Biosynthesis of Escherichia coli adenosine deaminase using cell-free protein synthesis
https://doi.org/10.29235/1029-8940-2021-66-3-271-276
Abstract
One of the recent perspective trends of molecular biotechnology is cell-free protein synthesis (CFPS). The procedure of CFPS is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes.
Previously, we constructed a strain Escherichia coli that produces homologous adenosine deaminase (ADase). In the present study, as an alternative to canonical submerged cultivation in a fermenter, the possibility of the ADase synthesis in the system of CFPS was studied. For synthesis of this enzyme we used the E. coli-30 cell extract, T7 bacteriophage RNA polymerase, and high-copy plasmid vector pET42mut with gene ADase inserted into it.
As a result of the work we have demonstrated for the first time the possibility of synthesis of ADase E. coli in the CFPS system. In a partially optimized process conditions, an experimental sample of recombinant AD with an activity of 530 U/ml of enzyme preparation was obtained.
Keywords
adenosine deaminase,
Escherichia coli,
cell-free protein synthesis,
high-copy pET42mut plasmid,
enzyme preparation
Supporting agencies: The work was supported by the grant 3.13 from the Belarus State Research Program “Microbial Biotechnologies”, 2016-2020.
About the Authors
I. S. Kazlouski
Institute of Microbiology of the National Academy of Sciences of Belarus
Belarus
Belarus
Illia S. Kazlouski - Master of Biology, Researcher, Institute of Microbiology of the National Academy of Sciences of Belarus.
2, Kuprevich Str., 220141, Minsk.
A. I. Zinchenko
Institute of Microbiology of the National Academy of Sciences of Belarus
Belarus
Belarus
Anatoli I. Zinchenko - Corresponding Member, D. Sc. (Biol.), Professor, Head of the Laboratory, Institute of Microbiology of the National Academy of Sciences of Belarus.
2, Kuprevich Str., 220141, Minsk.
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