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. 2008 Jun 11;3(6):e2387. doi: 10.1371/journal.pone.0002387

Figure 6. Directed evolution of protein folding using GFP insertion reporters and application to Rv0113.

Figure 6

(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.