Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Selection of peptide entry motifs by bacterial surface display.

ABSTRACT: Surface display technologies have been established previously to select peptides and polypeptides that interact with purified immobilized ligands. In the present study, we designed and implemented a surface display-based technique to identify novel peptide motifs that mediate entry into eukaryotic cells. An Escherichia coli library expressing surface-displayed peptides was combined with eukaryotic cells and the gentamicin protection assay was performed to select recombinant E. coli, which were internalized into eukaryotic cells by virtue of the displayed peptides. To establish the proof of principle of this approach, the fibronectin-binding motifs of the fibronectin-binding protein A of Staphylococcus aureus were inserted into the E. coli FhuA protein. Surface expression of the fusion proteins was demonstrated by functional assays and by FACS analysis. The fibronectin-binding motifs were shown to mediate entry of the bacteria into non-phagocytic eukaryotic cells and brought about the preferential selection of these bacteria over E. coli expressing parental FhuA, with an enrichment of 100000-fold. Four entry sequences were selected and identified using an S. aureus library of peptides displayed in the FhuA protein on the surface of E. coli. These sequences included novel entry motifs as well as integrin-binding Arg-Gly-Asp (RGD) motifs and promoted a high degree of bacterial entry. Bacterial surface display is thus a powerful tool to effectively select and identify entry peptide motifs.

SUBMITTER: Taschner S

PROVIDER: S-EPMC1222908 | biostudies-other | 2002 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Peptide amyloid surface display.

Project description:Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel ?-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells.

| S-EPMC4318585 | biostudies-literature

Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

Project description:A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

| S-EPMC1222007 | biostudies-other

Directed Evolution Using Stabilized Bacterial Peptide Display.

Project description:Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.

| S-EPMC7304070 | biostudies-literature

A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity.

Project description:Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. The E. coli protein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. A randomly mutated BirA library was used to identify novel variants. Bacteria displaying peptide sequences from 13 out of 14 tested proteins were strongly enriched after 3-5 selection rounds. Moreover, a clone selected for biotinylation of a C-terminal peptide from red-fluorescent protein TagRFP showed biotinylation of the native protein. Thus, active BirA variants with novel activity are effectively isolated with our bacterial display system and provides a basis for the development of BirA variants for site-selective biotinylation.

| S-EPMC6411976 | biostudies-literature

Surface display of metal fixation motifs of bacterial P1-type ATPases specifically promotes biosorption of Pb(2+) by Saccharomyces cerevisiae.

Project description:Biosorption of metal ions may take place by different passive metal-sequestering processes such as ion exchange, complexation, physical entrapment, and inorganic microprecipitation or by a combination of these. To improve the biosorption capacity of the potential yeast biosorbent, short metal-binding NP peptides (harboring the CXXEE metal fixation motif of the bacterial Pb(2+)-transporting P1-type ATPases) were efficiently displayed and covalently anchored to the cell wall of Saccharomyces cerevisiae. These were fusions to the carboxyl-terminal part of the sexual adhesion glycoprotein alpha-agglutinin (AGalpha1Cp). Compared to yeast cells displaying the anchoring domain only, those having a surface display of NP peptides multiplied their Pb(2+) biosorption capacity from solutions containing a 75 to 300 microM concentration of the metal ion up to 5-fold. The S-type Pb(2+) biosorption isotherms, plus the presence of electron-dense deposits (with an average size of 80 by 240 nm, observed by transmission electron microscopy) strongly suggested that the improved biosorption potential of NP-displaying cells is due to the onset of microprecipitation of Pb species on the modified cell wall. The power of an improved capacity for Pb biosorption was also retained by the isolated cell walls containing NP peptides. Their Pb(2+) biosorption property was insensitive to the presence of a 3-fold molar excess of either Cd(2+) or Zn(2+). These results suggest that the biosorption mechanism can be specifically upgraded with microprecipitation by the engineering of the biosorbent with an eligible metal-binding peptide.

| S-EPMC2849214 | biostudies-literature

Engineering Ag43 Signal Peptides with Bacterial Display and Selection.

Project description:Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

| S-EPMC9844295 | biostudies-literature

Bacterial display enables efficient and quantitative peptide affinity maturation.

Project description:A quantitative screening method was developed to enable isolation and affinity maturation of peptide ligands specific for a given target from peptide libraries displayed on the outer surface of Escherichia coli using multi-parameter flow cytometry. From a large, random 15-mer peptide library, screening identified a core motif of W-E/D-W-E/D that conferred binding to vascular endothelial growth factor (VEGF). One cycle of affinity maturation resulted in the identification of several families of VEGF-binding peptides having distinct consensus sequences, from which a preferred disulfide constraint emerged. In the second affinity maturation cycle, high affinity peptides were favored by the addition of a decoy protein that bound an adjacent epitope on the display scaffold. The decoy apparently reduced rebinding or avidity effects, and the resulting peptides exhibited consensus at 12 of 19 amino acid positions. Peptides identified and affinity matured using bacterial display were remarkably similar to the best affinity matured using phage display and exhibited comparable dissociation constants (within 2-fold; K(D) = 4.7 x 10(-7) M). Screening of bacterial-displayed peptide libraries using cytometry enabled optimization of screening conditions to favor affinity and specificity and rapid clonal characterization. Bacterial display thus provides a new quantitative tool for the discovery and evolutionary optimization of protein-specific peptide ligands.

| S-EPMC2791049 | biostudies-literature

Bacterial surface display of human lectins in Escherichia coli.

Project description:Lectin-glycan interactions sustain fundamental biological processes involved in development and disease. Owing to their unique sugar-binding properties, lectins have great potential in glycobiology and biomedicine. However, their relatively low affinities and broad specificities pose a significant challenge when used as analytical reagents. New approaches for expression and engineering of lectins are in demand to overcome current limitations. Herein, we report the application of bacterial display for the expression of human galectin-3 and mannose-binding lectin in Escherichia coli. The analysis of the cell surface expression and binding activity of the surface-displayed lectins, including point and deletion mutants, in combination with molecular dynamics simulation, demonstrate the robustness and suitability of this approach. Furthermore, the display of functional mannose-binding lectin in the bacterial surface proved the feasibility of this method for disulfide bond-containing lectins. This work establishes for the first time bacterial display as an efficient means for the expression and engineering of human lectins, thereby increasing the available toolbox for glycobiology research.

| S-EPMC10884992 | biostudies-literature

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins.

Project description:Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

| S-EPMC154567 | biostudies-literature

Thymoproteasomes produce unique peptide motifs for positive selection of CD8(+) T cells.

Project description:Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8(+) T cells requires cortical thymic epithelial cells that express β5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8(+) T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8(+) T cells by preferentially producing low-affinity TCR ligand peptides.

| S-EPMC4557289 | biostudies-literature

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