Cloning and heterological expression of cDNA fragment of mouse protein kinase gene akt1 in Escherichia coli

V. Zayets, K. Osmanova, I. Krupskaya
Taras Shevchenko National University of Kyiv, Kyiv; Institute of Molecular Biology and Genetics of NASU, Kyiv; Institute of Molecular Biology and Genetics of NASU, Kyiv

Abstract


Protein kinase Akt / PKB is serine / threonine-specific protein kinase B, which has three isoforms Akt1, Akt2 and Akt3 in the animal's body.
Protein kinase Akt / PKB is the focal point for many signaling pathways and plays a key role in regulating many cellular processes, such as
proliferation and cell survival, glucose metabolism, cell cycle, protein synthesis, and angiogenesis. Akt1 is more involved in the process of
proliferation and cell survival, Akt2 is more specific in the insulin-dependent signaling pathway, and Akt3 is predominantly expressed in the brain.
Disturbance of signaling pathways with the participation of Akt is directly related to the most common and incurable human diseases. It has been
established that hyperactivity Akt1 is a part of the pathological process in several types of the most common malignant tumors of a person.
Deregulation of Akt activity of nuclear transcription factor NF-kB causes the development of autoimmune diseases. Akt-kinase phosphorylation has
been detected in a number of neurodegenerative diseases of the brain such as Alzheimer's disease, Huntington's disease. Cardiovascular diseases
associated with Akt are characterized by a disturbance of the phosphorylation of Akt in signaling systems in various tissues and cells, in particular
in endothelial cells of the vascular system and cardiomyocytes, where Akt regulates cardiac growth, contractile function and coronary
angiogenesis. In our experimental work, using PCR, we obtained a cDNA fragment of the murine Akt1 protein kinase gene, cloned this fragment into
the expressing vector pET42a, and optimized expression of the recombinant protein in E.coli BL21 strain (DE3). Immunoblot analysis has showed
that the recombinant protein is a delineated Akt1 kinase and can be used after its development to produce polyclonal antibodies to the protein
kinase Akt / PKB to study its role in the molecular mechanisms of signaling pathways for apoptosis of human cardiomyocytes.


Keywords


protein kinase Akt , cloning, bacterial expression.

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