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| Status |
Public on Jun 09, 2017 |
| Title |
Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1 |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
|
| Summary |
We used computational models to design focused combinatorial libraries to identify BH3 peptides that bind tightly and selectively to Bfl-1. The final library was predicted to be Bfl-1 selective by PSSMSPOT and STATIUM (DeBartolo et. al. J Mol Biol. 2012). As a design control, we designed similar libraries to be selective for Bcl-xL and Mcl-1. The libraries were transformed into yeast for surface display, pooled, and screened for tight and selective binding to Bfl-1. Five rounds of positive, negative, and/or competition FACS screening were used to isolate cells that expressed the tightest and most selective peptides. To analyze enrichment trends and to assess the success of our library design, we deep sequenced samples from the naïve pool and from pools collected after 3, 4, 5, and 6 rounds of sorting. Additional detail is available in our corresponding publication.
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| |
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| Overall design |
DNA samples from yeast-surface display peptide libraries were deep sequenced before and after screening for Bfl-1 selective binding using Illumina NextSeq.
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| |
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| Contributor(s) |
Jenson JM, Keating AE |
| Citation(s) |
28594323 |
| |
| Submission date |
Feb 14, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Amy E Keating |
| E-mail(s) |
keating@mit.edu
|
| Organization name |
Massachusetts Institute of Technology
|
| Department |
Biology and Biological Engineering
|
| Lab |
Keating
|
| Street address |
MIT 68-622a
|
| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL19756 |
Illumina NextSeq 500 (Saccharomyces cerevisiae) |
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| Samples (7)
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| Relations |
| BioProject |
PRJNA374664 |
| SRA |
SRP099583 |
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