Dataset Information

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

ABSTRACT: Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

SUBMITTER: Pandya P

PROVIDER: S-EPMC7263589 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Pandya Priyanka P Sayers Robert O RO Ting Joey P JP Morshedian Shaghayegh S Torres Carina C Cudal Justine S JS Zhang Kai K Fitchett Jonathan R JR Zhang Qing Q Zhang Feiyu F FF Wang Jing J Durbin Jim D JD Carrillo Juan J JJ Espada Alfonso A Broughton Howard H Qian Yuewei Y Afshar Sepideh S

PloS one 20200601 6

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine varia ...[more]

PMID: 32479512

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Project description:PURPOSE:Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for the past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells. EXPERIMENTAL DESIGN:From the Ph.D.-12 phage display peptide library composed of 2.7 x 10(9) different displayed peptides, one peptide was enriched after four rounds of in vitro selection in 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue stainings. The peptide was further labeled with (18)F for positron emission tomography imaging studies. Cell uptake and efflux and ex vivo biodistribution were also done with (18)F-labeled osteosarcoma specific peptide. RESULTS:ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans. The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells, whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. (18)F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than (18)F-OSP-S. (18)F-OSP-1 also had higher uptake in 143B tumors than in UM-SCC-1 tumors. CONCLUSIONS:Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.

Project description:Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin.We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

Dataset Information

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Publications

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

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