The dangerous liaisons in innate immunity involving recombinant proteins and endotoxins: Examples from the literature and the Leptospira field.

Endotoxins, also known as lipopolysaccharides (LPS), are essential components of cell walls of diderm bacteria such as Escherichia coli. LPS are microbe-associated molecular patterns that can activate pattern recognition receptors. While trying to investigate the interactions between proteins and host innate immunity, some studies using recombinant proteins expressed in E. coli reported interaction and activation of immune cells. Here, we set out to provide information on endotoxins that are highly toxic to humans and bind to numerous molecules, including recombinant proteins. We begin by outlining the history of the discovery of endotoxins, their receptors and the associated signaling pathways that confer extreme sensitivity to immune cells, acting alone or in synergy with other microbe-associated molecular patterns. We list the various places where endotoxins have been found. Additionally, we warn against the risk of data misinterpretation due to endotoxin contamination in recombinant proteins, which is difficult to estimate with the Limulus amebocyte lysate assay, and cannot be completely neutralized (e.g., treatment with polymyxin B or heating). We further illustrate our point with examples of recombinant heat-shock proteins and viral proteins from severe acute respiratory syndrome coronavirus 2, dengue and HIV, for which endotoxin contamination has eventually been shown to be responsible for the inflammatory roles previously ascribed. We also critically appraised studies on recombinant Leptospira proteins regarding their putative inflammatory roles. Finally, to avoid these issues, we propose alternatives to express recombinant proteins in nonmicrobial systems. Microbiologists wishing to undertake innate immunity studies with their favorite pathogens should be aware of these difficulties.

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections.

Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.

In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases.

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors.

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.

Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising candidate for treating metabolic disorder diseases and has been used in phase II clinical trials. Currently, metabolic diseases are prevalent worldwide, underscoring the significant market potential of FGF21. Therefore, the production of FGF21 must be effectively improved to meet market demand. RESULTS: Herein, to investigate the impact of vectors and host cells on FGF21 expression, we successfully engineered strains that exhibit a high yield of FGF21. Surprisingly, the data revealed that vectors with various copy numbers significantly impact the expression of FGF21, and the results showed a 4.35-fold increase in expression levels. Furthermore, the performance of the double promoter and tandem gene expression construction design surpassed that of the conventional construction method, with a maximum difference of 2.67 times. CONCLUSION: By exploring engineered vectors and host cells, we successfully achieved high-yield production of the FGF21 strain. This breakthrough lays a solid foundation for the future industrialization of FGF21. Additionally, FGF21 can be easily, quickly and efficiently expressed, providing a better tool and platform for the research and application of more recombinant proteins.

Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants.

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species-Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus. KEY POINTS: • PVX-based expression vectors provide efficient transient expression of infα2b gene • N. benthamiana plants can produce human interferon alpha 2b at high levels • The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight.

Antifungal recombinant psoriasin of human origin effectively inhibits fungal growth on denture base.

OBJECTIVE: To evaluate the efficacy of recombinant psoriasin as a novel treatment for oral candidiasis by eliminating Candida albicans growth on polymethyl methacrylate denture base. MATERIALS AND METHODS: Recombinant psoriasin protein was expressed and purified from E. coli, and Candida growth was monitored in vitro with varying concentrations of psoriasin. Subsequently, denture-base polymethyl methacrylate was immersed in psoriasin's solution or voriconazole, and fungal growth on the acrylic base and in the medium was examined by scanning electron microscopy and optical density, respectively. Cellular viability of HeLa and human gingival fibroblast cells treated with psoriasin was measured by methylene blue assay. RESULTS: The findings reveal an effective antifungal activity of psoriasin, completely inhibiting Candida albicans growth in RPMI at a protein concentration above 400 nM. Immersing the polymethyl methacrylate with 50 µM psoriasin completely eradicates fungal growth. Psoriasin has low cytotoxicity in HeLa cells at a concentration higher than 12 µM and no toxic effect on human gingival fibroblasts. CONCLUSIONS: This study marks psoriasin as an effective alternative to conventional antifungal treatments for denture stomatitis and a safe alternative to chemical antifungals in dental medicine and beyond.

Comparative RNA-Seq of Ten Phaeodactylum tricornutum Accessions: Unravelling Criteria for Robust Strain Selection from a Bioproduction Point of View.

The production of biologics in mammalian cells is hindered by some limitations including high production costs, prompting the exploration of other alternative expression systems that are cheaper and sustainable like microalgae. Successful productions of biologics such as monoclonal antibodies have already been demonstrated in the diatom Phaeodactylum tricornutum; however, limited production yields still remain compared to mammalian cells. Therefore, efforts are needed to make this microalga more competitive as a cell biofactory. Among the seventeen reported accessions of P. tricornutum, ten have been mainly studied so far. Among them, some have already been used to produce high-value-added molecules such as biologics. The use of "omics" is increasingly being described as useful for the improvement of both upstream and downstream steps in bioprocesses using mammalian cells. Therefore, in this context, we performed an RNA-Seq analysis of the ten most used P. tricornutum accessions (Pt1 to Pt10) and deciphered the differential gene expression in pathways that could affect bioproduction of biologics in P. tricornutum. Our results highlighted the benefits of certain accessions such as Pt9 or Pt4 for the production of biologics. Indeed, these accessions seem to be more advantageous. Moreover, these results contribute to a better understanding of the molecular and cellular biology of P. tricornutum.

MS785-MS27 Reactive Misfolded/Non-Native Zn-Deficient SOD1 Species Exhibit Cytotoxicity and Adopt Heterozygous Conformations in Motor Neurons.

Misfolding of superoxide dismutase-1 (SOD1) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) with SOD1 mutations. The development of antibodies specific for misfolded SOD1 deepens our understanding of how the protein participates in ALS pathogenesis. Since the term "misfolding" refers to various disordered conformers other than the natively folded one, which misfolded species are recognized by specific antibodies should be determined. Here, we molecularly characterized the recognition by MS785-MS27, an antibody cocktail experimentally confirmed to recognize over 100 ALS-linked SOD1 mutants. Indirect ELISA revealed that the antibody cocktail recognized Zn-deficient wild-type and mutated SOD1 species. It also recognized conformation-disordered wild-type and mutated SOD1 species, such as unfolded and oligomeric forms, but had less affinity for the aggregated form. Antibody-reactive SOD1 exhibited cytotoxicity to a motor neuron cell model, which was blocked by Zn treatment with Zn-deficient SOD1. Immunohistochemistry revealed antibody-reactive SOD1 mainly in spinal motor neurons of SOD1G93A mice throughout the disease course, and the distribution after symptomatic stages differed from that of other misfolded SOD1 species. This suggests that misfolded/non-native SOD1 species exist as heterogeneous populations. In conclusion, MS785-MS27 recognizes various conformation-disordered SOD1 species lacking the Zn ion.

Factors affecting therapeutic protein purity and yield during chromatographic purification.

Therapeutic proteins are recombinant proteins generated through recombinant DNA technology and have attracted a great deal of interest in numerous applications, including pharmaceutical, cosmetic, human and animal health, agriculture, food, and bioremediation. Producing therapeutic proteins on a large scale, mainly in the pharmaceutical industry, necessitates a cost-effective, straightforward, and adequate manufacturing process. In industry, a protein separation technique based mainly on protein characteristics and modes of chromatography will be applied to optimize the purification process. Typically, the downstream process of biopharmaceutical operations may involve multiple chromatography phases that require the use of large columns pre-packed with resins that must be inspected before use. Approximately 20% of the proteins are assumed to be lost at each purification stage during the production of biotherapeutic products. Hence, to produce a high quality product, particularly in the pharmaceutical industry, the correct approach and understanding of the factors influencing purity and yield during purification are necessary.

Recent advancements in soluble expression of recombinant antibody fragments in microbial host systems.

Recombinant fabs dominate the pharmaceutical pipelines today with microbial host systems continuing to be a major contributor toward their production. Escherichia coli is a versatile host for recombinant protein expression due to its simplicity, affordability, and ability to be cultivated at high cell density. It is particularly suitable for non-glycosylated proteins and small proteins. Despite the aforementioned benefits, the use of E. coli as the host for the synthesis of recombinant antibody fragments often suffers from low yield and reduced activity. In most cases, proteins are expressed as inclusion bodies and need to undergo refolding to achieve their active forms and this refolding step is generally low-yielding. In this article, we review the various approaches that researchers have taken to enhance the production of recombinant antibody fragments in E. coli. Molecular biology-oriented approaches such as cloning, chaperone-mediated folding, and host cell screening as well as process optimization involving examination of process parameters, media, and feeding have been addressed.

Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin.

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins.

A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced Escherichia coli fed-batch cultures.

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

Thermoinduced recombinant E. coli grew less in a two-compartment scale-down model.Heat-inducible E. coli cultures at a large scale significantly decrease recombinant protein production.The accumulation of acetic and formic acid increases when E. coli is exposed to glucose and oxygen gradients.The axial flow pattern inside the PFB mimics glucose and dissolved oxygen gradients at the industrial scale.

Cell factory engineering: Challenges and opportunities for synthetic biology applications.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography.

BACKGROUND: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products. RESULTS: Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product. CONCLUSION: By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market.

Lactiplantibacillus plantarum: a new example of inclusion body producing bacteria.

BACKGROUND: Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. RESULTS: To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250-300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. CONCLUSIONS: These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.

The Influence of the Nucleotide Composition of Genes and Gene Regulatory Elements on the Efficiency of Protein Expression in Escherichia coli.

Recombinant proteins expressed in Escherichia coli are widely used in biochemical research and industrial processes. At the same time, achieving higher protein expression levels and correct protein folding still remains the key problem, since optimization of nutrient media, growth conditions, and methods for induction of protein synthesis do not always lead to the desired result. Often, low protein expression is determined by the sequences of the expressed genes and their regulatory regions. The genetic code is degenerated; 18 out of 20 amino acids are encoded by more than one codon. Choosing between synonymous codons in the coding sequence can significantly affect the level of protein expression and protein folding due to the influence of the gene nucleotide composition on the probability of formation of secondary mRNA structures that affect the ribosome binding at the translation initiation phase, as well as the ribosome movement along the mRNA during elongation, which, in turn, influences the mRNA degradation and the folding of the nascent protein. The nucleotide composition of the mRNA untranslated regions, in particular the promoter and Shine-Dalgarno sequences, also affects the efficiency of mRNA transcription, translation, and degradation. In this review, we describe the genetic principles that determine the efficiency of protein production in Escherichia coli.

Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines.

Carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in the disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification, and conjugation of already known and well-established proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying genetic constructs, use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives already being used or potentially suitable for use as carrier proteins in conjugate vaccines.

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.