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Expression of adenylate kinase fused MEK1R4F in Escherichia coli and its application in ERK phosphorylation
- Source :
- Biotechnology Letters. 39:1553-1558
- Publication Year :
- 2017
- Publisher :
- Springer Science and Business Media LLC, 2017.
-
Abstract
- 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.
- Subjects :
- 0301 basic medicine
Recombinant Fusion Proteins
MAP Kinase Kinase 1
Adenylate kinase
Bioengineering
Mitogen-activated protein kinase kinase
Protein Engineering
Applied Microbiology and Biotechnology
MAP2K7
03 medical and health sciences
Adenosine Triphosphate
0302 clinical medicine
Escherichia coli
Humans
c-Raf
Phosphorylation
Protein kinase A
biology
Chemistry
Adenylate Kinase
Cyclin-dependent kinase 2
General Medicine
ADK
Adenosine Diphosphate
030104 developmental biology
Biochemistry
030220 oncology & carcinogenesis
Biocatalysis
biology.protein
Cyclin-dependent kinase 9
Mitogen-Activated Protein Kinases
Biotechnology
Subjects
Details
- ISSN :
- 15736776 and 01415492
- Volume :
- 39
- Database :
- OpenAIRE
- Journal :
- Biotechnology Letters
- Accession number :
- edsair.doi.dedup.....826785d29c3d9684354dcb2e6c666022