ACS Synth. Biol. 10.1021/sb5002938

Functional complementation—in which polypeptide fragments are combined to regenerate active proteins, such as with split GFP—can provide a background-free readout for various cellular functions. However, identifying protein fragments that are sufficiently stable to withstand separation and that generate a functional change upon recombination is not straightforward. Pandey et al. now simplify the search for suitable constructs by using transposon mutagenesis to facilitate construction of protein libraries. The authors used either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins as fusion partners for fragments of near-infrared fluorescent protein (IFP) to both stabilize the isolated constructs and drive assembly. Testing 1,760 mutants of each system led to the identification of 13 sites where IFP could be split and successfully recombined. The relative fluorescence of the assembled fragments ranged from ∼15–150% of the signal generated by the wild-type IFP control, with the fusion partners required for optimal assembly. The mutations were found on the linker between IFP's two domains and secondary structure elements on either side of the linker but were not near the chromophore binding site, spurring questions regarding the mechanism of complementation. These results provide new tools for interrogation of cellular systems and useful guidelines for further fragment discovery.