To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK. We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased similar to 10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni-NTA and Q Sepharose fast flow (QFF) chromatography. The purified Adk-MEK1R4F protein was functional for ERK phosphorylation and could use ADP in addition to ATP. The Adk-MEK1R4F had higher catalytic activity than regular MEK1R4F both in vitro and in cell-free extracts system. Adenylate kinase was used as a soluble tag to facilitate MEK1R4F protein expression and its application in large-scale phosphorylated ERK1/2 preparation and purification.