Phosphorylation-Mediated Assembly of a Semisynthetic\nFluorescent Protein for Label-Free Detection of Protein Kinase Activity

Abstract

Protein\nphosphorylation catalyzed by protein kinases plays a critical\nrole in many intracellular processes, and detecting kinase activity\nis important in biochemical research and drug discovery. Herein, we\ndeveloped a novel fluorescent biosensor to detect protein kinase activity\nbased on phosphorylation-mediated assembly of semisynthetic green\nfluorescent protein (GFP). A chimaera S-peptide composed of the 10th\nβ-strand of GFP (s10) and a kinase substrate peptide was synthesized.\nKinase-catalyzed phosphorylation of the S-peptide can protect its\ns10 part against cleavage by carboxypeptidase Y (CPY). Then, the peptide\ncan bind the truncated GFP (tGFP, GFP without s10) to assemble intact\nGFP and recover fluorescence. Unphosphorylated S-peptide would be\ndegraded by CPY, and fluorescent protein assembly could not occur.\nThus, the kinase-catalyzed phosphorylation can switch on the fluorescence\nsignal. This platform has been successfully applied to detect the\nactivity of cAMP-dependent protein kinase with a low detection limit\nof 0.50 mU/μL and its inhibition of H-89 with an IC₅₀ value of 23.4 nM. The feasibility of this method has been further\ndemonstrated by assessment of the kinase activity and inhibition in\nthe cell lysate. Moreover, based on the reverse principle, this method\nwas expanded to detect the activity of protein phosphatase 1. Our\nmethod, using semisynthetic GFP as a readout, is facile, sensitive,\nlabel-free, and highly versatile, thus showing great potential as\na promising platform for protein kinase detection and inhibitor screening.