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| Status |
Public on May 09, 2016 |
| Title |
Computationally designed, high specificity inhibitors delineate the roles of BCL2 family proteins in cancer |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
In the related work, proteins were computationally designed and experimentally optimized to bind with high affinity and specificity to each human pro-survival BCL2 homolog. Site-directed saturation mutagenesis libraries were generated based on partially-specific computational designs 2CDP06 (for Bcl-2-targeting Computationally Designed Protein), XCDP07 (Bcl-xL), WCDP03 (Bcl-w), and FCDP01 (Bfl-1), and BCDP01 (Bcl-B) which were then transformed into yeast for surface display, as described in Chao et al (Nat Protoc. 2006). Binding to labeled target homolog was detected via FACS after co-incubation with unlabeled competitors to favor specific interactions. Similarly, an SSM library was generated based on partially-specific Mcl-1-targeting design MCDP02 and was sorted for specific interactions toward each Bcl-2 proteins (data labeled MCDP02_[homolog]). DNA was harvested from naïve libraries and sorted populations and deep sequenced. The frequency of each mutation in a sorted population was compared to its frequency in the naïve population to determine enrichment ratios for all possible single amino acid substitutions (frequency calculations based on counts, which can be found in analyzed data file along with enrichment ratios). The most enriching mutations were then combined in combinatorial libraries, sorted as described above, and variants present after four or five rounds of sorting exhibited excellent specificity. Please see our corresponding publication for additional detail.
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| Overall design |
For all samples except WCDP03, extracted DNA was amplified from two overlapping regions, generating two smaller fragments per target gene, in order to accommodate a 300-cycle MiSeq kit smaller than the length of our genes of interest (approximately 350 base pairs). For each paired-end sequencing experiment, the forward reads each fragment are combined in a single file (*R1*), as are reverse reads (*R2*). For WCDP03, a 600-cycle MiSeq kit was employed, so we instead amplified the entire gene of interest. SSMs based on 2CDP06, XCDP07, WCDP03, FCDP01 and BCDP01 underwent two rounds of FACS and thus three samples exist per construct: naïve library, sort 1 and sort 2. The MCDP02-based library underwent a single round of FACS toward specific binding for each indicated BCL2 homolog, thus there are seven related samples: naïve library plus six sorted populations. In total, there are 22 samples presented here.
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| Contributor(s) |
Berger S, Procko E, Baker D |
| Citation(s) |
27805565 |
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| Submission date |
Apr 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David Baker |
| E-mail(s) |
dabaker@uw.edu
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| Organization name |
University of Washington
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| Street address |
Molecular Engineering and Sciences, Box 351655
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-1655 |
| Country |
USA |
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| Platforms (1) |
| GPL17143 |
Illumina MiSeq (Saccharomyces cerevisiae) |
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| Samples (22)
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| Relations |
| BioProject |
PRJNA318254 |
| SRA |
SRP073190 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE80194_BCL2_inhibitors_processed_data.xlsx |
574.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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