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

Abstract

Protein phosphorylation catalyzed by protein kinases plays a critical role in many intracellular processes, and detecting kinase activity is important in biochemical research and drug discovery. Herein, we developed a novel fluorescent biosensor to detect protein kinase activity based on phosphorylation-mediated assembly of semisynthetic green fluorescent protein (GFP). A chimaera S-peptide composed of the 10th β-strand of GFP (s10) and a kinase substrate peptide was synthesized. Kinase-catalyzed phosphorylation of the S-peptide can protect its s10 part against cleavage by carboxypeptidase Y (CPY). Then, the peptide can bind the truncated GFP (tGFP, GFP without s10) to assemble intact GFP and recover fluorescence. Unphosphorylated S-peptide would be degraded by CPY, and fluorescent protein assembly could not occur. Thus, the kinase-catalyzed phosphorylation can switch on the fluorescence signal. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase with a low detection limit of 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 demonstrated by assessment of the kinase activity and inhibition in the cell lysate. Moreover, based on the reverse principle, this method was expanded to detect the activity of protein phosphatase 1. Our method, using semisynthetic GFP as a readout, is facile, sensitive, label-free, and highly versatile, thus showing great potential as a promising platform for protein kinase detection and inhibitor screening.

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

“…Moreover, the signal of P‐PKAtide slightly increased with the forskolin and IBMX amount increasing (Fig. ), indicating that higher PKA level would be induced with more stimulators in MCF‐7 cell, consistent with previous reports . However, no obvious peak of P‐CDK1tide could be observed, probably due to the two stimulators limited stimulation ability on CDK1 activity in MCF‐7 cell.…”

“…Although heterogeneous kinase assays based on electrochemistry , surface plasmon resonance have also been developed, homogeneous analysis exhibits high sensitivity, and more simplicity without tedious surface rinsing steps. Nowadays, some kinds of fluorophore such as green fluorescent protein (GFP) or nanomaterials such as quantum dots have been proposed, and multiple fluorescence techniques, for example, fluorescence polarization , fluorescence resonance energy transfer (FRET) , and fluorescence lifetime techniques , have also been developed for kinase sensitive analysis. However, they suffer from complicated probe preparation and difficulty for throughput analysis, as well as throughput screening of kinase inhibitors.…”

Integration of cell-free protein synthesis and purification in one microfluidic chip for on-demand production of recombinant protein

“…Previously, a microfluidic chip integrating positive and negative selection units for screening aptamers was developed by our group. 43,44 The microfluidic chip was uncomplicated and could be used repeatedly. Here, we integrated two functional units, i.e., a protein synthesis unit and a protein purification unit, into the microfluidic chip, allowing pure proteins to be obtained without offline purification (Fig.…”

“…Copyright 2015 American Chemical Society. 44 (DNase/RNase-free) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai). Other chemical reagents were purchased from Sinoreagent (Shanghai).…”

A label-free fluorescence assay for thrombin activity analysis based on fluorescent protein and gold nanoparticles

“…Green fluorescent protein (GFP) is widely used in modern life science because of its intrinsic fluorescence and genetically encoded properties. 13 Its chromophore is formed in an autocatalytic cyclization process that does not require a cofactor, which enable GFP being used in living systems with high biocompatibility. 14,15 In addition, its molecule structure has been rebuilt to obtain different kinds of mutants, which show different fluorescence emissions, exceptional fluorescence intensity, or extra stability against protein aggregation.…”