Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands
We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of lin...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/4/1634 |
| _version_ | 1797413935939321856 |
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| author | John Bowen John Schneible Kaitlyn Bacon Collin Labar Stefano Menegatti Balaji M. Rao |
| author_facet | John Bowen John Schneible Kaitlyn Bacon Collin Labar Stefano Menegatti Balaji M. Rao |
| author_sort | John Bowen |
| collection | DOAJ |
| description | We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[<i>E-</i>LYLAYPAH<i>-K</i>] featured a K<sub>D</sub> of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders. |
| first_indexed | 2024-03-09T05:24:56Z |
| format | Article |
| id | doaj.art-1098d58f96d54d5fbfa814751bf06a6e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T05:24:56Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-1098d58f96d54d5fbfa814751bf06a6e2023-12-03T12:37:08ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01224163410.3390/ijms22041634Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide LigandsJohn Bowen0John Schneible1Kaitlyn Bacon2Collin Labar3Stefano Menegatti4Balaji M. Rao5Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USADepartment of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USADepartment of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USADepartment of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USADepartment of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USADepartment of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USAWe present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[<i>E-</i>LYLAYPAH<i>-K</i>] featured a K<sub>D</sub> of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.https://www.mdpi.com/1422-0067/22/4/1634yeast display librarycyclic peptideYes Associated Protein (YAP) 65WW domaintransglutaminase |
| spellingShingle | John Bowen John Schneible Kaitlyn Bacon Collin Labar Stefano Menegatti Balaji M. Rao Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands International Journal of Molecular Sciences yeast display library cyclic peptide Yes Associated Protein (YAP) 65 WW domain transglutaminase |
| title | Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands |
| title_full | Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands |
| title_fullStr | Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands |
| title_full_unstemmed | Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands |
| title_short | Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands |
| title_sort | screening of yeast display libraries of enzymatically treated peptides to discover macrocyclic peptide ligands |
| topic | yeast display library cyclic peptide Yes Associated Protein (YAP) 65 WW domain transglutaminase |
| url | https://www.mdpi.com/1422-0067/22/4/1634 |
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