Abstract
Objective
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.
Results
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.
Conclusions
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.
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Acknowledgements
This work was supported by the Natural Science Foundation of China (30970630 and 31270831), the Outstanding Youth Science Foundation of Chongqing (cstc2011jjjq10003), and the Program for New Century Excellent Talents in University (NCET-08-0912), the Fundamental Research Funds for the Central Universities (XDJK2016C158) and the Doctoral Fund of Southwestern University (XJKJXM003338).
Supporting information
Supplementary Table 1—The primers used to construct the protein expression plasmids.
Supplementary Table 2—Summary of His-Adk-MEK1R4F purification.
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Yang, F., Li, H. Expression of adenylate kinase fused MEK1R4F in Escherichia coli and its application in ERK phosphorylation. Biotechnol Lett 39, 1553–1558 (2017). https://doi.org/10.1007/s10529-017-2385-3
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DOI: https://doi.org/10.1007/s10529-017-2385-3