Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control (original) (raw)
Figure 1
Flow cytometric analysis of GFP variants.
Fluorescence histograms for cells expressing: superfolder GFP (sf, gray fill), folding reporter GFP (fr, gray line), and GFPmut2 (mut2, black line) (a) without a signal peptide and a C-terminal 6xhis tag or (b) as an N-terminal fusion to the ssDsbA signal peptide; (c) an N-terminal ssDsbA signal peptide fused to GFPmut2 (black line) and TrxA-GFPmut2 (gray fill). The geometric mean is listed next to each histogram. There was no significant difference in growth rate between any of the cultures (data not shown). (d) Fluorescence (arbitrary units) and subcellular localization of GFP as measured for cytoplasmic (cyt, grey bars) and periplasmic (per, white bars) fractions generated from cells expressing the various ssDsbA-GFP fusions including sf, fr, mut2 and clones P1–P9. (e) Western blot analysis of the per and cyt fractions of cells expressing the same fusions probed with GFP antiserum. Blots were probed with anti-GroEL serum as a fractionation marker.