Location and Orientation of the Genetic Toxin–Antitoxin Element hok/sok in the Plasmid Affect Expression of Pharmaceutically Significant Proteins in Bacterial Cells

Location and Orientation of the Genetic Toxin–Antitoxin Element hok/sok in the Plasmid Affect Expression of Pharmaceutically Significant Proteins in Bacterial Cells

Genetic toxin–antitoxin element hok / sok from the natural Escherichia coli R1 plasmid ensures segregational stability of plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin are killed by the stable toxin. When introduced into bacterial expression ve...

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Bibliographic Details
Published in:Biochemistry (Moscow) Vol. 88; no. 9; pp. 1326 - 1337
Main Authors: Khodak, Yulia A., Shaifutdinov, Rolan R., Khasanov, Danila S., Orlova, Nadezhda A., Vorobiev, Ivan I.
Format: Journal Article
Language:English
Published: Moscow Pleiades Publishing 01-09-2023
Springer
Springer Nature B.V
Subjects:
Analysis
Antibiotics
Antitoxins
Asparaginase
Bacteria
Biochemistry
Biomedical and Life Sciences
Biomedicine
Bioorganic Chemistry
Biosynthesis
Cultivation
E coli
Expression vectors
Gene expression
Genes
Growth hormones
Identification and classification
Inclusion bodies
Kanamycin
Life Sciences
Localization
Microbiology
Orientation
Plasmids
Productivity
Protein biosynthesis
Proteins
Recombinant proteins
Severe acute respiratory syndrome coronavirus 2
Toxins
Toxins and antitoxins
Russia
nucleoprotein
toxin–antitoxin system
heterologous expression of recombinant proteins
asparaginase
somatotropin
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Summary:Genetic toxin–antitoxin element hok / sok from the natural Escherichia coli R1 plasmid ensures segregational stability of plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin are killed by the stable toxin. When introduced into bacterial expression vectors, the hok / sok element can increase the productive time of recombinant protein biosynthesis by slowing down accumulation of non-producing cells lacking the expression plasmid. In this work, we studied the effects of position and orientation of the hok / sok element in the standard pET28a plasmid with the inducible T7lac promoter and kanamycin resistance gene. It was found that the hok / sok element retained its functional activity regardless of its location and orientation in the plasmid. Bacterial cells retained the hok / sok -containing plasmids after four days of cultivation without antibiotics, while the control plasmid without this element was lost. Using three target proteins – E. coli type II asparaginase (ASN), human growth hormone (HGH), and SARS-CoV-2 virus nucleoprotein (NP) – it was demonstrated that the maximum productivity of bacteria for the cytoplasmic proteins (HGH and NP) was observed only when the hok / sok element was placed upstream of the target gene promoter. In the case of periplasmic protein localization (ASN), the productivity of bacteria during cultivation with the antibiotic decreased for all variants of the hok / sok location. When the bacteria were cultivated without the antibiotic, the productivity was better preserved when the hok / sok element was located upstream of the target gene promoter. The use of the pEHU vector with the upstream location of the hok / sok element allowed to more than double the yield of HGH (produced as inclusion bodies) in the absence of antibiotic and to maintain ASN biosynthesis at the level of at least 10 mg/liter for four days during cultivation without antibiotics. The developed segregation-stabilized plasmid vectors can be used to obtain various recombinant proteins in E. coli cells without the use of antibiotics.
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ISSN:0006-2979
1608-3040
DOI:10.1134/S0006297923090122