Cloning,Expression,Purification And Characterization Of Protein Kinase As Potential Herbicide Target

Herbicides play an important role in the agricultural production,however,because of the long-term and large-scale usage of the herbicides,weed resistance,non-target biological safety and environmental pollution have become increasingly serious problems.Therefore,finding novel herbicide targets has become the most important research topic to solve weed resistance.Plant protein kinases are mostly serine/threonine/tyrosine phosphotransferases,which are capable of transporting the y-phosphoryl group of adenosine triphosphate(ATP)to the substrate and phosphorylating the substrate protein.Studies have shown that most protein kinases are widely involved in a series of plant life activities,such as plant stress resistance,signal transduction,growth and development,therefore,protein kinases are regarded as novel potential herbicide targets.In this paper,glucokinase(GK)and riboflavin kinase/flavin mononucleotide hydrolase bifunctional protein(ATFMN/FHY)from Arabidopsis thaliana,which have not been reported with homologous structures,were selected as research targets.We cloned these protein kinase genes from Arabidopsis thaliana cDNA library by polymerase chain reaction(PCR),linked them to pET-28a vector,and constructed corresponding recombinant expression plasmids sucessfully.The protein kinases were expressed both as inclusion bodies,so we denatured the proteins with 6 M guanidine hydrochloride,refolded and purified them with Ni-NTA affinity chromatography,respectively.The kinase activities were determined by high performance liquid chromatography(HPLC).Results have shown that both two recombinant proteins can catalyze the phosphorylation reactions.The enzymatic properties of these two kinases were characterized at the same time.It was found that,the glucokinase showed max activity at 55?,with optimum pH of 9.0,and selectivity of divalent metal ion of Zn2+.The apparent Km of GK for ATP was determined as 0.337 mmol/L.ATFMN/FHY is a bifunctional protein,which contains riboflavin kinase and flavin mononucleotide hydrolase domains,respectively.The riboflavin kinase showed max activity at 42°C,with optimum pH of 8.5,and selectivity of divalent metal ion of Mg2+.The apparent Km of ATFMN for riboflavin and ATP were determined as 0.0694 mmol/L and 0.260 mmol/L,respectively.Whereas,flavin mononucleotide hydrolase showed max activity at 46?,with optimum pH of 6.0,and selectivity of divalent metal ion of Zn2+.The apparent Km of FHY for FMN was determined as 0.083 mmol/L.