Characterisation of a split GFP system: the effect of pH, salt and strand length on reintroduction kinetics of GFP’s strand 10

A circularly permutated variant of Green Fluorescent Protein (GFP), with strand 10 in the N- terminal of the protein, was successfully expressed and purified. Trypsin digest, followed by size exclusion chromatography under denaturing conditions lead to the successful removal of strand 10 from the remainder of GFP (truncated GFP). Upon complementation of truncated GFP with synthetic strand 10, the complex regained fluorescence, best explained by a double exponential association fit. The kinetics and fluorescence were pH dependent, but not affected significantly by up to 500 mM NaCl. A strand 10 that did not lead to fluorescence upon complementation was shown to still bind, and affect the kinetics upon adding a strand 10 that lead to fluorescence. Complementation was shown to be dependent on strand length, with three different lengths tested. The shortest strand exhibited the fastest kinetics. Based on the studies, a binding mechanism containing two parallel pathways was suggested.