anti-His Antibody Research Articles

Recombinant receptor-binding motif of spike COVID-19 vaccine candidate induces SARS-CoV-2 neutralizing antibody response

Introduction: The SARS-CoV-2 pandemic necessitates effective therapeutic solutions. The receptor-binding motif (RBM) is a subdomain of the spike protein's receptor-binding domain (RBD) and is critical for facilitating the binding of SARS-CoV-2 to the human ACE2 receptor. This study investigates the use of the receptor-binding motif (RBM) domain as an immunogen to produce potent neutralizing antibodies against SARS-CoV-2. Methods: The RBM gene was codon-optimized and cloned into the pET17b vector for expression in E. coli BL21 (DE3) cells, induced with 1 mM IPTG. The recombinant RBM protein was purified using Ni-NTA affinity chromatography. After validating the recombinant RBM by Western blotting with anti-His tag antibodies, BALB/c mice were immunized with 20 µg of the purified RBM protein. Anti-RBM IgG was subsequently purified using protein G resin, and its neutralizing capacity was assessed using the Pishtaz Teb Zaman Neutralization Assay Kit. Results: The recombinant RBM protein, with a molecular weight of 10 kDa, was expressed as inclusion bodies. the typical yield of purification was 27 mg/L of bacterial culture. The neutralization test demonstrated a concentration of 36 µg/mL of neutralizing antibodies in the immunized serum, preventing the spike protein from binding to ACE2. Conclusion: Our study demonstrated that anti-RBM antibodies exhibited neutralization effects on SARS-CoV-2. These findings provide evidence for the development of a vaccine candidate through the induction of antibodies against the RBM, necessitating further studies with adjuvants suitable for human use to evaluate its potential for human vaccination.

Bacterially expressed full length Hemagglutinin of Avian Influenza Virus H5N1 forms oligomers and exhibits hemagglutination

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development

Botulinum neurotoxins (BoNTs) serotypes A and B are the two most common of the four BoNTs that cause the high mortality botulism disease in individuals consuming contaminated foods. The gold standard assay for BoNT detection is the live mouse bioassay, which has several major disadvantages, including tedious procedures and animal sacrifice requirements. In this study, we developed an immuno-based assay using magnetic streptavidin nanoparticles (mSNP) functionalized with specific synthetic biotinylated, 6xHis-tagged peptide substrates (Peptides A, PA and B, PB) designed for BoNT/A and BoNT/B proteolytic reactions, respectively. In the presence of active toxins that possess endopeptidase activity, upon cleavage, the released fragments with His-tag were dotted on a blotting membrane, ultimately producing color signals after incubation with anti-His antibody, alkaline phosphatase (AP)-conjugated antibody, and then AP substrates. The results showed that the efficiency of peptide-mSNP complex formation reached up to 81%, and the dot blot immunoassay allowed peptide detection from 10 ng of His-tagged peptides. Preliminary testing with the extracellular extracts from the isolated Clostridium botulinum strains indicated that the botulinum toxin in the 2020 botulism outbreak in Vietnam belonged to serotype A, the most potent BoNT. The established assay could be applied to construct a portable biosensor for BoNT detection and a high throughput device to screen potential BoNT inhibitors for drug development.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Identification of antigens in the Trichinella spiralis extracellular vesicles for serological detection of early stage infection in swine

BackgroundSeveral studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis.MethodsExtracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA.ResultsIsolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis.ConclusionsThis study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.Graphical

Sample preparation of membrane proteins using anti-His tag antibody

Sample preparation of membrane proteins using anti-His tag antibody

Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system.

The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system. A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting. Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC). Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.

Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Anti–Integrin αvβ6 Antibody as a Diagnostic Marker for Pediatric Patients With Ulcerative Colitis

Anti–Integrin αvβ6 Antibody as a Diagnostic Marker for Pediatric Patients With Ulcerative Colitis

Recombinant human carbonic anhydrase VII: Purification, characterization, inhibition, and molecular docking studies.

Human carbonic anhydrase VII (hCA VII), a cytosolic enzyme, defends against oxidative stress by preventing reactive oxygen species from forming. In our study, first, hCA VII was cloned into Escherichia coli (One Shot Mach1-T1R) strain by using cDNA of the human brain and successfully expressed. The integrity of the plasmid generated by colony PCR was checked, and after, for protein expression, the plasmid was transformed into E. coli BL21 (DE-3) strain. hCA VII expression was observed after 6h of isopropyl-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein containing hexahistidine (6xHis) was purified with 7.02EU/mg of specific activity, had 48.07% of purification yield, and approximately 21-folds using a ProbondTM nickel chelating resin affinity column. Then, both molecular mass determination and purity control of the purified recombinant enzyme was done by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The mass of the SUMO-hCA VII fusion protein was calculated as 46.77kDa. As a result of Western blot analysis using anti-His G-HRP antibody, the fusion protein was detected as approximately 45kDa. Furthermore, the characterization assays and in vitro inhibition studies were done for the recombinant enzyme. KI values of these agents were found between 0.29μM and 157.6mM. Finally, molecular docking investigations of these antibiotics were undertaken to understand further the binding interactions on the active site of this recombinant enzyme.

Optimization of the heterologous expression and purification of Plasmodium falciparum generative cell specific 1 in Escherichia coli

Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.

Cell surface expression of γ-CGTase from Evansella caseinilytica on E. coli: Application in the enzymatic conversion of starch to γ-cyclodextrin

The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t1/2 of 24.75 min at 50ºC. The Km and Vmax values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min−1 g−1 DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and β-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at − 20 ºC even after 7 days.

Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae

ABSTRACT The mRNA turnover and ribosome assembly are facilitated by Mrt4 protein from Saccharomyces cerevisiae. In present study, we are reporting the cloning, expression and homogeneous purification of recombinant Mrt4. Mrt4 is a 236-amino-acid-long nuclear protein that plays a very crucial role in mRNA turnover and ribosome assembly during the translation process. mrt4 gene was amplified by polymerase chain reaction and cloned in expression vector pET23a (+) under the bacteriophage T7-inducible promoter and lac operator. Furthermore, protein was purified to homogeneity using immobilized metal affinity chromatography (IMAC) and its homogeneous purification was further validated by immunoblotting with anti-His antibody. The far-UV CD spectra represent that Mrt4 has a typical α helix with characteristic negative minima at 222 and 208 nm. At physiological pH, the fluorescence spectra and CD spectra showed properly folded tertiary and secondary structures of Mrt4, respectively. Saccharomyces Mrt4 protein possesses putative bipartite NLS (nuclear localization signal) at the N-terminal part followed by two well-conserved domains, rRNA-binding domains and translation factor (TF) binding domain. PIPSA analysis evaluates electrostatic interaction properties of proteins and concluded that Mrt4 protein can be used as a fingerprint for classifying Mrt4-like mRNA turnover protein from various species. The availability of an ample amount of protein may help in its biochemical and biophysical characterization, crystallization and identification of new interacting partners of Mrt4.

Expression of the First Recombinant Anti-Tumoral Snake Venom Kunitz-Type Serine Protease Inhibitor

PIVL is a Kunitz-type serine protease inhibitor that was previously characterized from Tunisian snake venom, Macrovipera lebetina transmediterranea. It reduced glioblastoma cells’ development and significantly blocked angiogenesis in in-vitro and ex-vivo models. PIVL exerted these effects by interfering with αvβ3 integrin. In order to produce a biological active recombinant, the cDNA cloning and expression of PIVL was performed in Escherichia coli (BL21)-DE3 cells using pET-22b (+) vector. The recombinant PIVL protein (rPIVL) was purified by nickel affinity chromatography and has recognized monoclonal anti-His antibody. Functionally, rPIVL exhibited potent anti-tumor cell effects as well as anti-angiogenesis properties. Interestingly, we found that both native PIVL (nPIVL) and rPIVL modulated PI3/AKT and MAPK signaling pathways. In all, our results showed that we have successfully expressed the first active anti-oncogenic snake venom Kunitz-type protease inhibitor that can be a potential therapeutic drug against glioblastoma, in its native or recombinant form.

Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1

Chitinases are glycosyl hydrolases, that cleave the ?-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes ? such as chitooligosaccharides with vast importance in the medical and biofuels industry ? lead to the necessity of creating chitinases using different strains of organisms. In this paper, the chiA gene from the Bacillus licheniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahistidine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chitinase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression continued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate?polyacrylamide gel electrophoresis (SDS-PAGE), as a heterogeneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cerevisiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl ?-D-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed.

Correction for Li et al., "Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis".

Volume 12, issue 5, e01192-21, 2021, https://doi.org/10.1128/mBio.01192-21. In Fig. 2B of the original paper, the image of the Western blot that shows the expression level of c15850 using anti-His monoclonal antibody was duplicated in the panel for c21220. This duplication issue happened at the figure assembly stage and was not identified at submission and proof stages. The authors provide here Fig. 2B with the correct Western blot image in the c21220 panel, which does not change results and conclusions of the article. Nonetheless, the authors apologize to the readers for any inconvenience that this error may have caused.

Cell death analysis of recombinant mature epsilon toxin on the kidney cell line.

Background and Objectives:Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombinant mature epsilon toxin to evaluate cell death impact on the kidney cell line.Materials and Methods:For this purpose, the sequence of mature epsilon toxin (46–328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different concentrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin.Results:Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 μg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percentage was shown after exposing to 100, 150, and 200 μg/mL of mature epsilon toxin (P≤0.05).Conclusion:The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.

B-Cell Receptor Antigens As the Targeting Moiety for Bispecific Constructs - a Novel Treatment Approach for B-Cell Lymphomas

BackgroundChronic antigenic stimulation of the B-cell receptor (BCR) has been suggested to play a role in the pathogenesis of B-cell lymphomas, however only few B-cell receptor antigens have been identified until recently, when we identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors from approximately 25% of DLBCLs of the ABC type and 45% of mantle cell lymphomas, respectively. These BCR antigens can be used to therapeutically target lymphoma cells in an approach we designated as BARs (B-cell receptor antigens for reverse targeting). To test whether BARs can substitute for the B-cell targeting antibody CD19, we designed T-cell engaging anti-CD3 single chain fragments (scFv) and NK-cell engaging anti-CD16 scFv with the respective BAR as the targeting moiety.Material and methodsVariable heavy- and light- chain genes of the anti-CD3 OKT3 hybridoma and the anti-CD16 3G8 hybridoma were cloned into a pcDNA 3.1 vector by standard cloning techniques, followed by DNA sequences of the autoantigens ARS2 or LRPAP1. VH and VL were linked by a glycine-serine linker, as was VL to the autoantigenic epitope resulting in a VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2/LRPAP1 DNA sequence. The final cloning product was transfected in HEK 293 cells for production of bispecific constructs. Binding capacity to lymphoma cell lines (DLBCL: OCI-Ly3, U2932, HBL-1; MCL : MAVER-1, GRANTA-519) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of bispecific constructs after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay.ResultsWe cloned, expressed, characterized and tested 4 bispecific constructs: αCD3-ARS2, αCD3-LRPAP1, αCD16-ARS2 and αCD16-LRPAP1. αCD3 bispecific constructs bound specifically to CD3 on T cells whereas αCD16 constructs bound exclusively to CD16-positive cells. Bispecific ARS2 constructs bound specifically to DLBCL cells with a B-cell receptor specific for ARS2, while LRPAP1 containing constructs bound exclusively to MCL with a B-cell receptor specific for LRPAP1. αCD3-ARS2 and αCD3-LRPAP1 induced dose-dependent T-cell mediated, rapid cytotoxicity exclusively in lymphoma cell lines with B-cell receptors specific for ARS2 and LRPAP1, respectively, starting at concentrations as low as 0,1 µg/ml. NK-cell mediated cytotoxicity against the respective DLBCL and MCL cell lines was observed with αCD16 constructs αCD16-ARS2 and αCD16-LRPAP1 at concentrations starting from 1 µg/ml. Control lymphoma cell lines with BCR specificities other than for ARS2 or LRPAP1 were not affected.ConclusionBARs can substitute for B-cell-directed antibodies (e. g. CD19) as the targeting moiety in bispecific constructs for the therapeutic targeting of B-cell lymphomas. Because approaches using the BCR antigen for targeting the malignant B cells have an exclusive specificity for the cells with the specific surface BCR (which are exclusively the respective lymphoma cells), they do not only represent a sensu stricto tumor-specific approach, they can also be expected to be more effective (due to the higher density of BCR molecules on the lymphoma cell surface), but less toxic than the currently available bispecific T-cell and NK-cell engaging antibodies, because they leave all other cells, in particular normal B-lymphocytes, unaffected. Supported by Wilhelm-Sander-Stiftung DisclosuresThurner:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Regitz:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Fadle:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent.