-EPMC3544275 | biostudies-literature

Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Project description:Cyclotides are naturally occurring microproteins (?30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

| S-EPMC7467683 | biostudies-literature

Water-soluble phosphinothiols for traceless staudinger ligation and integration with expressed protein ligation.

Project description:The traceless Staudinger ligation is an effective means to synthesize an amide bond between two groups of otherwise orthogonal reactivity: a phosphinothioester and an azide. An important application of the Staudinger ligation is in the ligation of peptides at a variety of residues. Here, we demonstrate that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent. Those reagents that provide a high yield of amide product discourage protonation of the nitrogen in the key iminophosphorane intermediate. The most efficacious reagent, bis(p-dimethylaminoethyl)phosphinomethanethiol, mediates the rapid ligation of equimolar substrates in water. This reagent is also able to perform a transthioesterification reaction with the thioester intermediate formed during intein-mediated protein splicing. Hence, the traceless Staudinger ligation can be integrated with expressed protein ligation, extending the reach of modern protein chemistry.

| S-EPMC2851833 | biostudies-literature

Analysis of protein kinase autophosphorylation using expressed protein ligation and computational modeling.

Project description:Protein kinases represent a family of enzymes that are critical in cell signaling. One mechanism by which protein kinases are regulated is via autophosphorylation. In the studies described here, we have examined the mechanism of autophosphosphorylation at serine 338 in the regulation of protein kinase A (PKA). Expressed protein ligation allowed for the covalent linkage of an ATP moiety to a Ser mimic at this phosphorylation site. Using a combination of size exclusion chromatography, fluorescence nucleotide binding, kinase measurements, and limited proteolysis assays on this semisynthetic ATP-linked protein, we have obtained unique evidence for an intramolecular autophosphorylation mechanism in PKA regulation. Computational analysis provided a plausible model for a PKA conformation consistent with intramolecular phosphoryl transfer. This approach could be applied to other autoprocessing enzymes by exploiting appropriate transition state analogue motifs in the context of protein semisynthesis.

| S-EPMC2654778 | biostudies-literature

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins.

Project description:In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.

| S-EPMC3357005 | biostudies-literature

Site-specific conjugation of RAFT polymers to proteins via expressed protein ligation.

Project description:Site-specific protein conjugates with RAFT polymers were synthesized using expressed protein ligation. Stable micelles were formed from both linear block copolymer and Y-shaped conjugates.

| S-EPMC4059823 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data