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 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.

High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors.

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the ß-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.

AFM study of Escherichia coli RNA polymerase σ7° subunit aggregation.

The self-assembly of Escherichia coli RNA polymerase σ7° subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ7° subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ7° polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ7° subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ7° subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. FROM THE CLINICAL EDITOR: In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ7°( subunit was investigated using atomic force microscopy and other complementary approaches.