Ni-NTA Affinity Chromatography Research Articles

Therapeutic efficacy of SipD/LptD-specific IgY entrapped in alginate nanoparticles against Salmonella Typhimurium infection

IntroductionSalmonella, a zoonotic pathogen causing gastroenteritis, lacks a preventive vaccine. Passive immunization with IgY antibodies derived from immunized chickens has shown potential for treating bacterial infections. This study investigated the therapeutic efficacy of entrapped IgY targeting recombinant SipD and LptD proteins from Salmonella Typhimurium. MethodsThe recombinant protein was expressed in E. coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Hens were immunized with the purified protein, and the resulting IgY was entrapped into alginate nanoparticles. The shape of spherical nanoparticles and their size in the nanometer range were determined by SEM and DLS analysis. The therapeutic efficacy of free and alginate-entrapped IgY against S. Typhimurium was evaluated in mice at 1, 50, and 100 LD50 bacterial doses. ResultsThe purified IgY concentration in each egg yolk was 6 mg/ml (35 mg/egg). Physicochemical and structural characterization revealed spherical nanoparticles with a diameter of 157.1 nm and a negatively charged surface (zeta potential of −35.6 mV). The loading efficiency of IgY into alginate nanoparticles was 95.5 %. In a challenge test with 100 LD50 of S. Typhimurium, all mice receiving alginate-entrapped IgY survived, whereas half of the mice receiving non-entrapped IgY died within 7 days. ConclusionOur results indicate that IgY antibodies entrapped in alginate nanoparticles may offer therapeutic effect against S. Typhimurium infection.

A multi-epitope protein vaccine encapsulated in alginate nanoparticles as a candidate vaccine against Shigella sonnei

Shigellosis, caused by the Gram-negative bacterium Shigella, is a major global health challenge. Despite extensive research over the past two decades, no commercial vaccine is available to prevent Shigella infection. Developing multi-epitope vaccines offers a promising and innovative approach to tackling infectious diseases. In this study, we produced a multi-epitope vaccine candidate using E. coli BL21 (DE3) plysS bacteria and purified the vaccine protein with Ni-NTA affinity chromatography. We then prepared alginate nanoparticles containing the vaccine protein, with a particle size of 122 ± 6 nm, PDI 0.17, SPAN 0.83, and zeta potential of -27 ± 2 mV. Successful protein loading was confirmed through nanodrop and ATR-FTIR analyses. To evaluate the immunogenicity of the encapsulated vaccine, mice were orally vaccinated, and their serum was analyzed for IgG, IL-4, and IFN-γ levels cytokines. The results showed a significant increase in IgG level in the vaccinated group compared to controls. Additionally, the vaccinated group exhibited a notable increase in IL-4 and IFN-γ cytokines, indicating a robust Th-cell-mediated immune response essential for combating Shigella. Our nano-vaccine demonstrated high efficacy in activating both humoral and cellular immunity, effectively protecting against the bacteria. The alginate-based oral vaccine candidate thus emerges as a promising strategy for developing a multi-epitope vaccine candidate against Shigella.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania donovani

Aim: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin (LTB) in the functional activity of L. donovani. Background: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease (gp63) has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival. Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani was fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. Results: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin (LTB) of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. Conclusion: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein (LTB) could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Enhanced Production and Therapeutic Evaluation of TP4 Peptide in Pichia pastoris Against Hepatocellular Carcinoma HUH-7 Cells

Tilapia piscidin 4 (TP4), a cationic antimicrobial peptide, is recognized for its diverse biological roles, including antibacterial, wound-healing, and anticancer properties. Herein, the codon-optimized sequence of TP4 peptide was expressed using the pPICZαA expression vector containing the AOX1 promoter, a strong and inducible promoter, in the Pichia pastoris KM71H expression system. Recombinant TP4 peptide was purified by Ni-NTA affinity chromatography. After purification, the anticancer activity of TP4 was assessed in HUH-7 hepatocellular carcinoma cells, and the underlying mechanisms were determined. In the present study, it was demonstrated for the first time that recombinant TP4 displayed strong anticancer activity in the human HUH-7 cell line. The TP4 antimicrobial peptide can be used as a competitive candidate for the treatment of cancer cells due to its anticancer effects.

Expression in Pichia pastoris of Thermostable Endo-1,4-β-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40kDa and ~ 35kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Recombinant expression and characterization of the endochitinase Chit36-TA from Trichoderma asperellum in Komagataella phaffii for chitin degradation of black soldier fly exuviae

The natural polymer chitin is an abundant source for valuable N-acetylchitooligosaccharides and N-acetylglucosamine applicable in several industries. The endochitinase Chit36-TA from Trichoderma asperellum was recombinantly expressed in Komagataella phaffii for the enzymatic degradation of chitin from unused insect exuviae into N-acetylchitooligosaccharides. Chit36-TA was purified by Ni–NTA affinity chromatography and subsequently biochemically characterized. After deglycosylation, the endochitinase had a molecular weight of 36 kDa. The optimum pH for Chit36-TA was 4.5. The temperature maximum of Chit36-TA was determined to be 50 °C, while it maintained > 93% activity up to 60 °C. The chitinase was thermostable up to 45 °C and exhibited ~ 50% activity after a 15 min incubation at 57 °C. Chit36-TA had a maximum specific enzyme activity of 50 nkat/mg with a Km value of 289 µM with 4-methylumbelliferyl-N,N′,N″-triacetyl-β-chitotrioside as substrate. Most tested cations, organic solvents and reagents were well-tolerated by the endochitinase, except for SDS (1 mM), Cu2+ (10 mM) and Mn2+ (10 mM), which had stronger inhibitory effects with residual activities of 3, 41 and 28%, respectively. With a degree of hydrolysis of 32% applying colloidal shrimp chitin (1% (w/v)) and 12% on insect larvae (1% (w/v)) after 24 h, the endochitinase was found to be suitable for the conversion of colloidal chitin as well as chitin from black soldier fly larvae into water-soluble N-acetylchitooligosaccharides. To prove scalability, a bioreactor process was developed in which a 55-fold higher enzyme activity of 49 µkat/l and a tenfold higher protein expression of 1258 mg/l were achieved.

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.

IgY-mediated protection against Vibrio cholerae infection: Efficacy of avian antibodies targeting a chimeric recombinant protein

Introduction: Vibrio cholerae, the etiologic pathogen of diarrheal disease, prevails mainly in developing countries, transmitted through contaminated water or food. The unique genetic makeup and remarkable competency has prompted intensive research to unravel the bacterium virulence properties. Egg yolk immunoglobulins (IgY) have emerged as innovative biotherapeutics for both passive immunotherapy and prophylactic strategies. Methods: In the present study, we generated avian antibodies against a chimeric recombinant protein comprising OmpW-TcpA-CtxB (OTC) antigens from V. cholerae, and examined its efficacy against bacterial toxins and infection. The chimeric protein was expressed in E. coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Leghorn chickens were intramuscularly immunized with the recombinant protein and the purity of extracted IgYs was assessed through SDS-PAGE analysis. The immunoreactivity and specificity of anti-OTC-IgYs were evaluated through protein and whole-cell ELISA, and their ability to neutralize cholera toxin (CT) of V. cholerae was evaluated in Y1 cell line. Finally, the protective efficacy of orally administered anti-OTC-IgY was investigated in V. cholerae-infected infant mice. Results: Anti-OTC-IgY successfully neutralized the cytotoxic effects of CT at a concentration of 250 µg/mL. Oral administration of two 100 µg doses of anti-OTC-IgY and resulted in 60% and 20% survival rates in suckling mice infected with LD and 10 LD of V. cholerae, respectively. Conclusion: The anti-OTC-IgY antibodies exhibited significant immunoreactivity, toxin-neutralizing potency, and protective effects, establishing their potential as promising antimicrobials against the bacterial pathogenicity through passive immunotherapy.

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

A thermostable xylanase hydrolyzes several polysaccharides from Bacillus altitudinis JYY-02 showing promise for industrial applications

Polysaccharides have attracted immense attention as the largest source of bioactive compounds. Its bioavailability and bioactivity can be improved by utilizing degradation enzymes to reduce their molecular weight and viscosity. In this study, a 654 bp gene encoding xylanase was screened from the genome of Bacillus altitudinis JYY-02 and overexpressed in Escherichia coli Rosetta (DE3). The recombinant xylanase with a molecular weight of 27.98 kDa was purified (11.7-fold) using Ni–NTA affinity chromatography, with a 43.6% final yield. Through molecular docking, Glu, Arg, Tyr, and Trp were found to be the main amino acids involved in the interaction between xylanase and xylobiose. The effects of pH, temperature, metal ions, and substrates on xylanase activity were determined, and the results showed that the highest catalytic activity was displayed at pH 6.5, 50 °C temperature, with Cu2+ as an activator and xylan as the substrate. The Km (substrate concentration that yields a half-maximal velocity) and Vmax (maximum velocity) of recombinant xylanase were 6.876 mg/mL and 10984.183 μmol/mg∙pr/min, respectively. The recombinant xylanase was thermostable, with 85% and 39% of the enzymatic activity retained after 1 h at 60 °C and 1 h at 90 °C, respectively. The recombinant xylanase demonstrated a significant clarifying effect on fruit juices.

A TonB dependent transporter YncD of Salmonella entericaSerovar Typhi possesses vaccine potential.

Multiple TonB dependent transporters (TBDTs) contribute to bacterial virulence due to the importance roles that their substrates play in bacterial growth, and possess vaccine potential. A putative TBDT, YncD, had been identified as one of in vivo induced antigens during human infection of typhoid fever, and is required for the pathogenicity of Salmonella enterica Serovar Typhi. The present study was aimed to determine the function and immunogenicity of YncD. Homologous recombination method was used to construct an yncD-deletion mutant and cirA-iroN-fepA-deletion mutant from the wild-type S. Typhi Ty2. The growth of mutants and the wild-type strain were assessed in iron-deficient medium, as well as in human macrophage cells. Recombinant YncD protein was expressed and purified using Ni-NTA affinity chromatography and anion exchange. A mouse model was then used to evaluate the immunogenicity and protection efficacy of the recombinant YncD. Antibody levels, serum bactericidal efficiency, passive immune protection, opsonophagocysis were assayed to analyse the immunoprotection mechanism of the recombinant YncD. Our results showed that YncD is associated with the iron-uptake of S. Typhi. The yncD-deletion mutant displayed impaired growth in iron-deficient medium, comparable to that the cirA-iroN-fepA-deletion mutant did. The mutation of yncD markedly decreased bacterial growth within human macrophage cells. Moreover, subcutaneous immunization of mice with recombinant YncD elicited high levels of specific anti-YncD IgG, IgG1 and IgG2a, which protected the immunized mice against the intraperitoneal challenge of S. Typhi, and decreased bacterial burdens in the livers and spleens of the infected mice. Passive immunization using the immunized sera also efficiently protected the mice from the challenge of S. Typhi. Moreover, the immunized sera enhanced in vitro bactericidal activity of complement, and opsonophagocytosis. Our results showed that YncD displays a role in the iron-uptake of S. Typhi and possesses immunogenicity.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

Monophosphoryl lipid A as a co-adjuvant in methicillin-resistant Staphylococcus aureus vaccine development: improvement of immune responses in a mouse model of infection.

To increase the effectiveness of methicillin-resistant Staphylococcus aureus vaccines (MRSA), a new generation of immune system stimulating adjuvants is necessary, along with other adjuvants. In some vaccines, monophosphoryl lipid A (MPLA) as a toll-like receptor 4 agonist is currently used as an adjuvant or co-adjuvant. MPLA could increase the immune response and vaccine immunogenicity. The current investigation assessed the immunogenicity and anti-MRSA efficacy of recombinant autolysin formulated in MPLA and Alum as co-adjuvant/adjuvant. r-Autolysin was expressed and purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE. Then, the vaccine candidate formulation in MPLAs and Alum was prepared. To investigate the immunogenic responses, total IgG, isotype (IgG1 and IgG2a) levels, and cytokines (IL-4, IL-12, TNF-α, and IFN-γ) profiles were evaluated by ELISA. Also, the bacterial burden in internal organs, opsonophagocytosis, survival rate, and pathobiology changes was compared among the groups. Results demonstrated that mice immunized with the r-Autolysin + Alum + MPLA Synthetic and r-Autolysin + Alum + MPLA Biologic led to increased levels of opsonic antibodies, IgG1, IgG2a isotype as well as increased levels of cytokines profiles, as compared with other experimental groups. More importantly, mice immunized with MPLA and r-Autolysin exhibited a decrease in mortality and bacterial burden, as compared with the control group. The highest level of survival was seen in the r-Autolysin + Alum + MPLA Synthetic group. We concluded that both MPLA forms, synthetic and biological, are reliable candidates for immune response improvement against MRSA infection.

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo

Shigella is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 μg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD50 of Stx and S. dysenteriae was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 μmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 μg IgY leads to complete survival of the mice against 1LD50 toxin and 4000 μg of IgY led to complete survival against 1LD50, also 70% and 30% survival against 10 and 50 LD50S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34mg of protein to be used in future functional studies.

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity – an integrated experimental and bioinformatic approach

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence. Communicated by Ramaswamy H. Sarma

Biophysical characterization and insights into the oligomeric nature of CD2-associated protein.

Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.

Molecular cloning, characterization and expression analysis of the Chinese soft-shelled turtle (Pelodiscus sinensis) chemokine CXCL11

Chemokines are small, secreted proteins with chemoattractive properties, which play an important role in the recruitment and activation of immune cells. CXCL11 is a CXC chemokine specific for the CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have bactericidal effects similar to defensins. Herein, we cloned the full-length cDNA of Chinese soft-shelled turtle (Pelodiscus sinensis) CXCL11, designated as PsCXCL11, which consist of an open reading frame (ORF) of 282 bp encoding 93 amino acids, with estimated molecular weight of 10.055 kDa and isoelectric point of 10.37. The deduced PsCXCL11 sequence had a signal peptide, a highly conserved family-specific small cytokine (SCY) domain, one putative N-glycosylation site and ten potential phosphorylation sites. Phylogenetic analysis showed a close relationship between P. sinensis and Chelydra Serpentina CXCL11. P. sinensis CXCL11 basal expression levels were higher in heart, kidney and spleen than in other organs of health turtles. Infections of Aeromonas hydrophila and Staphylococcus aureus led to significant upregulation of P. sinensis CXCL11 in the blood, while significant upregulation of PsCXCL11 were observed in liver and spleen after infection of A. hydrophila, but not S. aureus. PsCXCL11 recombinant protein with His-tag was successfully expressed by an auto-inducible expression system, and purified by Ni-NTA affinity chromatography. These findings laid a solid foundation for further research towards development of the Chinese soft-shelled turtle as a model for the role of CXCL11 in regulating inflammatory responses to stimulation by invading pathogens.