isopropyl-beta-D-thiogalactopyranoside Research Articles

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP3 [inositol(1, 4, 5)trisphosphate], IP4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24

To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Flagellin from Salmonella enteritidis Enhances the Immune Response of Fused F18 from Enterotoxigenic Escherichia coli.

F18 plays an important role in helping Enterotoxigenic Escherichia coli (ETEC) binds to specific receptors on small intestinal enterocytes, followed by secreting of toxins causing diarrhea in post-weaning piglets (post-weaning diarrhea, PWD). However, the F18 subunit vaccine is not sufficient to stimulate an immune response that can protect weaning pigs from F18-positive ETEC (F18+ETEC). Recently, a body of evidence shows that flagellin protein (FliC) helps to increase the immunity of fused proteins. Therefore, in this study, we combined FliC with F18 to enhance the immune response of F18. The f18 gene was obtained from F18+ETEC, then was fused with the fliC gene. The expression of recombinant FliC-F18 protein was induced by Isopropyl-beta-D-Thiogalactopyranoside (IPTG). The purified protein was tested in vivo in mouse models to evaluate the immunostimulation. Results showed that the fusion of FliC and F18 protein increased the production of anti-F18 antibodies. Besides, the anti-F18 antibody in the collected antiserum specifically identified F18+ETEC. This result provides proof-of-concept for the development of subunit vaccine to prevent PWD using F18 antigen.

Research Article: Characterization of a high-affinity polyclonal antibody against the Cyprinus carpio (Linnaeus, 1758) intestinal tight junction protein occluding

Tight junction protein, occludin, plays an important role in intestinal health of fish. To study the function of occludin in the intestinal barrier at the protein level, a rabbit occludin polyclonal antibody was prepared against heterologously expressed Cyprinus carpio A fragment of the occludin gene containing antigenic determinants was first ligated into the pET-21a, which is an expression vector and transformed into E. coli BL21 (DE3) strain. Expression of the target fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The purified fusion protein was used as an antigen to immunize New Zealand long-eared rabbits (Oryctolagus cuniculus) through ear margin vein and subcutaneous injection to obtain rabbit anti-carp polyclonal antibodies against occludin. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the antibody titre, and the antibody was used to determine the distribution and expression of occludin in the intestine of carp after infection with Aeromonas hydrophila. The target fusion protein had a molecular weight of approximately 31.7 ku, the antibody titre was 2.4 × 106, and the integrity of occludin protein was lower in various intestinal segments after A. hydrophila infection. The results indicated that the prepared antibody had a high titre, affinity, and specificity and can be applied to study the expression and distribution of occludin in C. carpio. The availability of this polyclonal antibody laid the foundation for the systematic study of the intestinal barrier of Cyprinus carpio . Additionally, this polyclonal antibody could also be used for explorative studies of the biological function of occludin in other fishes.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Construction and optimization of engineered Bacillus subtilis for surfactin production

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.

Molecular characterization, expression and functional analysis of TGFβ1-b in crucian carp (Carassius carassius)

Transforming growth factor β1 (TGFβ1) is a polyfunctional cytokine with important roles in growth, differentiation and immune function in various animals. In this study, PCR, bioinformatics, real-time quantitative PCR, prokaryotic expression, protein purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS) were applied to investigate the structural features and function of TGFβ1-b in crucian carp. The complete coding sequence (CDS) of TGFβ1-b was 1134 bp in length and was submitted to GenBank (ID: MH473141). TGFβ1-b encoded a putative protein of 377 amino acids and included a signal peptide consisting of 22 amino acids. TGFβ1-b was relatively conservative in fish and distant from mammals in terms of evolutionary relationship. TGFβ1-b was found to be expressed in various tissues, with the highest expression in the kidney. The expressions of TGFβ1-b in muscle, heart and liver were increased with the addition of Rhodopseudomonas palustris, Bacillus subtilis and Enterococcus faecium at 30 days (p < 0.01). While, the expressions of SMAD2, SMAD3 and SMAD7 were also up-regulated with the addition of R. palustris at 20 days (p < 0.01). The expression of TGFβ1-b could be affected by time and group factors (p < 0.05). Moreover, the expression vector TGFβ1-b-pDE2 was successfully constructed. Prokaryotic expression indicated that a 43 kDa target protein was obtained after induction with 1.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3.5 h at 37 °C for 200 r/h. The activities of alkaline phosphatase and lysozyme in injection TGFβ1-b protein group (ITg) and feeding broken bacterial liquid group (BTg) were significantly increased at 24 h (p < 0.01). And the activities of superoxide dismutase in ITg were significantly increased at 36 h (p < 0.01). Besides, the expressions of heat shock protein 30 and heat shock protein 47 in ITg and BTg were significantly increased (p < 0.01). Whereas, the expression of interleukin-11 was significantly reduced (p < 0.01). These results indicated that TGFβ1-b protein might play a role in immunity of crucian carp.

Molecular cloning, characterization of JunB in Schizothorax prenanti and its roles in responding to Aeromonas hydrophila infection

The transcription factor JunB can induce physiological or pathological responses to various stimuli, including immune stimulants and bacteria, and plays an important role in the immune response process. In this study, we identified a JunB family member in Schizothorax prenanti (S. prenanti), which was designated SpJunB. The complete coding sequence (CDS) of SpJunB was 930 bp in length, which was submitted to GenBank (ID: MN215886). SpJunB encodes a putative protein of 309 amino acids, which is highly homologous to JunB of common carp. The SpJunB protein contained a conserved JunB domain, and its 3D structure was also highly similar to (77.61%) the human SpJunB protein. SpJunB was found to be extensively expressed in various tissues, with the highest expression in the spleen. The expression of SpJunB was significantly upregulated after Aeromonas hydrophila (A. hydrophila) challenge. Prokaryotic expression indicated that a 51 kDa recombinant protein was obtained after induction with 1.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h at 37 °C. The expression levels of IL-1β, IL-6 and IL-8 were significantly upregulated (p < 0.01) after treatment of S. prenanti with the SpJunB protein. The activities of SOD, AKP and LZM were also significantly increased (p < 0.01) after the treatment of S. prenanti with the SpJunB protein. Simultaneously, the SpJunB protein reduced the infection rate of A. hydrophila in S. prenanti. In conclusion, SpJunB may improve the immune functions of S. prenanti. It will be beneficial to further study the immune mechanism of JunB in fish.

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.

Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir

Bacillus thuringiensis (Bt) is a gram positive bacterium which is effectively being used in pest management strategies as an eco-friendly bioinsecticide. In the present study a new cry2A gene was cloned from a promising indigenous B. thuringiensis SWK1 strain previously characterized for its toxicity against Spodoptera litura and Helicoverpa armigera larvae. The nucleotide sequence of the cloned cry2A gene pointed out that the open reading frame has 1902 bases encoding a polypeptide of 634 amino acid residues with a probable molecular weight of 70kDa. Homology comparisons showed that the deduced amino acid sequence of Cry2A had a similarity of 94% compared to that of the known Cry2Aa protein in the NCBI database and this gene has been named as cry2Al1 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry2Al1 was ligated into pET 22b vector and expressed in Escherichia coli BL21 (DE3) pLysS under the control of T7 promoter induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). SDS-PAGE analysis confirmed the expression of cry2Al1 as ∼65kDa protein. Insect pest bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 are toxic to S. litura and H. armigera with LC50 2.448μg/ml and H. armigera with 3.374μg/ml respectively.

Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine.

The purpose was to design a new construction containing influenza virus (H1N1) M2e gene and HA2 gene by bioinformatics approach, cloning the construct in to Escherichia coli and produce M2e-HA2 peptide. The procedure was done by virus cultivation in SPF eggs, hemagglutination assay (HA), RNA isolation, RT-PCR, primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (Ensemblegly-Iowa), complete open reading frame (ORF), stop codon studied (NCBI ORF Finder), rare codon determination (GenScript), Solvent accessibility of epitopes (Swiss-PdbViewer), antigenic sites prediction (Protean), fusion PCR of M2e-HA2 gene, sequence analysis, nested PCR, gel electrophoresis, double digestion of pET22b(+) plasmid and the fusion construct, ligation of them, transformation of the ligated vector (pET22b-M2e-HA2) to E.coli (BL21), mass culture the cloned bacterium ,induction the expression by isopropyl-beta-D-thiogalactopyranoside (IPTG), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), purification the fusion peptide by Ni-NTA column, western blot to verify the purification. In this study we developed a new approach for fusion of Influenza virus M2e (96 nucleotides) and HA2 (663 nucleotides) genes based on fusion PCR strategy and produced a fused fragment with 793 nucleotides. The construct was successfully cloned and expressed. This construct is a 261 amino acid chimeric fusion peptide with about 30 KD molecular weight. According on the latest information; this is the first case of expression and purification M2e-HA2 fusion chimeric peptide, which could be used for development of a recombinant M2e-HA2 fusion protein vaccine.

Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies

At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H.pylori infections, the detection of anti-CagA antibodies done by non-invasive commercial ELISA or WB methods, is important in diagnosis. Recombinant CagA protein produced in this study could easily be used in the ELISA tests to be developed for screening anti-CagA antibodies in the patients' sera. However, since an ELISA method using this antigen has not been developed yet, its diagnostic performance could not be evaluated in this study. In conclusion, the construction of such a system for recombinant CagA antigen expression may be a pilot study for the development of our own ELISA tests in Turkey, and also will help the clinicians for the prediction of disease outcome and decision of the appropriate antimicrobial treatment by the help of anti-CagA antibody detection.

English

Repeated use of L-asparaginase II enzyme, in the treatment of acute lymphoblastic leukaemia, is commonly needed because of the enzyme&rsquo;s instability and relatively short half-life which leads to more serious side effects on patients. In the present study, we report on the cloning and expression of L-asparaginase from a thermotolerant strain of Escherichia coli (KH027) which was isolated from camel manure and could grow at 45&deg;C. Expression of recombinant asparaginase was conducted by fusion asparaginase gene to pelB leader sequence and 6His residues at the C-terminus under the inducible T7 promoter in DH5&alpha; cells. Induction of the cells with 0.1 mM isopropyl-&beta;-D-thiogalactopyranoside (IPTG) at late log phase of growth resulted in 0.6-fold (2111 UI) higher to that obtained in early log phase induction (1319 UI) and 0.3-fold compared with mid log phase induction (1623 UI). The recombinant asparaginase protein was purified from the culture supernatant through nickel affinity chromatography. The apparent molecular weight of the tetramer enzyme was found to be ~141 kDa. Overall yield (87 mg/L) of the purified recombinant asparaginase was achieved at the shake flask level. The purified protein showed optimum activities at a temperature of 43&deg;C and pH 6. The Km and Kcat parameters were 3.8 mM-1 and 2.92 &times; 103s&minus;1, respectively. The enzyme retained around 57 and 30% of its initial activity after 30 and 60 min of incubation at 50&deg;C, respectively. Recombinant L-asparaginase was evaluated for its antiproliferative effect in the leukemia cell lines of RS4; 11 and HL60 after 96 and 72 h of incubation. The doses of 100 &mu;g/mL and time-response effect of 96 h caused a reduction value of 50% in cell viability of RS4. However, cell viability of 50% in the leukemic cells HL-60 was noticed with a concentration of 200 &mu;g/mL with an incubation period of 72 h. In vitro antiproliferative results in the leukemia cell lines encourage for making in vivo investigation to increase the possibility of using this thermostable enzyme in leukaemia therapy. &nbsp; Key words: L-Asparaginase II, leukemia cell, recombinant asparaginase.

HETEROLOGOUS EXPRESSION AND PURIFICATION OF DERMASEPTIN S4 FUSION IN Escherichia coli AND RECOVERY OF BIOLOGICAL ACTIVITY

Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system.

Molecular cloning of Reteplase and its expression in E. coli using tac promoter.

Background and Aims:This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression.Materials and Methods:Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE.Results:Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG.Conclusion:In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.

English

Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH) from Staphylococcus aureus ATCC 12600. Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR) product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH) was overexpressed with 1 mM isopropyl beta-D-1thiogalactopyranoside (IPTG); Michaelis constant (Km), maximum enzyme velocity (Vmax) and catalytic constant (Kcat) of expressed IMPDH were determined. Results: The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 μM, Vmax of 0.247±0.84/μM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ μM, Vmax of 0.378±0.036 μM/mg/min and Kcat of 4.78±0.021/min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP) correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa. Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis.

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library

Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.

Expression, Purification, Characterization and &lt;i&gt;In Vitro&lt;/i&gt; Activity of Recombinant Mouse Cu/Zn-Binding Superoxide Dismutase (mSOD1)

Purpose: To express, purify and characterize recombinant mouse Cu/Zn-binding superoxide dismutase (mSOD1), and investigate its activity in vitro.Methods: The protein, mSOD1, was expressed after induction with isopropyl-beta-Dthiogalactopyranoside (IPTG). The target protein was purified by Ni-NTA affinity chromatography. The identity of the recombinant protein was confirmed by Western-blot and peptide mass fingerprinting(PMF) analysis. Protein activity in vitro was investigated by SOD activity assay kit and DNA damage protective assay.Results: mSOD1 protein was expressed with a final yield of about 60 mg of pure protein per liter of culture medium. After purification, the target protein was &gt; 95 %. The identity of the recombinant protein was confirmed. SOD activity assay showed that the highest activity of the mSOD1 was 3789.0 ± 80.5 U/mg. The present work showed that mSOD1 was effective in protecting DNA from oxidative damage.Conclusion: High purity recombinant mSOD1 was obtained and characterized, and had high activity in vitro. The study indicates that the mSOD1 may serve as a potential therapeutic agent for those diseasescaused by oxidative stress.Keywords: Cu/Zn-binding Superoxide dismutase, Expression, Purification, Metal ions, DNA damage

Enhanced production of CoQ10 by constitutive overexpression of 3-demethyl ubiquinone-9 3-methyltransferase under tac promoter in Rhodobacter sphaeroides

Coenzyme Q10 (CoQ10), as an essential electron carrier in the aerobic respiratory electron transfer system, has been proven to be an effective compound in health care and disease prevention. With the aim of improving the production of CoQ10, the biosynthetic pathway of CoQ10 was engineered in Rhodobacter sphaeroides by overexpressing 3-demethyl ubiquinone-9 3-methyltransferase (UbiG), which catalyzes the two steps of O-methylations of the quinonoid ring. For expressing the UbiG, a constitutive expression vector harboring tac promoter and ubiG gene was firstly developed. After cultivated in dark conditions, the transcriptional level of ubiG of the recombinant R. sphaeroides without inducer, which approached to that of with inducer, was about 133 times that of the wild type. The production was correspondingly improved up to 65.8mg/L compared to 47.6mg/L for the wild type. In addition, it was found that isopropyl-beta-d-thiogalactopyranoside (IPTG) had a negative effect on CoQ10 production when added to the medium, although it showed no significant influence on the growth rate of the microbes.