Study Of A New Labeling Enzyme And A Method For Detection Of Protein Kinase Activities

This study developed a new labeling enzyme and a method for detection ofprotein kinases activities. Methyl parathion hydrolase (MPH), isolated from the soildwelling bacterium Pseudomonas sp. WBC-3 by our lab, is an enzyme that catalyzesdegradation of methyl parathion, which generates yellow product with a maximumabsorption at 405 nm. In the study of a new labeling enzyme, we identified thepotential capacity of MPH as a new labeling enzyme by analysis of its characteristics,including the molecular weight, state in solution, thermostability, storage stability, theinitial reaction speed. In order to validate the feasibility of MPH as a new labelingenzyme, MPH was ligated to the A1E scFv against White Spot Syndrome Virus(WSSV) with insertion of a [-(Gly-Ser)₅-] linker through gene splicing. With theELISA format, the resulting fusion protein was successfully used to detect WSSV. Inthe study of a method for detection of protein kinase activities, we developed aFe³⁺-NTA modified chip which is a kind of universal phosphorylated peptide (orphosphorylated protein) recognition element. This method could overcome thedisadvantages of methods of protein kinase assay based on phosphospecific antibodies.Phosphopeptides and their corresponding non-phosphopeptides containing differentphosphoamino acid, which were acting as the substrates and products of differentkinds protein kinases, were detected by the Fe³⁺-NTA chip to identified the universalcapacity of recognizing the phosphopeptides. All phosphopeptides were successfullydetected from nanomole to micromole concentrations with the Fe³⁺-NTA modifiedchip. Furthermore, using three different protein kinase, including Glycogen synthasekinase3β(GSK3β), Epidermal growth factor receptor (EGFR) and Cyclin-dependentKinase (CDK), and their specific peptide substrate as model, we performed theprotein kinase reaction in vitro. Phosphorylation efficiency of different potein kinaseswas evaluated with the Fe³⁺-NTA modified chip.