Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination.

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor-mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA-mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. "Toxin-based therapy" targets diseased cells using three strategies. Targeted cancer therapy, like antibody-toxin conjugates, fusion toxins, or "suicide gene therapy", can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection's critical role.

Diphtheria toxin-derived, anti-PD-1 immunotoxin, a potent and practical tool to selectively deplete PD-1+ cells.

Programmed death-1 (PD-1), an immune checkpoint receptor, is expressed on activated lymphocytes, macrophages, and some types of tumor cells. While PD-1+ cells have been implicated in outcomes of cancer immunity, autoimmunity, and chronic infections, the exact roles of these cells in various physiological and pathological processes remain elusive. Molecules that target and deplete PD-1+ cells would be instrumental in defining the roles unambiguously. Previously, an immunotoxin has been generated for the depletion of PD-1+ cells though its usage is impeded by its low production yield. Thus, a more practical molecular tool is desired to deplete PD-1+ cells and to examine functions of these cells. We designed and generated a novel anti-PD1 diphtheria immunotoxin, termed PD-1 DIT, targeting PD-1+ cells. PD-1 DIT is comprised of two single chain variable fragments (scFv) derived from an anti-PD-1 antibody, coupled with the catalytic and translocation domains of the diphtheria toxin. PD-1 DIT was produced using a yeast expression system that has been engineered to efficiently produce protein toxins. The yield of PD-1 DIT reached 1-2 mg/L culture, which is 10 times higher than the previously reported immunotoxin. Flow cytometry and confocal microscopy analyses confirmed that PD-1 DIT specifically binds to and enters PD-1+ cells. The binding avidities between PD-1 DIT and two PD-1+ cell lines are approximately 25 nM. Moreover, PD-1 DIT demonstrated potent cytotoxicity toward PD-1+ cells, with a half maximal effective concentration (EC50 ) value of 1 nM. In vivo experiments further showed that PD-1 DIT effectively depleted PD-1+ cells and enabled mice inoculated with PD-1+ tumor cells to survive throughout the study. Our findings using PD-1 DIT revealed the critical role of pancreatic PD-1+ T cells in the development of type-1 diabetes (T1D). Additionally, we observed that PD-1 DIT treatment ameliorated relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), a mouse model of relapsing-remitting multiple sclerosis (RR-MS). Lastly, we did not observe significant hepatotoxicity in mice treated with PD-1 DIT, which had been reported for other immunotoxins derived from the diphtheria toxin. With its remarkable selective and potent cytotoxicity toward PD-1+ cells, coupled with its high production yield, PD-1 DIT emerges as a powerful biotechnological tool for elucidating the physiological roles of PD-1+ cells. Furthermore, the potential of PD-1 DIT to be developed into a novel therapeutic agent becomes evident.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Comparative whole-genome analysis of diphtheria toxin-producing Corynebacterium ulcerans isolates from sheltered cats in Osaka, Japan.

Diphtheria toxin-producing Corynebacterium ulcerans is a zoonotic pathogen that causes human diphtheria-like symptoms. After performing whole-genome analysis of the five isolates from sheltered cats in Osaka, Japan, we compared them with genome sequences of 25 strains of C. ulcerans from a public database. The five isolates from cats harbored 14 genes encoding possible virulence factors in diphtheria-toxin-producing C. ulcerans. These isolates also had diphtheria toxin gene-encoding prophage in their chromosome, although differences were found in other prophages possession. Whole-genome single-nucleotide polymorphism analysis showed that cats' isolates belonged to ST337 branch, as were strains from Japanese human patients, with 41 or more single-nucleotide polymorphisms variations. High-resolution single-nucleotide polymorphism analysis of C. ulcerans was sufficient to distinguish cats' isolates clearly as not different by conventional genotyping methods.

Genomic analysis of an outbreak of toxin gene bearing Corynebacterium diphtheriae in Northern Queensland, Australia reveals high level of genetic similarity.

Toxigenic diphtheria is rare in Australia with generally fewer than 10 cases reported annually; however, since 2020, there has been an increase in toxin gene-bearing isolates of Corynebacterium diphtheriae cases in North Queensland, with an approximately 300% escalation in cases in 2022. Genomic analysis on both toxin gene-bearing and non-toxin gene-bearing C. diphtheriae isolated from this region between 2017 and 2022 demonstrated that the surge in cases was largely due to one sequence type (ST), ST381, all of which carried the toxin gene. ST381 isolates collected between 2020 and 2022 were highly genetically related to each other, and less closely related to ST381 isolates collected prior to 2020. The most common ST in non-toxin gene-bearing isolates from North Queensland was ST39, an ST that has also been increasing in numbers since 2018. Phylogenetic analysis demonstrated that ST381 isolates were not closely related to any of the non-toxin gene-bearing isolates collected from this region, suggesting that the increase in toxigenic C. diphtheriae is likely due to the expansion of a toxin gene-bearing clone that has moved into the region rather than an already endemic non-toxigenic strain acquiring the toxin gene.

Analysis of Corynebacterium silvaticum genomes from Portugal reveals a single cluster and a clade suggested to produce diphtheria toxin.

Background: Corynebacterium silvaticum is a pathogenic, gram-positive bacterial species that causes caseous lymphadenitis in wild boars, domestic pigs and roe deer in Western Europe. It can affect animal production and cause zoonosis. Genome analysis has suggested that one strain from Portugal and one from Austria could probably produce the diphtheria toxin (DT), which inhibits protein synthesis and can cause death. Methods: To further investigate the species genetic diversity and probable production of DT by Portuguese strains, eight isolates from this country were sequenced and compared to 38 public ones. Results: Strains from Portugal are monophyletic, nearly identical, form a unique cluster and have 27 out of 36 known Corynebacterium virulence or niche factors. All of them lack a frameshift in the tox gene and were suggested to produce DT. A phylogenetic analysis shows that the species has diverged into two clades. Clade 1 is composed of strains that were suggested to have the ability to produce DT, represented by the monophyletic strains from Portugal and strain 05-13 from Austria. Clade 2 is composed of strains unable to produce DT due to a frameshifted tox gene. The second clade is represented by strains from Austria, Germany and Switzerland. Ten genome clusters were detected, in which strains from Germany are the most diverse. Strains from Portugal belong to an exclusive cluster. The pangenome has 2,961 proteins and is nearly closed (α = 0.968). Exclusive genes shared by clusters 1 and 2, and Portuguese strains are probably not related to disease manifestation as they share the same host but could play a role in their extra-host environmental adaptation. These results show the potential of the species to cause zoonosis, possibly diphtheria. The identified clusters, exclusively shaded genes, and exclusive STs identified in Portugal could be applied in the identification and epidemiology of the species.

A Novel Immunotoxin Targeting Epithelial Cell Adhesion Molecule Using Single Domain Antibody Fused to Diphtheria Toxin.

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl ß-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy.

Ongoing toxin-positive diphtheria outbreaks in a federal asylum centre in Switzerland, analysis July to September 2022.

Two diphtheria outbreaks occurred in a Swiss asylum center from July to October 2022, one is still ongoing. Outbreaks mainly involved minors and included six symptomatic respiratory diphtheria cases requiring antitoxin. Phylogenomic analyses showed evidence of imported and local transmissions of toxigenic strains in respiratory and skin lesion samples. Given the number of cases (n = 20) and the large genetic diversity accumulating in one centre, increased awareness and changes in public health measures are required to prevent and control diphtheria outbreaks.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker.

Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)2-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)2-YP7 immunotoxin is expected to function well in inhibiting HCC.

Structures of distant diphtheria toxin homologs reveal functional determinants of an evolutionarily conserved toxin scaffold.

Diphtheria toxin (DT) is the archetype for bacterial exotoxins implicated in human diseases and has played a central role in defining the field of toxinology since its discovery in 1888. Despite being one of the most extensively characterized bacterial toxins, the origins and evolutionary adaptation of DT to human hosts remain unknown. Here, we determined the first high-resolution structures of DT homologs outside of the Corynebacterium genus. DT homologs from Streptomyces albireticuli (17% identity to DT) and Seinonella peptonophila (20% identity to DT), despite showing no toxicity toward human cells, display significant structural similarities to DT sharing both the overall Y-shaped architecture of DT as well as the individual folds of each domain. Through a systematic investigation of individual domains, we show that the functional determinants of host range extend beyond an inability to bind cellular receptors; major differences in pH-induced pore-formation and cytosolic release further dictate the delivery of toxic catalytic moieties into cells, thus providing multiple mechanisms for a conserved structural fold to adapt to different hosts. Our work provides structural insights into the expanding DT family of toxins, and highlights key transitions required for host adaptation.

Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line.

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 µg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.

Rapid detection and discrimination of potentially toxigenic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis by multiplex real-time PCR and amplicon melting curve analysis.

We developed a multiplex real-time PCR assay with amplicon melting curve analysis to rapidly discriminate Corynebacterium ulcerans from Corynebacterium pseudotuberculosis and detect the bacterial diphtheria toxin gene. This assay should be a valuable tool for identification of potentially toxigenic C. ulcerans.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

BACKGROUND: CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. OBJECTIVE: As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. METHODS: CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. RESULTS: When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 µM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 µM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 µg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. CONCLUSION: These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Proteins from the core genome of Corynebacterium ulcerans respond for pathogenicity and reveal promising vaccine targets for diphtheria.

Corynebacterium ulcerans is an emerging pathogen able to transmit the acute infection diphtheria to humans. Although there is a well-established vaccine based on the toxin produced by Corynebacterium diphtheriae, another species of this genus known to cause the disease, there is still no vaccine formulations described for C. ulcerans; this fact contributes to the increase in cases of infection that has been observed. In this study, we want to provide information at the genomic level of this bacterium in order to suggest proteins as possible vaccine targets. We carried out an in silico prospection of vaccine candidates through reverse vaccinology for targets that exhibit antigenic potential against diphtheria. We found important virulence factors, such as adhesion-related ones, that are responsible for pathogen-host interaction after infection, but we did not find the diphtheria toxin, which is the main component of the currently available vaccine. This study provides detailed information about the exoproteome and hypothetical proteins from the core genome of C. ulcerans, suggesting vaccine targets to be further tested in vitro for the development of a new vaccine against diphtheria.

Control of axillary bud growth in tobacco through toxin gene expression system.

The control of axillary bud development after removing the terminal buds (topping) of plants is a research hotspot, and the control of gene expression, like switching on and off, allows us to further study biological traits of interest, such as plant branching and fertility. In this study, a toxin gene control system for plants based on dexamethasone (DEX) induction was constructed, and the positive transgenic tobacco exhibited growth retardation in the application area (axillary bud). The expression level of the lethal Diphtheria toxin A (DTA) gene under different DEX concentrations at different application days was analyzed. The highest expression levels appeared at 5 days after the leaf injection of DEX. The DTA transcripts were induced by 5 µM DEX and peaked in response to 50 µM DEX at 5 days after leaf injection. Here, a chemical induction system, combined with a toxin gene, were used to successfully control the growth of tobacco axillary buds after topping. The DTA expression system under DEX induction was sensitive and efficient, therefore, can be used to control axillary bud growth and development in tobacco.

In Vivo Tumor Therapy with Novel Immunotoxin Containing Programmed Cell Death Protein-1 and Diphtheria Toxin.

Immunotoxins, as a class of antitumor agents, consist of tumor-selective ligands linked to highly toxic protein molecules. This type of modified antibody has been designed for the therapy of cancers and a few viral infections. In this study, we designed immunotoxin consisting of mouse programmed cell death protein-1 (PD1), which genetically fused to diphtheria toxin (DT) subunit A (DT386). DNA construct was cloned, expressed in a bacterial system, purified, and confirmed by western blotting. The immunotoxin potency in the treatment of tumorous C57BL/6 mice was evaluated. Immunotoxin was injected intratumoral to mice, and through eight injections, 67% of the tumor volume of the test group started shrinking dramatically. On the contrary, the tumor size of the control group, treated with phosphate-buffered saline, continued its growth. The successful targeting of solid tumor cells by PD1-DT immunotoxin demonstrates the potential therapeutic utility of these conjugates.

[Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli].

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.