Recombinant Protein Expression Research Articles

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host’s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5ʹ proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5ʹ coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 UmL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 UmL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

Design and Expression of Recombinant cell Penetrating Protein Based on Tat and pertussis-like Toxin A and Evaluation of its Effects on the lung cancer

Design and Expression of Recombinant cell Penetrating Protein Based on Tat and pertussis-like Toxin A and Evaluation of its Effects on the lung cancer

Hydrogen Sulfide Exerted a Pro-Angiogenic Role by Promoting the Phosphorylation of VEGFR2 at Tyr797 and Ser799 Sites in Hypoxia-Reoxygenation Injury.

The protective effects of hydrogen sulfide (H2S) against ischemic brain injury and its role in promoting angiogenesis have been established. However, the specific mechanism underlying these effects remains unclear. This study is designed to investigate the regulatory impact and mechanism of H2S on VEGFR2 phosphorylation. Following expression and purification, the recombinant His-VEGFR2 protein was subjected to LC-PRM/MS analysis to identify the phosphorylation sites of VEGFR2 upon NaHS treatment. Adenovirus infection was used to transfect primary rat brain artery endothelial cells (BAECs) with the Ad-VEGFR2WT, Ad-VEGFR2Y797F, and Ad-VEGFR2S799A plasmids. The expression of VEGFR2 and recombinant Flag-VEGFR2, along with Akt phosphorylation, cell proliferation, and LDH levels, was assessed. The migratory capacity and tube-forming potential of BAECs were assessed using wound healing, transwell, and tube formation assays. NaHS notably enhanced the phosphorylation of VEGFR2 at Tyr797 and Ser799 sites. These phosphorylation sites were identified as crucial for mediating the protective effects of NaHS against hypoxia-reoxygenation (H/R) injury. NaHS significantly enhanced the Akt phosphorylation, migratory capacity, and tube formation of BAECs and upregulated the expression of VEGFR2 and recombinant proteins. These findings suggest that Tyr797 and Ser799 sites of VEGFR2 serve as crucial mediators of H2S-induced pro-angiogenic effects and protection against H/R injury.

Recombinant expression and characterization of Canine circovirus capsid protein for diagnosis.

Canine circovirus (CanineCV) is a contagious virus that causes severe gastroenteritis, diarrhea, respiratory disease, and vasculitis, often resulting in fatality among infected dogs. In this study, a recombinant Capsid protein (rCap) of CanineCV was expressed in the Escherichia coli (E. coli) Rosetta (DE3) pLysS host cell, followed by affinity purification, and then analyzed by SDS-PAGE, revealing a molecular weight of approximately 31 kDa. The antigenicity of the CanineCV rCap protein was confirmed through recognition by a rabbit anti-CanineCV rCap protein polyclonal antibody (PoAb). Additionally, the reactivity and specificity of this PoAb were assessed using indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis before applying in an immunohistochemistry (IHC), namely, immunoperoxidase detection. The immunoperoxidase assay using rabbit anti-CanineCV rCap protein PoAb demonstrated that the CanineCV Cap protein was predominantly located in immune cells, especially lymphocytes and macrophages, within the spleen, lung, tracheobronchial lymph nodes, small intestine, and kidney. Similarly, the Cap protein was also found in pneumocytes in the lung and renal tubular epithelial cells in the kidney. These findings reflected the biological activity and cell tropism of the virus. Therefore, the recombinant Cap protein and its PoAb could be used for the development of a valuable diagnostic tool for CanineCV detection.

Sodium acetate increases the productivity of HEK293 cells expressing the ECD-Her1 protein in batch cultures: experimental results and metabolic flux analysis.

Human Embryonic Kidney cells (HEK293) are a popular host for recombinant protein expression and production in the biotechnological industry. This has driven within both, the scientific and the engineering communities, the search for strategies to increase their protein productivity. The present work is inserted into this search exploring the impact of adding sodium acetate (NaAc) into a batch culture of HEK293 cells. We monitored, as a function of time, the cell density, many external metabolites, and the supernatant concentration of the heterologous extra-cellular domain ECD-Her1 protein, a protein used to produce a candidate prostate cancer vaccine. We observed that by adding different concentrations of NaAc (0, 4, 6 and 8mM), the production of ECD-Her1 protein increases consistently with increasing concentration, whereas the carrying capacity of the medium decreases. To understand these results we exploited a combination of experimental and computational techniques. Metabolic Flux Analysis (MFA) was used to infer intracellular metabolic fluxes from the concentration of external metabolites. Moreover, we measured independently the extracellular acidification rate and oxygen consumption rate of the cells. Both approaches support the idea that the addition of NaAc to the culture has a significant impact on the metabolism of the HEK293 cells and that, if properly tuned, enhances the productivity of the heterologous ECD-Her1 protein.

A flexible ‘plug and play’ bicistronic construct and its application in the screening of protein expression system in Escherichia coli

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.

Fast and Accurate Disulfide Bridge Detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.

A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Overexpression of YTHDF3 increases the specific productivity of the recombinant protein in CHO cells by promoting the translation process.

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

Manipulation Strategy to Increase Expression Level of Soluble Recombinant Protein Penicillin G Acylase (PGA) in Bacterial Host Escherichia coli: A Review Article

The strategy of producing PGA on a massive scale with high levels of soluble protein can be through recombinant genetic techniques and expressed in a certain host. E. coli is still a popular bacterial host to produce a recombinant protein which has advantages such as fast growth, low production cost, and high expression rate. Apart from its advantages, E. coli as a production host also has disadvantages including the expression of recombinant proteins often failing to form the proper folding conformation which makes the protein biologically inactive. Many strategies can be developed to overcome these problems, such as the selection of the host strain (E. coli HB101 & JM109), fusion protein to enhance the recovery of soluble protein (MBP & NusA), optimization of fermentation (low-temperature incubation), and optimization of the protein isolation process for the recovery of active PGA (Freeze-thawing method).

A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater.

The development of innovative methods for highly efficient production of recombinant proteins remains a prominent focus of research in the biotechnology field, primarily due to the fact that current commercial protein expression systems rely on expensive chemical inducers, such as isopropyl β-D-thiogalactoside (IPTG). In our study, we designed a novel approach for protein expression by creating a plasmid that responds to copper. This specialized plasmid was engineered through the fusion of a copper-sensing element with an optimized multiple cloning site (MCS) sequence. This MCS sequence can be easily customized by inserting the coding sequences of target recombinant proteins. Once the plasmid was generated, it was introduced into an engineered Escherichia coli strain lacking copA and cueO. With this modified E. coli strain, we demonstrated that the presence of copper ions can efficiently trigger the induction of recombinant protein expression, resulting in the production of active proteins. Most importantly, this expression system can directly utilize copper-containing industrial wastewater as an inducer for protein expression while simultaneously removing copper from the wastewater. Thus, this study provides a low-cost and eco-friendly strategy for the large-scale recombinant protein production. To the best of our knowledge, this is the first report on the induction of recombinant proteins using industrial wastewater.

Robotic Actuation-Mediated Quantitative Mechanogenetics for Noninvasive and On-Demand Cancer Therapy.

Cell mechanotransduction signals are important targets for physical therapy. However, current physiotherapy heavily relies on ultrasound, which is generated by high-power equipment or amplified by auxiliary drugs, potentially causing undesired side effects. To address current limitations, a robotic actuation-mediated therapy is developed that utilizes gentle mechanical loads to activate mechanosensitive ion channels. The resulting calcium influx precisely regulated the expression of recombinant tumor suppressor protein and death-associated protein kinase, leading to programmed apoptosis of cancer cell line through caspase-dependent pathway. In stark contrast to traditional gene therapy, the complete elimination of early- and middle-stage tumors (volume ≤ 100mm3) and significant growth inhibition of late-stage tumor (500mm3) are realized in tumor-bearing mice by transfecting mechanogenetic circuits and treating daily with quantitative robotic actuation in a form of 5min treatment over the course of 14days. Thus, this massage-derived therapy represents a quantitative strategy for cancer treatment.

Modifications of the 5' region of the CASPONTM tag's mRNA further enhance soluble recombinant protein production in Escherichia coli.

Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPONTM tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae.

Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli.

Producing recombinant proteins is a major accomplishment of biotechnology in the past century. Heterologous hosts, either eukaryotic or prokaryotic, are used for the production of these proteins. The utilization of microbial host systems continues to dominate as the most efficient and affordable method for biotherapeutics and food industry productions. Hence, it is crucial to analyze the limitations and advantages of microbial hosts to enhance the efficient production of recombinant proteins on a large scale. E. coli is widely used as a host for the production of recombinant proteins. Researchers have identified certain obstacles with this host, and given the growing demand for recombinant protein production, there is an immediate requirement to enhance this host. The following review discusses the elements contributing to the manifestation of recombinant protein. Subsequently, it sheds light on innovative approaches aimed at improving the expression of recombinant protein. Lastly, it delves into the obstacles and optimization methods associated with translation, mentioning both cis-optimization and trans-optimization, producing soluble recombinant protein, and engineering the metal ion transportation. In this context, a comprehensive description of the distinct features will be provided, and this knowledge could potentially enhance the expression of recombinant proteins in E. coli.

Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-sourceused, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

Comparison of Five Escherichia coli Strains to Achieve the Maximum Yield of a Recombinant Immunotoxin Consisting of an Antibody against VEGF Conjugated with MAP30 Toxin in a Benchtop Bioreactor.

Cancer is among the leading causes of death worldwide, imposing high costs on the health systems of all societies. Extensive biological studies are required to discover appropriate therapies. Escherichia coli has long been regarded as one of the main biotechnological bio-factories to produce recombinant protein-based therapeutics. In the present study, five strains of E. coli were compared to achieve the maximum production of a previously designed recombinant immunotoxin-carrying MAP30 toxin against VEGF-overexpressed cancer cells in a benchtop bioreactor. The recombinant immunotoxin coding gene sequence was extracted from the NCBI database. The host used to produce the recombinant immunotoxin were five E. coli strains of BL21 (DE3), DH5α, SHuffle®T7, XL1-Blue, and Rosetta-gamiTM (DE3). CaCl2 method was used for bacterial transformation. Bacterial growth measurements were performed using optical density measurements at 600 nm. The immunotoxin production was measured using SDS-PAGE analysis. The best-producing strain was cultivated in a 10-L benchtop stirred tank bioreactor. Recent patents on this field were also studied. The results demonstrated that the BL21 (DE3) strain had the highest expression of recombinant protein in comparison to other strains. Moreover, the cell growth of E. coli BL21 (DE3) and SHuffle®T7 strains before transformation in the LB medium, were significantly higher in comparison to other strains. Additionally, the transformation of Rosettagami was associated with decreased cell proliferation. The transformation of the XL1-Blue strain did not effect cell growth. Analysis of the growth kinetics demonstrated appropriate proliferation of the transformed BL21 (DE3) cells in the laboratory benchtop bioreactor. Based on the results of this study, the BL21 (DE3) strain could be used as a suitable host for the production of the recombinant immunotoxin against VEGF in stirred tank bioreactor, which can be employed for the treatment of tumors. Yet, its precise mechanism must be explored in extensive studies.