Recombinant Protein Expression Research Articles

The Potential of Plant-Produced Virus-like Particle Vaccines for African Horse Sickness and Other Equine Orbiviruses

African horse sickness is a devastating viral disease of equids. It is transmitted by biting midges of the genus Culicoides with mortalities reaching over 90% in naïve horses. It is endemic to sub-Saharan Africa and is seasonally endemic in many parts of southern Africa. However, outbreaks in Europe and Asia have occurred that caused significant economic issues. There are attenuated vaccines available for control of the virus but concerns regarding the safety and efficacy means that alternatives are sought. One promising alternative is the use of virus-like particles in vaccine preparations, which have the potential to be safer and more efficacious as vaccines against African horse sickness. These particles are best made in a complex, eukaryotic system, but due to technical challenges, this may cause significant economic strain on the developing countries most affected by the disease. Therefore, this review also summarises the success so far, and potential, of recombinant protein expression in plants to reduce the economic strain of production.

Seed plastids: A novel platform for recombinant protein expression.

Seed plastids: A novel platform for recombinant protein expression.

Tandem gene duplication selected by activation of horizontally transferred gene in bacteria

Horizontal gene transfer occurs frequently in bacteria, but the mechanism driving activation and optimization of the expression of horizontally transferred genes (HTGs) in new recipient strains is not clear. Our previous study found that spontaneous tandem DNA duplication resulted in rapid activation of HTGs. Here, we took advantage of this finding to develop a novel technique for tandem gene duplication, named tandem gene duplication selected by activation of horizontally transferred gene in bacteria (TDAH), in which tandem duplication was selected by the activation of horizontally transferred selectable marker gene. TDAH construction does not contain any reported functional elements based on homologous or site-specific recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection and 9-bp-microhomology border sequences for precise illegitimate recombination. One transformation and 3 days were enough to produce a high-copy strain, so its procedure is simple and fast. Without subsequent knockout of the endogenous recombination system, TDAH could also generate the relatively stable high-copy tandem duplication for plasmid-carried and genome-integrated DNA. TDAH also showed an excellent capacity for increase gene expression and worked well in different industrial bacteria. We also applied TDAH to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5 times and remained stable even after 12 subcultures. TDAH is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.Key points• We develop a novel and efficient technique (TDAH) for tandem gene duplication in bacterium. TDAH is based on the mechanism of HTG rapid activation. TDAH does not contain any reported functional elements based on homologous recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection, so its construction and procedure are very simple and fast.• TDAH is the first reported selected and stable tandem-gene-duplication technique in which the selected high-copy plasmid-carried and genome-integrated DNA could remain stable without the subsequent knockout of recombination system.• TDAH showed an excellent capacity for regulating gene expression and worked well in different industrial bacteria, indicating it is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.• TDAH was applied to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5-fold and remained stable even after 12 subcultures.

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.

Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.

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.

Sodium valproate promotes low metabolism and high protein expression in CHO-engineered cell lines

Chinese hamster ovary cells are widely used in various biopharmaceutical industries as preferred host cells for the production of recombinant proteins. In an effort to increase yield and reduce production costs, the incorporation of small molecule additives into the culture medium has emerged as an effective approach to augment the expression of recombinant proteins, thus increasing overall efficiency. Here, we examined the impact of five small molecules on protein expression in CHO-engineered cell lines. These molecules included sodium valproate, alanyl-L-tyrosine, ascorbic acid, sodium citrate and ferrous sulphate. Their optimal concentrations were determined through univariate analysis aimed at enhancing cell growth and protein production. Our findings revealed that inoculating cells at a density of 1.0×106 cells/mL with the simultaneous addition of sodium valproate at a concentration of 2mM for 0h yielded the most favorable results. Notably, while valproate inhibited cell growth in a dose-dependent manner, it simultaneously extended cell culture longevity. Additionally, sodium valproate was observed to decrease glucose consumption, collectively increasing protein expression. In conclusion, our data suggest that sodium valproate is a potent chemical agent capable of increasing the production of recombinant protein in CHO cells. This enhancement is achieved by the modulation of metabolic pathways in CHO-engineered cell lines, thereby facilitating a state of low metabolism and high protein expression.

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.

High-level expression of codon-optimized Taq DNA Polymerase under the control of rhaBAD promoter

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the L-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

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.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.