E-score

Activity

Ligase Acitivity

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Sanger Sequencing , Illumina Sequencing , Deep Mutational Scanning , Phage Display , PCR , barcode-directed subassembly

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Using 163,829 unique protein variants identified from high-throughput sequencing and subassembly, we performed successive rounds of selection for Ub ligase activity. Each round of selection consisted of in vitro phage auto-ubiquitination reactions with an E1, the E2 UbcH5c, and Flag-Ub, followed by enrichment of the phage on anti-Flag agarose beads and reamplification of the phage in Escherichia coli. We determined the frequency at which each variant was present in the starting population and in the population after three rounds of selection. The ratio of the selected frequency of each variant vs. its starting frequency, called here the “enrichment ratio” or E, provides a measure of the performance of each variant during the selection for E3 function. All E scores in this study are normalized to the performance of the WT U-box (E = 1.06).

The Enrich software package was used to determine the locations and identity of mutations in the assemblies, as well as the frequency at which that each variant appeared in the population. A nonspecific carryover correction factor was applied to the tally of each variant in the selected population (refer to Jomla A, et al, Genome Res, 2010, 20:861-873). The corrected frequencies were used to calculate E scores and WT normalized E scores for variants found in both the input and selected populations.