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. 2021 Oct 19;12:6093. doi: 10.1038/s41467-021-26337-1

Fig. 7. Identification of protein variants with improved folding properties from a PARP1-BRCT-I33N mutant library using the dual-reporter system.

Fig. 7

A random PARP1-BRCT-I33N mutant library was co-expressed with the protein folding sensor. The cell populations were analyzed using FACS 1 h after IPTG induced protein expression. a FACS analysis of PARP1-BRCT WT, PARP1-BRCT-I33N, and the PARP1-BRCT-I33N mutant library, where the mCherry signal correlates with the translation level of PARP1-BRCT, while the GFP fluorescence is a measure of folding properties. Two gates were defined for sorting populations with high translation (Gate 1) and low GFP fluorescence (Gate 2), thus with improved folding properties compared to PARP1-BRCT-I33N. A shift is observed in GFP signal distribution and intensities between the two rounds of sorting, showing that it is possible to enrich the population with low GFP clones after multiple rounds of sorting. b Single clones analyzed after each round of sorting, resulted in 1.5% or 12.5% of the clones overlapping with the PARP1-BRCT WT GFP signal (n = 5 biologically independent samples for PARP1-BRCT WT, PARP1-BRCT-I33N and n = 1 for each of the 75 individual clones isolated from the library). Data are presented as individual data points with the mean value indicated. Source data are provided as a Source Data file.