Directed evolution to improve protein folding in vivo

Abstract

Recently, several innovative approaches have been developed that allow one to directly screen or select for improved protein folding in the cellular context. These methods have the potential of not just leading to a better understanding of the in vivo folding process, they may also allow for improved production of proteins of biotechnological interest.

Figure 1

Selection and screening systems for improved protein folding in vivo. (a) An inherent, measurable function (e.g. enzymatic, chromogenic reaction) of a protein of interest (POI) is exploited to screen for stabilized protein variants. (b) A screen or selection for stabilized POI variants is enabled by fusion to a reporter protein with a measurable function. (c) Tight interaction of two portions of POI variants leads to proper complementation of fused fluorescent reporter protein portions and thereby allows screening for stabilized protein variants. Unstable protein variants will be depleted from the screen by degradation. (d) Split halves of a reporter protein will only interact and confer its inherent selectable/screenable function if the inserted protein variant folds well and doesn’t get proteolyzed or aggregated. (e) The twin arginine translocation (Tat) system relies on translocation of exclusively well-folded protein variants into the periplasm where proper folding is additionally selected for by a fused β-lactamase tag. POI, protein of interest; POI_a/POI_b, POI_a half or POI_b half, respectively, of split POI; R_a/R_b, R_a half or R_b half, respectively, of split reporter protein R; P, proteolysis; Tat, twin arginine translocase; ss, Tat signal sequence; β-lac, β-lactamase resistance marker.