Yang Fang, Li Hongtao
The State Key Laboratory Breeding Base of Bioresources and Eco-environments, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Southwest University, Beibei, 400715, Chongqing, People's Republic of China.
Biotechnol Lett. 2017 Oct;39(10):1553-1558. doi: 10.1007/s10529-017-2385-3. Epub 2017 Jul 26.
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 ~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.
通过融合可溶性腺苷酸激酶（Adk）标签构建高表达且活性高的MEK1R4F（丝裂原活化蛋白激酶激酶1的组成型活性形式），从而获得适用于制备磷酸化细胞外信号调节激酶（ERK）的Adk-MEK1R4F蛋白。
我们通过重叠PCR将Adk融合到MEK1R4F的N端。Adk-MEK1R4F融合蛋白在大肠杆菌中的表达增加了约10倍，并通过镍-次氮基三乙酸（Ni-NTA）和Q Sepharose快速流动（QFF）层析两步纯化至95%。纯化后的Adk-MEK1R4F蛋白对ERK磷酸化具有功能活性，除了ATP外还能利用ADP。在体外和无细胞提取物系统中，Adk-MEK1R4F均比常规MEK1R4F具有更高的催化活性。
腺苷酸激酶被用作可溶性标签，以促进MEK1R4F蛋白的表达及其在大规模磷酸化ERK1/2制备和纯化中的应用。
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