Ni-NTA Affinity Chromatography Research Articles

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5mM IPTG after 4h incubation at 37°C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35kDam.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of β-pleated sheets and β-turns in RbL protein structure. β-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

In this study, Rhizomucor miehei -1,3-1,4-glucanase gene was expressed under the regulation of AOX1 promoter in the Pichia pastoris expression system, and the clone providing the highest production level was determined. The codon-optimized gene was ligated into expression vector pPICZA and transferred into competent P. pastoris KM71H cells. Thirty transformants were selected from plates containing different concentrations of zeocin and cultured in test tubes for protein production. Glucanase enzyme activities in supernatant samples were measured and ten clones showing the highest glucanase activity were determined. The proteins in supernatant samples were also analyzed by SDS-PAGE and the glucanase enzyme was observed in 2 bands, approximately 34 and 38 kDa. Protein production was performed for 72 hours in 200 mL induction medium (BMMY) with the clone providing the highest glucanase enzyme production and the recombinant glucanase enzyme was purified by Ni-NTA affinity chromatography. After purification, it was determined that the analyzed clone reached 796 mg/L glucanase production level. SDS-PAGE analysis of samples from each step of the purification procedure showed that the two protein bands also observed in the supernatant samples represent glucanase enzyme.

Production of bioactive recombinant ovine cysteine-rich secretory protein 1 in Escherichia coli

ABSTRACT Ovine cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymal origin under CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The aim of the present study was the optimization of bacterial production and partial characterization of putative mature ovine CRISP-1 protein. The cDNA corresponding to T23 – C242 peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using E. coli DH5α. Protein expression was carried out in E. coli BL21(DE3) by inducition with 1 mM IPTG at 37°C for 4 h. The recombinant protein was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography using a pH gradient. Further purification of the protein was carried out by gel extraction following zinc sulfate negative staining. SDS-PAGE analysis of the purified recombinant CRISP-1 protein revealed a 43.8 kDa band. Bioactivity of the purified CRISP-1 protein was examined on sperm motility and capacitation. The recombinant ovine CRISP-1 protein at 5 µg/ml caused significant inhibition of sperm motility, and the activity was lost following heating the protein at 100°C for 5 min. The protein also demonstrated decapacitation activity, and at a concentration of 2 µg/ml, it caused a significant (P < 0.05) reduction in sperm capacitation. In conclusion, the thioredoxin-tagged ovine CRISP-1 protein was successfully produced in E. coli and purified in the soluble form by a combination of Ni-NTA affinity chromatography, gel purification, and dialysis. The recombinant protein exhibited both motility-inhibiting and decapacitating activities. Further study is needed to elucidate the mechanism of action and evaluate it’s possible use in semen preservation. Abbreviations: CRISP-1: Cysteine-rich secretory protein-1; PCR: polymerase chain reaction; IPTG: isopropyl-β-D-thiogalactopyranoside; LB: Luria Bertani; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA: ethylene diamine tetraacetic acid; Ni-NTA: Nickel nitrilotriacetic acid.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

Design, expression, and purification of a multi-epitope vaccine against Helicobacter Pylori based on Melittin as an adjuvant

Helicobacter Pylori, a Gram-negative bacterium in the human stomach, causes adenocarcinoma and MALT (mucosa-associated lymphoid tissue) lymphoma in addition to infection and gastric ulcer. With regard to Helicobacter Pylori prevalence rate and widespread, producing an effective vaccine against this bacterium appears reasonable and necessary. Today, vaccine design by immunoinformatics is a promising solution in vaccine field. In the present study, potential immunodominant CD4⁺ T cell epitopes of UreB, HpaA, and NapA antigens were selected with a focus on IFN-γ secretion inducing ability. After joining the selected epitopes with KK and GPGPG linkers, sequence of Melittin, the major active protein of honey bee venom, was put in C-terminal by DPRVPSS linker as adjuvant. After reverse translation and codon optimization, the designed vaccine was cloned into pET-23a vector. The final construct was estimated as antigenic (71 & 74%) and non-allergenic with molecular weight of 36.785KD. The instability index (II) and codon frequency distribution were predicted to be 26.5 and 92%, respectively. The pET-23a vector transformed to the E.coli BL21 (DE3) strain. The evaluation of expression by SDS-PAGE analysis showed that the optimized expression is in SOB medium 8 h after induction by 0.5 mM IPTG. Finally, purification was performed by Ni-NTA affinity chromatography and Western blot analysis validated the purified protein. Future research is needed to investigate the designed vaccine efficiency against H. pylori, and also it's potential as a gastric cancer-preventive candidate.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50°C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22mM and 417.1min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

Structural Characterization and Binding Studies of the Ectodomain G Protein of Respiratory Syncytial Virus Reveal the Crucial Role of pH with Possible Implications in Host–Pathogen Interactions

Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host–pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host–pathogen interaction.

&lt;p&gt;&lt;strong&gt;Cloning and expression of cultural filtrate proteins from novel and native strains of &lt;em&gt;Mycobacterium avium &lt;/em&gt;subspecies &lt;em&gt;paratuberculosis &lt;/em&gt;and their application in ELISA based sero-diagnosis of Johne's disease&lt;/strong&gt;&lt;/p&gt;

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA.

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Evaluation of sporozoite and macroschizont antigen (Spm2) of Theileria annulata for its diagnostic potential

Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle.

Bioinformatic and immunological investigation of Per a 5 (delta class GST) allergen from Periplaneta americana

IntroductionGSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. MethodsComputational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitive patient’s sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. ResultsPer a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50–58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitive patient’s sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. ConclusionRecombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitive patient’s sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.

Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue.

The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

Characterizing the Structure and Function of Rhodoquinone Biosynthesis Enzymes

Rhodoquinone (RQ) is essential to the bioenergetics of many organisms that survive in low oxygen environments because it allows the electron transport chain to function with fumarate as a final electron acceptor instead of oxygen. Rhodoquinone biosynthesis protein a (RquA) is a putative methyltransferase-like enzyme that is essential for RQ biosynthesis in many bacteria and protists, making RquA a drug target specific to pathogens that require RQ. RquA uses ubiquinone as a substrate; however, the enzymatic mechanism of RquA is unknown. In this study we used Escherichia coli to produce RquA from Rhodospirillum rubrum, Blastocystis spp., Pygsuia biforma, and Euglena gracilis and purified it using Ni-NTA affinity chromatography under denaturing conditions. The recombinant RquA was initially refolded by shock dilution in Brij-35 (0.1% w/v) and we are further optimizing this refolding condition prior to protein crystallization. In addition, a computational approach was used to identify the possible cofactors and substrates required for RQ biosynthesis. The structure of RquA proteins were predicted using trRosetta to have a Rossmann-fold similar to that of S-adenosyl-L-methionine (SAM) dependent methyltransferases. Molecular dynamics simulation and the I-TASSER server predicted that SAM binds with RquA from Rhodospirillum via two aspartic acid residues. Mutation of those residues to alanine disrupted RQ production, confirming that SAM is required for RquA activity. Future computational studies will investigate possible mechanisms by which RquA localizes to cell membranes as well as devise strategies to increase the solubility of recombinantly produced RquA, in order to aid the in vitro characterization of RquA. Keywords: RquA; rhodoquinone; protein refolding; MD simulation; structure prediction.

Production of recombinant human G-CSF from non-classical inclusion bodies in Escherichia coli.

Recombinant granulocyte colony-stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.

Effect of mutation at oxyanion hole residu (H110F) on activity of Lk4 lipase

Mutant of lipase at oxyanion hole (H110 F) was constructed. The gene was highly expressed in Eschericia coli BL21 (DE3) and the recombinant protein was purified using Ni-NTA affinity chromatography. The activity of mutant enzyme was significantly increased compared to that the wild type. Further comparison showed that both of the enzymes exhibited same optimum pH and temperature, and showed highest lipolytic activity on pNP-decanoate (C10). The wild type appeared lost of activity on C14 and C16 substrates meanwhile the mutant still showed activity up to 20 %. In the presence of non polar organic solvent such as n-hexane, the wild type became inactive enzyme meanwhile the mutant still remained 50 % of its activity. The results suggested that mutation at oxyanion hole (H110 F) caused enzyme-substrate interaction change resulting on elevation of activity, better activity toward longer carbon chain substrate and improving the activity in the present of non polar organic solvent.

Soluble expression of recombinant active cellulase in E.coli using B.subtilis (natto strain) cellulase gene

BackgroundCellulases are well known for their various industrial applications. They are naturally produced by different species of bacteria and fungi. Fermentation process of cellulase producers has limitation due to the high substrate cost required for cellulase induction and challenges to maintain the suitable condition for the respective cellulase production. Recombinant cellulase production could be the potential solution to these problems. In the current study, we investigated recombinant cellulase expression in Escherichia coli using cellulase gene from Bacillus subtilis. ResultsExtracellular cellulase production from B. subtilis strain was first confirmed on CMC agar and then the cellulase gene (1500 bp) was amplified from this strain and was further cloned in pET21a expression vector. In initial experimental studies, recombinant cellulase expression was achieved in inclusion bodies through shake flask level fermentation of transformed E. coli expression host BL21DE3. Attempts were made to express this 55 KDa His tagged recombinant cellulase into soluble form by modifications in fermentation conditions. Partially purified recombinant cellulase was obtained using Ni-NTA affinity chromatography. The activity of the purified enzyme was confirmed by 3,5-dinitrosalicylic acid (DNS) qualitative assay. ConclusionSoluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.

Ni-NTA Affinity Chromatography to Characterize Protein-Protein Interactions During Fe-S Cluster Biogenesis.

Affinity chromatography can be repurposed to provide useful information about the specific partner protein(s) to which a protein of interest may bind as well as the relative binding affinity of that partner protein for the protein of interest. Here, we provide a protocol for an Ni-NTA affinity chromatography assay that may be utilized to uncover insightful information about the nature of protein-protein interactions during iron-sulfur (Fe-S) cluster biogenesis reactions.

Development and Evaluation of an Indirect Capripoxvirus ELISA Based on Truncated P32 Protein Expressed in E. coli.

As notifiable diseases, lumpy skin disease (LSD), sheep pox (SPP), and goat pox (GTP) are associated with a profound effect on cattle, sheep, and goat farming industries. Development of the ELISA method could effectively facilitate serodiagnosis of the infected animals. This study aimed to develop an ELISA system based on the recombinant full-length and truncated P32 protein (Tr.P32) of goat pox virus. The P32 protein was expressed in Rosetta strain of E. coli using pET24a+ vector and evaluated by SDS-PAGE and Western blotting. Then, Tr.P32 was purified by Ni-NTA affinity chromatography under denaturing conditions and used to develop a capripoxvirus-specific ELISA. Checkerboard titration and receiver-operating characteristic (ROC) analysis were used to optimize the ELISA system and determine diagnostic specificity and sensitivity, respectively. The diagnostic potential of the developed ELISA was evaluated using positive and negative control sera collected from goat, sheep, and cattle. Results showed that the expression level of full-length P32 recombinant protein was negligible, while Tr.P32, a ~ 31 kDa recombinant protein, was expressed up to 0.270-0.300 mg/200 mL of culture media. The results of checkerboard titration revealed that 675 ng/well of Tr.P32 antigen and 1:10 dilution of control sera (anti GTPV HIS and healthy goat sera) caused maximum difference in absorbance between positive and negative goat sera. The recombinant Tr.P32 showed good reactions with antibodies against GTP virus (GTPV), SPP virus (SPPV), and LSD virus (LSDV), whereas no cross-reactions with anti-Orf virus antibodies were detected. By comparing with the neutralization index (NI), cut off, diagnostic sensitivity and specificity of the developed indirect-ELISA were estimated, 0.397, 94% and 96.6%, respectively. These findings indicate that the ELISA system based on Tr.P32 protein could potentially be used in sero-surveillance of all capripoxviruses; however, further investigations are required.

The Role of Helicobacter pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model.

Background: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. Materials and Methods: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 μg /mL) and the propolis ethanolic extract (5-40 μg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. Results: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 μg/ml (P<0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 μg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 μg/mL (P=0.0001 and P=0.0004). Conclusion: We found that an OipA concentration of 10 μg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis’s capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response.