Fig. 5: Quantification of degraded library fractions following cell-free expression in the presence of Lon protease.
From: Experimental characterization of de novo proteins and their unevolved random-sequence counterparts
a,b, Total (T) and soluble (S) expression with cotranslational addition of DnaK and/or Lon protease at 37 °C: triplicate western blots shown for libraries DN (a) and R (b). Non-specific cleavage of hydrophobic regions by Lon protease results in preferential degradation of disordered proteins, with a visible net reduction in yield for Lon+ samples. c, Quantification of degraded fractions with respect to solubility reveals a greater IDP-like (soluble/degraded) fraction for putative de novo proteins versus ‘true’ random sequences. DnaK addition, however, results in a greater increase in the soluble/undegraded fraction than the IDP-like fraction (for both DN and R). d, Summary of degraded versus undegraded fractions, regardless of solubility (sum of dark and light bars in c, respectively). Library R is marginally less degradable than DN, suggesting slightly higher structural propensity (one-tailed t-test, R/R + DnaK versus DN/DN + DnaK, P = 1.67 × 10−2).
