Phosphorylation-mediated Assembly Of Semisynthetic Fluorescent Protein For Label-free Detection Of Protein Kinase And Protein Phosphatase Activity

Protein phosphorylation catalyzed by protein kinases plays a critical role in many intracellular processes, and the abnormal of phosphorylation process is closely associated with various human diseases such as cancer, diabetes and heart disease.Protein kinase and protein phosphatase take important roles in protein phosphorylation.Thus, developing new and label-free assays for detecting kinase and phosphatase are not only helpful for the knowledge of life but have significant roles in biochemical research and drug discovery.Green fluorescent protein is a 238 aa protein which can be used as fusion tag and indicator in molecular biology and cell biology owing to its auto-fluorescent property.Bimolecular fluorescence complementation(BiFC) techniques based on GFP has the advantages of nontoxic and biocompatible which has been used in many fields such as the detection of protein and protein interaction, soluble expression of protein and so on.However, the application of BiFC in phosphorylation assays are relative sacre, thus developing a novel method for phosphorylation on the basis of BiFC is of great significance.In this paper, we developed a novel fluorescent biosensor to detect protein kinase and protein phosphatase activity based on phosphorylation-mediated assembly of green fluorescent protein. These methods are summarized as below.1. Truncated GFP(tGFP) can be obtained by two methods. One is called molecular cloning method which is based on construction of a tGFP recombinant plasmid. The other is based on the digestion of entire GFP and removing S10 after urea denaturation. We compared the quality of tGFP obtained by these two assays as well as the complementation outcome of tGFP and the synthetic S10 peptide.2. Based on the complementation of tGFP and S10 pairs, we developed a novel method for PKA activity detection by adding an PKA substrate peptide at the C-terminal of S10 peptide(S-peptide). CPY can recognize the phosphorylated peptide incubated by PKA and unphosphorylated peptide. Phosphorylation hinder the CPY digestion of S-peptide which enables S-peptide attach to tGFP and fluorescence recovery. Hence, the fluorescence intensity is positive to the PKA activity.3. Based on the same principle of second work, we modified the S-peptide with a phosphate group(P-peptide) for detection of protein phosphatase 1(PP1). AfterP-peptidedephosphorylated by PP1, CPY digested the peptide leading the fail reassembly of P-peptide and tGFP. Therefore, the fluorescence intensity of the complex presents negative correlation to PP1 activity.