Fig. 2.

Directed evolution of dual-loop-inserted GFPM mutants that resist thermal denaturation. (A) Flow cytometric evaluation of resistance to thermal denaturation for non-loop-inserted GFPM (i and ii) and dual-loop-inserted mutant 37-2-7 (iii and iv). Full-length expression was monitored using the C-terminal c-myc epitope (PE fluorescence). Expression and GFP fluorescence were monitored both at the permissive temperature of 20°C and after 30 min at the denaturing temperature of 70°C. For panels i–iv, inset GFP values are the geometric mean GFP fluorescence of the entire displaying population. Also depicted are flow cytometric plots of the mutagenic dual-loop-inserted library after denaturation for 30 min at 70°C (v) and after four rounds of enrichment (vi) using a sorting gate similar to that depicted on the panels to recover mutants with template-like expression (high PE) and improved resistance to thermal denaturation (high GFP). Insets indicate the GFP mean of the entire high PE population. (B) Quantified flow cytometric data denoting full-length expression (c-myc) and yeast cell surface-localized GFP fluorescence per molecule (GFP/c-myc). It is important to note the GFP fluorescence comprises both intracellular and yeast cell surface contributions. For the GFP/c-myc quantity, only the cell surface GFP contribution is quantified so that per molecule fluorescence properties (per cell surface c-myc) can be monitored during the evolution process. The resistance to denaturation is presented as the ratio of GFP fluorescence after 30 min at 70°C to that at the permissive temperature of 20°C. Data are derived from triplicate yeast transformants and normalized to non-loop-inserted GFPM for comparison. (C) GFP structure denoting newly added mutations found in the evolved, denaturation resistant dual-loop-inserted GFPM molecules. Mutant 70C-3 only carries the Y151C mutation denoted in 70C-9 structure. Loop insertion sites are indicated at positions 101–102 (red) and 172–173 (blue).