Fusion proteins are multi-domain proteins typically created by linking two genes end-to-end through a sequence coding for a peptide linker. Usually, it is expected and desired that the domains function independently. However, fusion proteins in which the functions of the two domains are coupled have potential applications as molecular sensors and selective therapeutics. Previously, we have found that creating fusions in which one domain is inserted into a second can result in proteins with such switch-like behavior. Creation of a switch is depends upon the target protein's ability to accept insertion of another domain. Here, we have explored maltose binding protein's (MBP) tolerance to accept internal insertions of the green fluorescent protein (GFP) and circular permutations of GFP. Several protein libraries were created. The diversity of these libraries derived from random circular permutation of GFP and random or targeted insertions of GFP into MBP. Library members with functional GFP domains were isolated through fluorescent activated cell sorting (FACS). Knowledge of the locations of insertions in functional fusions will guide future efforts to create fusions of MBP and GFP that behave as switches (i.e. maltose alters protein fluorescence).

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