isopropylthio-β-D-galactoside Research Articles

Recombinant HNP-1 Produced by Escherichia coli Triggers Bacterial Apoptosis and Exhibits Antibacterial Activity against Drug-Resistant Bacteria.

ABSTRACTHuman neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical applications have been hampered by challenges during mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that Escherichia coli expressing full-length preproHNP-1 secretes a soluble form of HNP-1, which can be recovered from the total cell lysate after isopropyl thio-β-d-galactoside (IPTG) induction and ultrafiltration. Label-free quantitative proteomics and co-immunoprecipitation experiments revealed that HNP-1 induces cell apoptosis in bacteria by causing DNA and membrane damage. Notably, we found that HNP-1 disrupts the DNA damage response pathway by interfering with the binding of RecA to single-stranded DNA (ssDNA). Further experiments demonstrated that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results indicated that recombinant protein expression may be a simple and cost-effective solution to produce HNP-1 and that RecA inhibition via HNP-1 may serve as an alternative strategy to counteract antibiotic resistance.IMPORTANCE Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical application has been hampered by the difficulty of mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that recombinant protein expression combined with ultrafiltration may be a simple and cost-effective solution to HNP-1 production. We further found that HNP-1 induces bacterial apoptosis and prevents its SOS repair pathway from binding to the RecA protein, which may be a new antibacterial mechanism. In addition, we showed that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results provide new insights into the production and antibacterial mechanism of HNP-1, both of which may promote its clinical application.

Expression and physicochemical characterization of an N-terminal polyhistidine-tagged phosphotriesterase from the soil bacterium Brevundimonas diminuta

Abstract In this study, a truncated organophosphate-degrading (opd) gene encoding a Brevundimonas diminuta phosphotriesterase (BdPTE) without its leader peptide was artificially synthesized and cloned into the expression vector pQE-30 to yield pQE-BdPTE. The His6-tagged 37.8-kDa enzyme was purified from the cell-free extract of isopropyl thio-β- d -galactoside (IPTG)-induced E. coli M15 (pRep4) cells harboring pQE-BdPTE to near homogeneity by metal chelate affinity chromatography. The assay conditions for His6-tagged BdPTE activity were optimized at 60 °C, pH 8.0, and 1.0 mM Co2+ ion. Kinetic analysis of His6-tagged BdPTE-catalyzed hydrolysis of diethyl p-nitrophenyl phosphate (paraoxon-ethyl) showed that its Km and kcat values were 21.9 ± 1.4 μM and 311.6 ± 12.3 s−1, respectively. Also, the recombinant enzyme had the ability to efficiently degrade the two most commonly used OP pesticides, ethion and fenthion, in Taiwan. His6-tagged BdPTE with 1 mM Co2+ was very stable during the storage period of 35 days at 4 °C. Urea- and guanidine hydrochloride (GdnHCl)-induced denaturation studies showed that the purified enzyme had [denaturant]0.5,N–U values of 6.7 and 3.4 M, respectively. Notably, His6-tagged BdPTE did exhibit some degree of tolerance to a variety of water-miscible organic co-solvents. Dimethyl sulfoxide (DMSO), acetone, dimethylformamide (DMF), and ethanol were obviously harmful to the hydrolytic activity of the enzyme at concentrations above 50% (v/v), while acetonitrile was the highest tolerated co-solvent. The organic solvent-tolerant feature verifies His6-tagged BdPTE highly suitable as a remediation agent for the biodegradation of non-polar OP pesticides.

Enhanced periplasmic expression of human activin A in Escherichia coli using a modified signal peptide

Activin A is a member of the transforming growth factor-beta (TGF-β) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-β-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.

One-step orientated immobilization of nanobodies and its application for immunoglobulin purification

Affinity chromatography technologies play an important role in the purification of antibodies. To prepare affinity materials, prior isolation and purification of affinity ligands are required before coupling onto solid supports, which is quite expensive and laborious in large-scale applications. In this study, a one-step approach which circumvents the ligand purification procedures was developed to fabricate affinity gel for purifying immunoglobulin G (IgG). A self-labeling tag, haloalkane dehalogenase, was fused to the C-terminal of an anti-Fc variable domain of the heavy chain of the heavy-chain antibody (AFV) which was isolated in previous work. The AFV binds to various sources of IgG and is highly thermal stable. The fusion protein, namely HAFV, was expressed in Escherichia coli as a soluble protein. The binding affinity of HAFV to the Fc region of IgG was characterized and compared with the untagged anti-Fc nanobody. Next, the HAFV was immobilized directly from the crude cell lysate of isopropylthio-β-D-galactoside (IPTG) induced E. coli. The effects of NaCl concentrations and pH on the capacity of the HAFV resin were investigated. In addition, the one-step coupled HAFV resin was compared with the AFV resin and commercial resins (Protein A and Protein G) by evaluating the static capacity and stability. Though the Protein A (8.34 ± 0.37 mg/ml) and Protein G (9.19 ± 0.28 mg/ml) showed higher static capacity, the static capacity of HAFV resin (8.21 ± 0.30 mg/ml) was better than that of the untagged AFV gel (6.48 ± 0.56 mg/ml). The recovery results calculated for the reusability and stability show that there is no significant difference between the results obtained for the HAFV gel with those of the untagged AFV gel and commercial Protein A and G. After stored at 37 ℃ for 7 days and recycled 10 times, the static capacity of HAFV gel remains above 78%. Our strategy is site-specific, cost-effective, reproducible, and has the potential to dramatically cut down the costs of affinity materials for IgG purification.

Optimization of Vasopressin Type 2 Receptor Expression in Escherichia coli BL21

Background: Vasopressin type 2 receptor (V 2 R) is a G-protein-coupled receptor (GPCRs) located in nephrons mediating water reabsorption in kidneys. Vasopressin acts at these receptors to enhance water reabsorption in the kidneys while by acting on V1 receptors in blood vessels increases blood pressure. To study V 2 R better, it must be produced in high quantities. Objectives: The current study aimed to optimize expression of V 2 R in Escherichia coli. Materials and Methods: In this investigation, V 2 R gene was cloned in E. coli. The recombinant plasmid pEt15b/V 2 R (rpET-BL21) was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropyl-thio-β-D-galactoside (IPTG). The effects of different IPTG concentrations, temperatures and sampling times on the expression of V 2 R were examined. Samples were analyzed by SDS-PAGE. Results: The maximum amount of protein production was obtained by the addition of 0.75 mM IPTG at 37°C after three hours. The most important factor was harvesting time with P value < 0.0001. In statistical analysis of data, the normal plot shows the normality of distribution of the studentized data. Conclusions: Full factorial design experimental approach helped to identify the critical culture condition parameters in heterologous expression of V 2 R. It would allow studying the parameters affecting the function of this protein in future experiments.

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BackgroundMutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.MethodsSuppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-rasval12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7–4) harboring the inducible Ha-rasval12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.ResultsRetinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-rasval12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells “7-4” by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.ConclusionsWe confirmed that RbAp46 is a Ha-rasval12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1155-7) contains supplementary material, which is available to authorized users.

Efficient expression of myo-inositol oxygenase in Escherichia coli and application for conversion of myo-inositol to glucuronic acid

Glucuronic acid is an important biochemical with wide applications in the food and medical industries. The myo-inositol oxygenase (MIOX) gene was synthesized and expressed in Escherichia coli BL 21(DE3). After optimization of induction conditions and the culture temperature, the highest MIOX activity (45.46 kU/L) was achieved when 0.1 mM isopropyl-thio-β-d-galactoside (IPTG) was added to cell cultures with an OD600 value 1.0 followed by induction at 26°C for 8 hours. The purified MIOX enzyme exerted characteristics similar to the native form. Conversion of myo-inositol to glucuronic acid was performed using whole cells in a pH 8.5 buffer system. Whole cells harboring myo-inositol oxygenase were used as a biocatalyst to produce 2.13 g/L of glucuronic acid with a conversion rate of 99%. A promising novel process for glucuronic acid production from abundant agricultural byproducts is presented.

English

Escherichia coli 16 has potential probiotic properties including antimicrobial activity due to extracellular secretion of colicins E1/1a1b. Inulosucrase (InuJ) enzyme catalyses the polymerization of a fructose moiety of sucrose leading to the formation of fructooligosaccharides. The present investigation compared the activity of InuJ enzymes cloned into pMAL-p2ΔlacI Q a deletion vector and transformed into E. coli 16 and standard strain that is, E. coli BL21. Specific activities of InuJ enzyme were estimated in supernatant, periplasm and lysate. Specific activities of InuJ activity in cell lysate were similar in E. coli 16 and E. coli BL21 without induction of tac promoter with isopropyl thio-β-Dgalactoside (IPTG). InuJ activity is mainly present in the periplasm of E. coli BL21 whereas in E. coli 16, most of the activity is in the supernatant. Superantant of E. coli 16 strain also showed good antibacterial activity due to colicin E1/Ia1b. Colicin E1/1a1b transport system could allow extracellular secretion of InuJ proteins in probiotic E. coli 16. Key words: Colicin, extracellular, E. coli , fructooligosaccharide, inulosucrase, prebiotic, probiotic.

Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-β-D-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap™ HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 ± 0.01 U/ml for C12 LA isomerase and 8.12 ± 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 °C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.

Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells

With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β- d-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of rabavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.

A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20–25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-β-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19–CD50, with CD27–28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.

Periplasmic expression and one-step purification of urease subunit B of Helicobacter pylori

UreB is one of the urease subunits of Helicobacter pylori and can be used as an excellent antigen candidate for H. pylori vaccination. Easy access to highly purified UreB protein, facilitate advances in therapeutic or preventive strategies. To achieve a simplified purification procedure, the present report represents a novel method of producing recombinant urease subunit B extracellularly. ureB gene from 26,695 standard strain was amplified by PCR and cloned into pET-26b(+) expression vector. UreB was expressed as a soluble, N-terminal pelB and C-terminal hexahistidine-tagged fusion protein (UreB-6His) and secreted into the periplasmic space of Escherichia coli. Expression of the recombinant UreB in E. coli BL21 (DE3) was induced by isopropylthio-β-d-galactoside (IPTG). Expression of UreB was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis using anti-His monoclonal antibody. UreB-6His protein was extracted from the periplasm by osmotic shock treatment and was purified in one step by Nickel affinity chromatography. In conclusion, the present protocol is easier to perform; more time effective and low cost than earlier methods.

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

Correlaion between serum Golph2 protein and hepatocellular carcinoma

To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein. Golph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein. There were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40. The polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.

Molecular Cloning, Recombinant Expression, and Characterization of Lysozyme from Chinese Shrimp Fenneropenaeus chinensis

Lysozyme catalyzes the hydrolysis of bacterial cell walls and acts as an innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named it as Fc-lysozyme ( FcLyz in short). The full length of the gene is of 709 bp, and the open reading frame (477 bp) encodes 158 amino acids. The predicted protein has a signal peptide (–1∼–18 residue) and the molecular weight of the mature protein (residue 1–140) is of 16.2 kD. A Lyz 1 domain (residue 1–130) in the FcLyz was identified by SMART analysis. The results of semi-quantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-β-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria and the MIC reached 3.43 μM, while it showed relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important effector molecules against pathogens in innate immunity of the shrimp.

Expression and Purification of ZNF191(243–368) in Three Expression Systems

AbstractZNF191(243–368), a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain adequate amount of ZNF191(243–368) for the study of its property, structure and function, three different expression systems of inclusion‐body, glutathione S‐transferase (GST), and hexahistidine (6×His) were used and compared. Among these systems, the expression level of ZNF191(243–368) was increased in inclusion body system under a higher isopropylthio‐β‐D‐galactoside (IPTG) concentration, but the non‐target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His‐tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

CLONAMIENTO, EXPRESIÓN Y SEROREACTIVIDAD DEL ANTÍGENO RECOMBINANTE FLAGELINA DE Bartonella bacilliformis

Objectives: To clone the Bartonella bacilliformis flagellin gene (flaA), and to preliminarily express and assess reactivity of the recombinant protein against B. bacilliformis bartonellosis patients’ sera. Materials and Methods: A couple of initiating oligonucleotides was designed –BbFlaA1 and BbFlaA2– for complete B. bacilliformis flagelline A gene amplification. The amplification product obtained was cloned in pEGM, and later it was subcloned in pGEX4T1 expression vector. Fusion protein rBbFlaA-GST expression was induced with isopropyl thio-β-D-galactoside (IPTG). The fusion protein produced was digested with thrombin in order to release its GST contents. Finally, an ELISA test was standardized in order to detect IgG antibodies against fusion protein rBbFlaA-GST and rBbflaA GST-free. Sera from patients with bartonellosis caused by B. bacilliformis (n= 30), sera from healthy individuals (n= 20), and sera from patients with a possible cross-reactivity; i.e., brucellosis (n= 3), leptospirosis (n= 3), and salmonellosis (n= 7) were assessed. Results: It was determined that for optimal expression of fusion protein rBbFlaA in E. coli BL21, it is required that the culture grows in LB/ampicillin broth at a 30o C temperature, supplemented with 2% glucose from a 100 µL pre-inoculum (left to grow overnight), until it reaches a 1 optical density (OD 600 ), and being induced for two hours at 2,5 mM IPTG. Finally, 57,6% (17 of 30) sera from patients with a confirmed bartonellosis diagnosis reacted with BbFlaA recombinant protein in an ELISA format. Conclusions: B. bacilliformis BbFlaA recombinant protein was successfully expressed in E. coli, and an rBbFlaA expression and purification protocol was determined for producing this protein. Also, rBbFlaA antigen is recognized by antibodies present in sera from bartonellosis patients infected with B. bacilliformis.

Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-β- d-galactoside (IPTG) was found to be 5 μM. The fusion protein was purified in a single step by Ni–IDA affinity chromatograph to a specific activity of 35.3 U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 °C, respectively. The K m and V max are 17.6 μM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.