New Molecular Reporters for Rapid Protein Folding Assays
Figure 6
Directed evolution of protein folding using GFP insertion reporters and application to Rv0113.
(A) General strategy for improving protein folding from multiple reporters with increased stringency (see text for detailed explanation). (B) Directed evolution of Rv0113 variant in GFPi 9/8 vector family. Constructs evaluated in X-FR (row 1) and the four GFPi 9/8 constructs (rows 2–5). Images of E. coli colonies expressing wild type Rv0113 in the indicated reporters (column A), brightest optimum after three round of evolution in GFPi 9/8_FR/SF expressed in the indicated reporters (column B), and brightest optimum after two additional rounds in GFPi 9/8_FR/FR (column C). All constructs expressed at 37°C. Exposure time is 2 s. The SDS-PAGE gel (bottom) shows soluble (S) and pellet (P) fractions for each variant (columns A, B, C) cloned without the fused GFP domains in a C-terminal polyhistidine pET vector, expressed in shake cultures at 37°C.