Fig. 4: Evolved PylB mutants are present at elevated levels inside cells, and show increased protease resistance.

A Quantitative western blot assays were performed on clarified lysates of cells expressing different His-tagged PylB variants. Prior to cell lysis, cultures all found to exhibit similar OD₆₀₀ values (3.1–3.3), colony-forming units (1–3 × 10⁹ CFUs/mL), and cell pellet wet weights (1.2–1.4 g). For each sample, His-tag antibodies were used to quantify PylB, and samples were normalized using the housekeeping gene GAPDH (see “Methods”). These experiments show two evolved mutants, PylB.3f2 and PylB.JM10.1, are present at a higher concentration compared to the ancestral variant SUMO-PylB. These results further show that two codon-deoptimized evolved mutants, PylB.3f2_deopt and PylB.JM10.1_deopt, are also present at higher concentrations compared to the codon-optimized ancestral variant SUMO-PylB, suggesting that the elevated levels of evolved mutant proteins likely do not results from improved codon usage. B Proteolytic degradation assays were performed using purified PylB variants. Protein samples were incubated with the protease enzyme chymotrypsin, and the concentration of PylB was evaluated over time (see “Methods”). Evolved mutants PylB.3f2 and PylB.JM10.1 were found to degrade at a slower rate compared to the ancestral variant SUMO-PylB, indicating increased protease resistance in the evolved mutants. Samples were tested in biological sextuplicate; data shown represent mean values ± s.d. For both figures A and B, p values were calculated using a one-tailed Student’s t-test, without adjustments for multiple comparisons. Source data are provided as a Source Data file.