Location and orientation of the genetic toxin-antitoxin element hok/sok in the plasmid affects the expression level of pharmaceutically significant proteins

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Тек жазылушылар үшін

Аннотация

The genetic toxin-antitoxin element hok/sok from the natural Escherichiacoli R1 plasmid ensures the segregation stability of the plasmids. Bacterial cells that have lost all copies of the plasmid encoding the short-lived antitoxin die under the action of the long-lived toxin. The hok/sok element in vector plasmids for bacterial expression can increase the productive time of biosynthesis of recombinant proteins, slowing down the accumulation of non-producing cells lacking the target plasmid in the population. In this work, we studied various variants of the position and orientation of the hok/sok element in the standard plasmid pET28a with the inducible T7lac promoter and the kanamycin resistance gene. It was found that the hok/sok element retained functional activity regardless of location on the plasmid and orientation, bacterial cells retained hok/sok plasmids after four days of cultivation without antibiotics and lost the control plasmid without this element. Using the example of three target proteins - E. coli type II asparginase, human growth hormone, and the SARS-CoV-2 virus nucleoprotein, it was demonstrated that for cytoplasmic target proteins, the maximum productivity of bacteria is maintained only when the hok/sok element is located on the plasmid upstream of the target gene promoter. In the case of periplasmic localization of the protein, the productivity of bacteria decreases for all variants of the hok/sok location during cultivation with an antibiotic, and in the case of periodic cultivation of bacteria without an antibiotic, productivity is also better preserved when the hok/sok element is located upstream of the target gene promoter. This variant of the pEHU vector plasmid makes it possible to more than double the biosynthesis of human growth hormone, which is insoluble in the cytoplasm of bacteria, when bacteria are cultivated without antibiotics, and also to maintain asparaginase biosynthesis during periodic cultivation without antibiotics for four days at a level of at least 10 mg/liter. The developed segregation-stabilized plasmid vector can be used to obtain various recombinant proteins in E. coli cells without the use of antibiotics.

Авторлар туралы

Yu. Khodak

Institute of Bioengineering, FRC Biotechnology, Russian Academy of Sciences

117312 Moscow, Russia

R. Shaifutdinov

Institute of Bioengineering, FRC Biotechnology, Russian Academy of Sciences

117312 Moscow, Russia

D. Khasanov

Institute of Bioengineering, FRC Biotechnology, Russian Academy of Sciences

117312 Moscow, Russia

N. Orlova

Institute of Bioengineering, FRC Biotechnology, Russian Academy of Sciences

117312 Moscow, Russia

I. Vorobiev

Institute of Bioengineering, FRC Biotechnology, Russian Academy of Sciences

Email: ptichman@gmail.com
117312 Moscow, Russia

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