Figure 5
Schematic representation of the sequential steps to obtain MBP:GFP1-10fast from ffGFP. (A) The fast-folding variant GFP (ffGFP) was fused with GFP, and it was ascertained that the fusion retained the same properties as the original ffGFP; (B) Production of the MBP:GFP1-10 fragment, which demonstrated an inability to complement with GFP11; (C) After introducing the amino acid substitutions indicated in MBP:GFP1-10s1 through site-directed mutagenesis, in vivo fluorescence was achieved within our split-GFP system; (D) The first round of system evolution was conducted through epPCR, resulting in the selection of a MBP:GFP1-10s2 variant exhibiting an approximate 70% increase in fluorescence; (E) The second round of evolution was carried out via ePCR, leading to the identification and selection of two variants displaying an additional 10% enhancement in fluorescent signal. The preferred variant, designated MBP:GFP1-10s4, was ultimately named MBP:GFP1-10fast.
