Expression Of Adenylate Kinas E Fused MEK1R4F In Escherichia Coli And Its Application In ERK Phosphorylation

MAPK signal transduction pathways mainly transduce extracellular signals(such as growth factors and cytokines)to elicit a variety of cellular responses including growth,differentiation,inflammation and apoptosis.Three parallel MAPKs signaling pathways have been found in mammalian cells,including ERK(extracellular signal-regulated kinase)signaling pathway,JNK / SAPK pathway and p38 MAPK pathway.Each pathway is composed of three classes of protein kinases: MAPK,MAPK kinase and MAPK kinase kinase.The best charatecrized is Ras/Raf/MEK/ERK pathway.Mitogen activated protein kinase kinase 1(MKK1 or MEK1)is a member of the MAP kinase pathway.Because it is an important factor governing cellular proliferation and differentiation,MEK1 is an attractive target for cancer therapy.It can phosphorylate its downstream substrats ERK1 and ERK2 at conserved threonine and tyrosine residues.Activation of ERK is one of the immediate responses of mitogen stimulation and displays a significant role on ERK itself localization in nucleus and carcinoma cell migration.Most protein kinases have activity until they are activated through phosphorylation.A number of upstream kinases can be able to phosphorylate MEK1 in vitro: Mos,MEKK,and members of the Raf family.The physiological upstream kinase of MEK1 is Raf-1.Raf-1 is composed of two functionally distinct domains,an amino-terminal regulatory domain and a carboxy-terminal kinase domain.Deletion of the amino-terminal domain of Raf-1(Raf-BXB)results in the constitutive activation of the catalytic domain.Raf BXB can also activate MEK1.In this study,we use the constitutively active form of MEK1,MEK1R4 F as object of study.The aim of present study is the expression,purification and characterization of recombinant human MEK1 protein.In the previous study,MEK1 expression was reported in insect cells and Escherichia coli.While the expression levels of MEK1 were very low in Escherichia coli.Here we show that fusion a soluble tag adenylate kinase(Adk)to the N-terminus of MEK1R4F(constitutively active form of MEK1)can enhance its expression level and downstream purification.Previous studies showed that adenylate kinase(Adk)is an essential phosphotransferase enzyme expressed in all living cells from bacteria to mammals and it plays an important role in cellular energy homeostasis and in adenine nucleotide metabolism.It can catalyze the reversible transfer of the terminal phosphate group between ATP and AMP.Adk from E.coli not only be expressed in soluble and active form at high level with little toxicity to the host cells,but also the enzyme could be easily purified via affinity elution off Ni-TED beads.The resulting Adk-MEK1R4 F fusion protein was highly expressed in E.coli,and can be purified at 95% purity via two purification steps including Ni-NTA and QFF chromatography.The purified Adk-MEK1R4 F protein kinase not only is fully active for ERK phosphrylation,but also can use ADP as energy source in addition to ATP.Moreover,we also found that the Adk-MEK1R4 F has higher catalytic activity than MEK1R4 F kinase in vitro and in cell-free extract system,so we can acquire more phosphrylated ERK using Ni-NTA column.Thus the method may be applied to promote recombinant protein expression as well as their downstream purification.