Browse
Submit Data
Databases
API
Help

Dataset Information

38 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proximity-Based Sortase-Mediated Ligation.

ABSTRACT: Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase?A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95?% by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.

SUBMITTER: Wang HH

PROVIDER: S-EPMC5537000 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Proximity-Based Sortase-Mediated Ligation.

Wang Hejia Henry HH Altun Burcin B Nwe Kido K Tsourkas Andrew A

Angewandte Chemie (International ed. in English) 20170404 19

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein ...[more]

PMID: 28374553

Similar Datasets

Sortase-Mediated Ligation of Purely Artificial Building Blocks.

Project description:Sortase A (SrtA) from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML) is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs), poly(ethylene glycol) and poly(N-isopropyl acrylamide) are chosen as synthetic building blocks. As a proof of concept, NP⁻polymer, NP⁻NP, and polymer⁻polymer structures are formed by SrtA catalysis. Therefore, the building blocks are equipped with the recognition sequence needed for SrtA reaction-the conserved peptide LPETG-and a pentaglycine motif. The successful formation of the reaction products is shown by means of transmission electron microscopy (TEM), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS), and dynamic light scattering (DLS). The sortase catalyzed linkage of artificial building blocks sets the stage for the development of a new approach to link synthetic structures in cases where their synthesis by established chemical methods is complicated.

| S-EPMC6414994 | biostudies-literature

End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation.

Project description:We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity.

| S-EPMC3310247 | biostudies-literature

Efficient Sortase-Mediated Ligation Using a Common C-Terminal Fusion Tag.

Project description:Sortase-mediated ligation is a powerful method for generating site-specifically modified proteins. However, this process is limited by the inherent reversibility of the ligation reaction. To address this, here we report the continued development and optimization of an experimentally facile strategy for blocking reaction reversibility. This approach, which we have termed metal-assisted sortase-mediated ligation (MA-SML), relies on the use of a solution additive (Ni2+) and a C-terminal tag (LPXTGGHH5) that is widely used for converting protein targets into sortase substrates. In a series of model systems utilizing a 1:1 molar ratio of sortase substrate and glycine amine nucleophile, we find that MA-SML consistently improves the extent of ligation. This enables the modification of proteins with fluorophores, PEG, and a bioorthogonal cyclooctyne moiety without the need to use precious reagents in excess. Overall, these results demonstrate the potential of MA-SML as a general strategy for improving reaction efficiency in a broad range of sortase-based protein engineering applications.

| S-EPMC7357393 | biostudies-literature

Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.

Project description:Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.

| S-EPMC4613866 | biostudies-literature

Proximity Ligation-Based Fluorogenic Imaging Agents for Neuraminidases.

Project description:Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase-imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus-specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti-neuraminidase antibodies.

| S-EPMC6311531 | biostudies-literature

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

Project description:BackgroundThere is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.MethodologySolid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.ConclusionsThe Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

| S-EPMC2063460 | biostudies-literature

Sensitive plasma protein analysis by microparticle-based proximity ligation assays.

Project description:Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

| S-EPMC2830843 | biostudies-other

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.

Project description:NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is approximately 40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

| S-EPMC3111449 | biostudies-literature

Protein ligation in living cells using sortase.

Project description:Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²?-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); we used proteins with an N-terminal (oligo)glycine as nucleophiles. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30?min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein-based nucleophiles in different intracellular compartments.

| S-EPMC3490390 | biostudies-literature

Detection of RNA-DNA association by a proximity ligation-based method.

Project description:We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30-45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis.

| S-EPMC4891662 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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