Peptide gene sequences of substrate phage for different proteases
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ABSTRACT:
PROVIDER: PRJNA647886 | ENA |
REPOSITORIES: ENA
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Project description:We present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical 6 to 8 residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases 2, 3, 6, 7 and 8, and the ectodomains of the membrane sheddases, ADAMs 10 and 17. The first library (Lib 10AA) was used to determine substrate cleavage motifs. Lib 10AA contains a highly diverse randomized 10-mer substrate peptide sequences (10^9 unique members) that was displayed mono-valently on filamentous phage and bound to magnetic beads via an N-terminal biotin. The protease was allowed to cleave the SPD beads, and the released phage subjected to up to three total rounds of positive selection followed by next generation sequencing (NGS). This allowed us to identify from 10^4 to 10^5 unique cleavage sites over a broad dynamic range of NGS counts (ranging from 3-5000), and produced consensus and optimal cleavage motifs based positional sequencing scoring matrices and state-of the-art machine learning algorithm that closely matched synthetic peptide data. A second SPD-NGS library (Lib hP) was constructed that allowed us to identify candidate human proteome sequences. Lib hP displayed virtually the entire human proteome tiled in contiguous 49AA sequences with 25AA overlaps (nearly 1 million members). After three rounds of positive selection we identified up to 10^4 natural linear cut sites depending on the protease and captured most of the examples previously identified by proteomics (ranging from 30 to 1000) and predicted 10 to 100-fold more.
2020-07-23 | GSE154923 | GEO
Project description:Plasticity in AAA+ proteases reveals substrate specificity niches
| PRJNA701919 | ENA
Project description:Purpose: The goal of this study is to compare RNA-seq libraries of wildtype Caulobacter crescentus with two lon deletion strains (delta lon and delta lon clpX*). Methods: See Methods section of "Plasticity in AAA+ proteases reveals substrate specificity niches" for information regarding methods or contact lead correspondence. Briefly, Samples for RNAseq were extracted from wt and lon deletion strains grown to stationary phase. Conclusions: Our study represents the first detailed analysis of lon deletions (delta lon and delta lon clpX*) comparison to wt caulobacter transcriptomes, with biologic replicates, generated by RNA-seq technology in stationary phas
2022-02-12 | GSE166765 | GEO
Project description:RNA sequencing (RNA-seq) of phage infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, we performed RNA-seq of P. aeruginosa PAO1 cultures infected with phage LUZ19 in a mammalian cell culture medium (MCCM) to better simulate a phage therapy event, and the data were compared to LB medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection (t= 5 min) and gradually shuts down bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. Our data allowed us to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations of the mammalian cell culture medium. From a phage therapy perspective, this study contributes towards a better understanding of phage-host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.
2020-11-28 | GSE162278 | GEO
Project description: Not available
| S-EPMC5643515 | biostudies-literature
Project description:By using a phage displayed peptide library we identified peptide P60 as able to bind Foxp3 and inhibit Treg activity. To gain insight into the effect of P60 in Foxp3+ve cells we used Karpas 299 human cell line derived from a human lymphoma with a regulatory T cell profile. Microarray analysis of Karpas cell line incubated with or without P60 peptide was carried out using the Affymetrix human U133 Plus 2.0 array.
2024-01-21 | GSE165260 | GEO
Project description: Not available
| S-EPMC3080479 | biostudies-literature
Project description:We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with these proteases. Next, biotinylated peptides were enriched after digestion with the three combinations of different proteases.
2023-03-04 | PXD032061 | JPOST Repository
Project description:We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with these proteases. Next, biotinylated peptides were enriched after digestion with the three combinations of different proteases. We assessed reproducibility of each combination using three technical replicates.
2022-08-25 | PXD035242 | JPOST Repository
Project description:Genomic material isolated from purified phage YerA41 lysate was shown to contain RNA. YerA41 phage lysate was RNase treated to remove phage-external RNA and total RNA was then isolated from the phage preparate using Qiagen Rneasy mini kit. The isolated RNA was sequenced to elucidate its origin. The results suggested that the RNA originated from intact ribosomes of the host bacterium that contaminated the phage lysate.
2022-04-04 | GSE199969 | GEO