IPTG Research Articles

Directed evolution of alditol oxidase for the production of optically pure D-glycerate from glycerol in the engineered Escherichia coli.

D-glycerate is an attractive chemical for a wide variety of pharmaceutical, cosmetic, biodegradable polymers, and other applications. Now several studies have been reported about the synthesis of glycerate by different biotechnological and chemical routes from glycerol or other feedstock. Here, we present the construction of an Escherichia coli engineered strain to produce optically pure D-glycerate by oxidizing glycerol with an evolved variant of alditol oxidase (AldO) from Streptomyces coelicolor. This is achieved by starting from a previously reported variant mAldO and employing three rounds of directed evolution, as well as the combination of growth-coupled high throughput selection with colorimetric screening. The variant eAldO3-24 displays a higher substrate affinity toward glycerol with 5.23-fold than the wild-type AldO, and a 1.85-fold increase of catalytic efficiency (kcat/KM). Then we introduced an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 expression system in E. coli to overexpress the variant eAldO3-24, and deleted glucosylglycerate phosphorylase encoding gene ycjM to block the consumption of D-glycerate. Finally, the resulting strain TZ-170 produced 30.1 g/l D-glycerate at 70 h with a yield of 0.376 mol/mol in 5-l fed-batch fermentation.

Engineering the permeability of Halomonas bluephagenesis enhanced its chassis properties

Bacterial outer membrane (OM), an asymmetric lipid bilayer functioning as a self-protective barrier with reduced permeability for Gram-negative bacteria, yet wasting nutrients and energy to synthesize, has not been studied for its effect on bioproduction. Here we construct several OM-defected halophile Halomonas bluephagenesis strains to investigate the effects of OM on bioproduction. We achieve enhanced chassis properties of H. bluephagenesis based on positive cellular properties among several OM-defected strains. The OM-defected H. bluephagenesis WZY09 demonstrates better adaptation to lower salinity, increasing 28%, 30% and 12% on dry cell mass (DCM), poly(3-hydroxybutyrate) (PHB) accumulation and glucose to PHB conversion rate, respectively, including enlarged cell sizes and 21-folds reduced endotoxin. Interestingly, a poly(3-hydroxybutyrate-co-21mol%4-hydroxybutyrate) (P(3HB-co-21mol%4HB)) is produced by H. bluephagenesis WZY09 derivate WZY249, increasing 60% and 260% on polyhydroxyalkanoate (PHA) production and 4HB content, respectively. Furthermore, increased electroporation efficiency, more sensitive isopropyl β-D-1-thio-galactopyranoside (IPTG) induction, better oxygen uptake, enhanced antibiotics sensitivity and ectoine secretion due to better membrane permeability are observed if OM defected, demonstrating significant OM defection impacts for further metabolic engineering, synthetic biology studies and industrial applications.

Expression of heterologous Polymerase n (PolN) using fusion partner protein maltose‐binding protein

Polymerase n (PolN) is a poorly understood error-prone polymerase that is involved in DNA repair pathways. PolN-deficient cells are more sensitive to interstrand cross-links (ICL) agents, suggesting that PolN is involved in repairing ICL. PolN has also been linked to cancer, including amplification in neuroendocrine prostate cancers, but its role is unknown. One of the challenges that hinders PolN studies is the poor heterologous expression of PolN. Here we seek to systematically improve the expression and purification of PolN heterologously expressed in E. coli. Plasmids with fusion partners that enhance high expression of insoluble proteins have been widely used. We employed the maltose-binding protein (MBP) as a solubility-enhanced protein and His tag to facilitate bacterial expression and purification. The Gateway plasmid was transformed to BL21 DE3 bacterial strain and the expression was induced by adding 0.5mM isopropyl β- d-1-thiogalactopyranoside (IPTG) after an OD600 of 0.6 was reached and kept constant at cold temperature overnight. The expression was optimized further to increase soluble protein production, including addition of arabinose and varying concentrations of both arabinose and IPTG. The induction temperature was kept at 37 degree Celsius and was kept constant for a duration of less than 10 hours. Finally, the cells were harvested and analyzed by SDS-PAGE to assess expression levels. The higher expression level was achieved at a cold temperature in the presence of both IPTG and 1% of arabinose. To achieve higher concentration of PolN harvested, we will increase the volume of cultures that will be expressed and purification will be done using affinity chromatography.

Chimeric Virus-like Particles of Universal Antigen Epitopes of Coronavirus and Phage Qβ Coat Protein Trigger the Production of Neutralizing Antibodies.

Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qβ) coat protein presenting the universal epitope of the coronavirus. The RNA phage Qβ coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl β-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. The successful construction of the chimeric VLPs of the phage Qβ coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.

Potato virus Y recombinant coat protein production in Escherichia coli: Applications in antibody production and virus detection

AbstractThe coat protein coding region of Potato virus Y (PVY) was cloned into pGEM‐T, sub‐cloned into plasmid pET‐28a (+) then transformed into Escherichia coli strain BL21 (DE3) pLysS to express the protein. Induction of the recombinant protein was made with isopropyl‐β‐d‐thiogalactopyranoside (IPTG), which has been tested in a variety of concentrations and for varying periods of time. The highest expression level of the protein was achieved with 1 mM IPTG for 4 hr. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting has been used to confirm the identity of the expressed protein. The PVY coat protein (30 kDa) was purified under denaturing conditions by affinity chromatography and identity reconfirmed by Western blotting. The expressed protein was used as a recombinant antigen to raise polyclonal antibodies in mice. Purified anti‐PVYCP immunoglobulin (IgG) reacted positively with PVY‐infected leaf samples and the purified expressed CP in plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA). The currently produced antiserum detects the more prevailing strains in the region: PVY N, NTN and Wilga.

BIOACTIVITY CHARACTERIZATION OF PURIFIED RECOMBINANT HYPOTHETICAL PROTEIN CODED BY OPEN READING FRAME-112 OF STREPTOMYCES.

This study was aimed to investigate the Open Reading Frame-112 (ORF-112) gene, which encoded for a hypothetical 218 amino acids protein in Streptomyces bacteria. A complete ORF-112 gene was synthesized, with addition of a 6xHis-Tag at the N-terminal location. The synthesized DNA nucleotides were sub-cloned into bacterial expression plasmid pBAT4. The pBAT4-ORF-112 plasmid transformed in bacterial cells BL21(De3)pLysS, intended for protein over expression, induced by isopropyl β- d-1-thiogalactopyranoside (IPTG). The IMAC affinity chromatography technique was deployed for protein purifications. Highly-purified fractions of ORF-112 were achieved by using affinity Ni++-columns. The purified ORF-112 protein was tested for possible biological activity. The SDS-PAGE analysis exhibited a soluble 30 kDa size purified ORF-112 protein which showed a slight gel shifting from the predicted size. The virulent activity test on purified fractions of ORF-112 was measured using the Disk Diffusion Test and it disclosed a clear zone formed in response to fungi Candida albicans growth. The data implies that the ORF-112 protein has an acceptable protective effect against the fungus C. albicans as compared to positive control ketoconazole (KCZ) (P < 0.05) while the protein has a significantly lower protective effect against the fungus than Itraconazole (ICZ) (P > 0.05). The results clarify the hypothetical ORF-112 protein is a novel protein with protective response effect against fungi cells C. albicans on disk-diffusion.test.

Identification of a Candidate Gene for the Novel Cytoplasmic Male Sterility Derived from Inter-Subspecific Crosses in Rice (Oryza sativa L.).

Tetep-cytoplasmic male sterility (CMS) was developed through successive backcrosses between subspecies indica and japonica in rice (Oryza sativa L.), which showed abnormal anther dehiscence phenotypes. Whole genome sequencing and de novo assembly of the mitochondrial genome identified the chimeric gene orf312, which possesses a transmembrane domain and overlaps with two mitotype-specific sequences (MSSs) that are unique to the Tetep-CMS line. The encoded peptide of orf312 was toxic to Escherichia coli and inhibited cell growth compared to the control under isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction. The peptide of orf312 contains COX11-interaction domains, which are thought to be a main functional domain for WA352c in the wild abortive (WA-CMS) line of rice. A QTL for Rf-Tetep (restorer-of-fertility gene(s) originating from Tetep) was identified on chromosome 10. In this region, several restorer genes, Rf1a, Rf1b, and Rf4, have previously been reported. Collectively, the interactions of orf312, a candidate gene for Tetep-CMS, and Rf-Tetep, a restorer QTL, confer male sterility and fertility restoration, respectively, which enables a hybrid rice breeding system. Further studies on orf312 and isolation of Rf-Tetep will help to identify the underlying molecular mechanism of mitochondrial ORFs with the COX11-interaction domains.

Immunological detection of AcAMP antimicrobial peptide secreted by Aspergillus clavatus.

Background and Objectives:Aspergillus clavatus antimicrobial peptide (AcAMP) is a fungi-derived peptide with a broad spectrum of activity against pathogenic bacteria and fungi. Natural antimicrobial peptides, including AcAMP, have attracted many attentions in the development of new natural antibiotics against pathogenic bacteria, especially multidrug resistant ones.Materials and Methods:In the present study, acamp gene was codon-optimized and chemically synthesized in pUC57 cloning vector, subcloned into pET28a (+) expression vector and transferred into competent Escherichia coli BL21 (DE3) cells. The expression of AcAMP was induced by addition of Isopropyl β- d-1-thiogalactopyranoside (IPTG) and the expressed peptide was purified by Ni-NTA. BALB/c mice were immunized with the purified peptide and the ability of the immunized mice sera for the detection of the native AcAMP secreted by A. clavatus IRAN 142C was examined through ELISA and Western blotting techniques.Results:Both ELISA and Western blotting demonstrated the ability of the sera of the immunized mice to detect the native AcAMP.Conclusion:The results of the present work show that the raised antibody against recombinant AcAMP can be used to detect AcAMP peptide, an issue which paves the way to develop detection kits for the detection of AcAMP-producing organisms, purification of this valuable peptide for further investigations.

Permeabilization-free β-galactosidase-induction-based electrochemical detection of Escherichia coli

Abstract Escherichia coli (E. coli) is recognized as an indicator of fecal bacterial contamination, and the induction of intracellular β-galactosidase (Gal) is commonly used for sensitive and selective E. coli detection. However, this method requires a permeabilization process to ensure that the Gal substrate and product are highly permeable through the E. coli membrane, making it unsuitable for simple point-of-care detection. Here, we report a sensitive electrochemical method for E. coli detection that does not require a permeabilization process. Intracellular Gal expression is increased via induction by isopropyl-β- d -thiogalactopyranoside (IPTG). 4-Methoxyphenyl-β- d -galactopyranoside (MPGP) and 4-methoxyphenol (MP) are used as the highly cell-permeable Gal substrate and product, respectively. Externally-added tyrosinase (Tyr) converts MP into electrochemically active 4-methoxycatechol, which is then electrochemically oxidized via electrochemical-chemical redox cycling involving an electrode, 4-methoxycatechol, and tris(2-carboxyethyl)phosphine. The presence of MPGP during the induction of intracellular Gal led to amplified MP production. The results of four different detection methods using three Gal substrates (MPGP, phenyl-β- d -galactopyranoside, and 4-aminophenyl-β- d -galactopyranoside) were compared. The detection limit for both drinking water and tap water spiked with E. coli, obtained following a 2-h-long IPTG treatment and 5-min-long Tyr reaction, was ∼2 × 103 colony-forming units (CFU)/mL, indicating that this method can be applied to monitoring E. coli contamination in water samples. Importantly, the present method does not require the electrode modification, affinity binding, washing, and filtration steps.

Overproduction and Purification of Organomercuric Lyase (MerB) from Mercury-resistant Bacteria Pseudomonas aeruginosa Isolate 4B2

Mercury is a very toxic element even though there is very little concentration in the body. Although all chemical forms of mercury are toxic, public health attention is focused on organic mercury which is the most toxic form of mercury. Organic mercury can, however, be detoxified by organomercuric lyase (MerB) protein derived from mercury resistant bacteria. This study aims to overproduce of MerB protein by transforming merB gene into E. coli BL-21(DE3). Nucleotide sequence of merB gene of mercury resistant bacteria Pseudomonas aeruginosa isolates 4B2, optimized by using gene program designers (www.dna20/com) then commercially synthesized and cloned in pET16b expression plasmid vector. Plasmid pET16b_merB (syntetic gene) was transformed into E. coli BL21(DE3) to produce MerB protein recombinant, induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and purified by imidazol. Overproduction and purification of MerB protein was successfully performed in E. coli BL21 mediated by plasmid pET16b, resulting MerB protein with a molecular weight of 25.6 kDa, with the optimum at 37°C incubation temperature, incubation time of 3 hours and 0.1 mM IPTG induction. MerB protein obtained can be used in further research on the enzymatic detoxification of organic mercury.

English

Staphylococcus aureus (S. aureus) is a versatile bacterium which exhibits multiple antibiotic resistances. To ameliorate the undesirable diseases causing potential, there is a need to design a protective vaccine capable of stimulating immune response against this pathogen. In a similar study in our laboratory, reverse vaccinology approach was used to nominate potential vaccine candidate genes against S. aureus. Zinc Metalloproteinase Aureolysin (aur) gene was one of the nominated genes based on that previously published in-silico study. The objective of this study is the cloning, expression, purification of aur gene and testing the aur protein reactivity with serum antibodies collected from groups of human patients with confirmed Staphylococcal disease. Cloning was done in pH6HTN His6HaloTag® vector and it was expressed in E. coli BL21 (DE3) using these conditions; 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37°C. Purification was carried out by Immobilized Metal Affinity Chromatography (IMAC). The his-tag aur protein was detected at ~86 KDa as a single band after western blot assay and was successfully reacted with antibodies obtained from humans infected with S. aureus. The results encourage further testing of aur protein as a potential vaccine candidate for S. aureus. Key words: Staphylococcus aureus, Zinc Metalloproteinase Aureolysin (aur), cloning, expression.

Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl β-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (1–4%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/μg pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.

Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only

BackgroundPrecise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production.ResultsHere, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting.ConclusionsIn all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

Expression of Cloned Genes in E. coli Using IPTG-Inducible Promoters.

Many Escherichia coli expression vectors make use of the lac operon. In general, the lac operator (lacO) is located downstream from the promoter of the target gene, so that binding of the lac repressor blocks transcription initiation until lactose or the isopropyl-β-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the ara operon offer an alternative to expression systems based on the lac operon; guidance for their use is also provided.

DNA photolyase from Antarctic marine bacterium Rhodococcus sp. NJ-530 can repair DNA damage caused by ultraviolet.

The online version contains supplementary material available at 10.1007/s13205-021-02660-8.

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes

This study aimed to produce and characterize a recombinant Kluyveromyces sp. β-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl β-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant β-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.

Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

For pilot-scale production of chito-oligosaccharides, it must be cost-effective to prepare designable recombinant chitosanase. Herein, an efficient method for preparing recombinant Bacillus chitosanase from Escherichia coli by elimination of undesirable substances as a precipitate is proposed. After an optimized culture with IPTG (Isopropyl β-d-1-thiogalactopyranoside) induction, the harvested cells were resuspended, disrupted by sonication, divided by selective precipitation, and stored using the same solution conditions. Several factors involved in these procedures, including ion types, ionic concentration, pH, and bacterial cell density, were examined. The optimal conditions were inferred to be pH = 4.5, 300 mM sodium dihydrogen phosphate, and cell density below 1011 cells/mL. Finally, recombinant chitosanase was purified to >70% homogeneity with an activity recovery and enzyme yield of 90% and 106 mg/L, respectively. When 10 L of 5% chitosan was hydrolyzed with 2500 units of chitosanase at ambient temperature for 72 h, hydrolyzed products having molar masses of 833 ± 222 g/mol with multiple degrees of polymerization (chito-dimer to tetramer) were obtained. This work provided an economical and eco-friendly preparation of recombinant chitosanase to scale up the hydrolysis of chitosan towards tailored oligosaccharides in the near future.

Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

The present study describes the cloning of the cellobiohydrolase gene from a thermophilic bacterium Clostridium clariflavum and its expression in Escherichia coli BL21(DE3) utilizing the expression vector pET-21a(+). The optimization of various parameters (pH, temperature, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, time of induction) was carried out to obtain the maximum enzyme activity (2.78 ± 0.145 U ml−1) of recombinant enzyme. The maximum expression of recombinant cellobiohydrolase was obtained at pH 6.0 and 70 °C respectively. Enzyme purification was performed by heat treatment and immobilized metal anionic chromatography. The specific activity of the purified enzyme was 57.4 U mg−1 with 35.17% recovery and 3.90 purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cellobiohydrolase was 78 kDa. Among metal ions, Ca2+ showed a positive impact on the cellobiohydrolase enzyme with increased activity by 115%. Recombinant purified cellobiohydrolase enzyme remained stable and exhibited 77% and 63% residual activity in comparison to control in the presence of n-butanol and after incubation at 80 °C for 1 h, respectively. Our results indicate that our purified recombinant cellobiohydrolase can be used in the biofuel industry.

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea.

SummaryBy screening 27,000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase highlighting the vast potential of prokaryotes. Caspian water with similar salt composition to the human serum was targeted for in silico L-asparaginase screening. We screened ca. three million predicted genes of its assembled metagenomes that resulted in annotation of 87 putative L-asparaginase genes. The L-asparagine hydrolysis was experimentally confirmed by synthesizing and cloning three selected genes in E. coli. Catalytic parameters of the purified enzymes were determined to be among the most desirable reported values. Two recombinant enzymes represented remarkable anti-proliferative activity (IC50 <1IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Similar salinity and ionic concentration of the Caspian water to the human serum highlights the potential of secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

Cloning and Expression of virulent proteins CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis

Aims and objects: Bovine tuberculosis caused by Mycobacterium bovis is an important disease that has negative effects in public health and economic losses. Traditional controlling program in cattle is based on test and slaughter strategy, but false positive is disadvantage of tuberculin skin test. To overcome the limitation, there is a need of using proteins with high specificity. The objective of this study was to clone and express of virulent protein CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis. Methods: Briefly, full-length genes of cfp-10 and esat-6 were amplified by PCR technique. In parallel pET23a (+) and PCR product has been double digested by EcoRI and HindIII. After Ligation, transformation was done into competent E.coli DH5α. Then the cloned vector was transformed into E.coli BL21. Induction was performed by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed proteins almost entirely in the inclusion body form. To dissolution of expressed protein, urea 8M was used. Purification of recombinant proteins was done by Nickle-Resin. Urea eliminated by decreasing gradient. Delayed hyper sensitivity of two proteins beside bovine tuberculin, avian tuberculin and human tuberculin studied in guinea-pig model. Results: Protein expression was confirmed by Western Blotting (WB) using 6X His-tag antibodies. The results showed that CFP-10 and ESAT-6 molecules has been successfully cloned and expressed in prokaryotic system. Results showed that CFP-10 has satisfactory relative potency but it is not in range about ESAT-6. Conclusion: The recombinant CFP-10 can play a significant role in reducing the false positive in diagnosis of bovine tuberculosis by improving the specificity of the tuberculin skin test