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Sun, Huigang, Xiaomei Bie, Fengxia Lu, Yaping Lu, Yundailai Wu y Zhaoxin Lu. "Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter". Canadian Journal of Microbiology 55, n.º 8 (agosto de 2009): 1003–6. http://dx.doi.org/10.1139/w09-044.
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Bacillus subtilis fmbR-1 was obtained from wild-type B. subtilis fmbR by replacement of the native promoter of the surfactin operon with the inducible promoter Pspac. The recombinant B. subtilis fmbR-1 produced more antibacterial substances than the wild-type strain. The overproducing phenotype was related to the enhancement of antagonistic activities against Bacillus cereus . HPLC peaks of surfactin for recombinant fmbR-1 showed patterns of lipopeptides similar to those of the wild-type strain, and surfactin production of the recombinant fmbR-1 was up to about fivefold greater than that of the wild-type strain without induction by isopropyl β-d-1-thiogalactopyranoside. However, the production of surfactin increased to about 10-fold more than that of the wild-type strain when it was induced by isopropyl β-d-1-thiogalactopyranoside.
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Lee, Sung Kuk, Howard H. Chou, Brian F. Pfleger, Jack D. Newman, Yasuo Yoshikuni y Jay D. Keasling. "Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters". Applied and Environmental Microbiology 73, n.º 18 (20 de julio de 2007): 5711–15. http://dx.doi.org/10.1128/aem.00791-07.
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ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.
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HUBCHYK, K. A., R. N. BIRUKOU, А. М. НLUSHEN, I. S. KAZLOUSKI y A. A. KASTSIANEVICH. "GENETIC ENGINEERING OF STRAIN ESCHERICHIA COLI BL21.BT1 CAPABLE TO SYNTHESIZE RECOMBINANT BETA-1,3-NACETYLGLUCOSAMINE TRANSFERASE". Микробные биотехнологии: фундаментальные и прикладные аспекты 13 (21 de octubre de 2021): 52–65. http://dx.doi.org/10.47612/2226-3136-2021-13-52-65.
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A strain of Escherichia coli BL21.Bt1, a producer of the recombinant beta-1,3-N-acetylglucosamine transferase Bacillus thuringiensis BIM B-180, has been constructed. The cultivation conditions of the producer strain are optimized: the initial pH value of the nutrient medium is 7.2; cultivation temperature after induction – 20 °C; constant stirring at an intensity of 200 rpm; the use of 1 mM isopropyl-β-D-1-thiogalactopyranoside as an inducer; introduction of 10 mM lactose 3 h after induction.
It was shown that the yield of the target enzymatic protein after 24 h of cultivation of the E. coli BL21.Bt1 strain under optimized conditions reaches 63 μg/ml.
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Sitasiwi, Agung Janika, Wayan Tunas Artama, Agung Budiyanto y Edy Dharmana. "MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21". Jurnal Kedokteran Hewan - Indonesian Journal of Veterinary Sciences 11, n.º 1 (7 de abril de 2017): 11–14. http://dx.doi.org/10.21157/j.ked.hewan.v11i1.5891.
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This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully.
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Muttar, Arafat y Intesar Tarik Numan. "Cloning & expression of SAK enzyme from Staphylococcus aureus in E. coli BL21-CodonPlus". Journal of Medicine and Life 15, n.º 6 (junio de 2022): 768–71. http://dx.doi.org/10.25122/jml-2021-0335.
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Staphylokinase (SAK), also known as staphylococcal fibrinolysin, is a protein with a molecular mass of about 15 kDa produced by Staphylococcus aureus. Staphylokinase is synthesized in the late exponential phase, similar to streptokinase. The current study identified and predicted the protein SAK from Staphylococcus aureus. SAK is a fibrinolytic enzyme of the third generation that acts as an indirect activator of plasminogen. The current study cloned and expressed SAK protein isolated from Staphylococcus aureus and used in the form of a grid for enhancement of SAK Catalyst with PCR, disengagement, and change into articulation vector PET24b(+). The recombinant plasmid was changed into E. coli strain BL21 (codon additionally to 440) acceptance with isopropyl β-D-1-thiogalactopyranoside (IPTG).
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Shafat, Zoya, Abu Hamza, Farah Deeba, Md Imam Faizan, Nazim Khan, Asimul Islam, Anwar Ahmed, Salman F. Alamery y Shama Parveen. "Optimization of parameters for expression and purification of G glycoprotein ectodomain of respiratory syncytial virus". Future Virology 15, n.º 4 (abril de 2020): 225–35. http://dx.doi.org/10.2217/fvl-2019-0157.
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Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was expressed and purified to achieve maximum yield of the protein. Materials & methods: We optimized different parameters like strains, temperature, inducer concentration and post induction time period for efficient protein expression in Escherichia coli. The protein was purified using affinity chromatography and confirmed by western blotting. Results: It was concluded that a 5-h induction with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C in BL21(DE3) cells was the most favorable condition for maximal protein expression. The far-UV circular dichroism spectroscopy suggested that it is an α-helical protein. Conclusion: The purified Ge protein can be characterized by antigenic and biophysical methods in future studies, which will probably assist in vaccine development.
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Grinanda, Dita y Takashi Hirasawa. "Effectiveness of the Bacillus subtilis genome-reduced strain as an ethanol production host". Bioscience, Biotechnology, and Biochemistry 86, n.º 4 (31 de enero de 2022): 543–51. http://dx.doi.org/10.1093/bbb/zbac017.
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ABSTRACT We investigated the performance of a genome-reduced strain of Bacillus subtilis MGB874, whose 0.87 Mbp of genomic DNA was cumulatively deleted, as an ethanol production host. A recombinant strain A267_EtOH was constructed by introducing the pdc and adhB genes from Zymomonas mobilis, both of which were expressed from an isopropyl-β-d-1-thiogalactopyranoside-inducible spac promoter, into the A267 strain, a tryptophan prototrophic derivative of the MGB874 with disruption of metabolic pathways for producing lactic acid, acetic acid, and acetoin. Focusing on the stationary phase in fed-batch fermentation, 1.6 g L−1 ethanol was produced by the A267_EtOH strain after 144 h. Moreover, its ethanol production further increased by approximately 3.7-fold (5.9 g L−1) at 80 h through replacing the spac promoter for expressing pdc and adhB genes with the lytR promoter and the yield was about 112%. These results indicate that the MGB874 is an effective host for ethanol production during the stationary phase.
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Zhen-Hui, Song, Guo Wan-Zhu y Zhang Ying-Jun. "Prokaryotic expression of nucleoprotein gene of Transmissible gastroenteritis virus and development of ELISA based on the expressed protein". Chinese Journal of Agricultural Biotechnology 6, n.º 3 (diciembre de 2009): 249–52. http://dx.doi.org/10.1017/s1479236209990520.
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AbstractThe recombinant PET-N plasmid, which includes the N gene of the Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21 (DE3), and induced at 37°C with 1.0 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside). An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the TGEV nucleocapsid protein antibody was developed after the reactogenicity of the recombinant protein was demonstrated by Western blot. The operating conditions for the ELISA, an antigen concentration of 15 μg/ml, serum dilution of 1:40, blocking solution of 0.5% fetal bovine serum (FBS), serum sample incubation for 90 min, a concentration of horseradish peroxidase (HRP)-spa of 1:5000, incubated for 60 min, incubation of the substrate at room temperature for 10 min, and a cutoff value for the ELISA OD450⩾0.35, were carried out using a checkerboard titration method. The sensitivity and specificity of this method relative to the Svanova TGEV/PRCV antibody diagnosis kit were 93.5% and 93.8%, respectively.
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Lipničanová, Sabina, Barbora Legerská, Daniela Chmelová, Miroslav Ondrejovič y Stanislav Miertuš. "Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli". Biomolecules 12, n.º 2 (19 de febrero de 2022): 331. http://dx.doi.org/10.3390/biom12020331.
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Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.
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Jie, Jing, Xiao Chu, Dan Li y Zhaoqing Luo. "A set of shuttle plasmids for gene expression in Acinetobacter baumannii". PLOS ONE 16, n.º 2 (10 de febrero de 2021): e0246918. http://dx.doi.org/10.1371/journal.pone.0246918.
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Infections caused by the emerging opportunistic bacterial pathogen Acinetobacter baumannii are occurring at increasingly alarming rates, and such increase in incidence is further compounded by the development of wide spread multidrug-resistant strains. Yet, our understanding of its pathogenesis and biology remains limited which can be attributed in part to the scarce of tools for molecular genetic analysis of this bacterium. Plasmids based on pWH1277 originally isolated from Acinetobacter calcoaceticus are the only vehicles currently available for ectopic gene expression in Acinetobacter species, which restricts experiments that require simultaneous analysis of multiple genes. Here, we found that plasmids of the IncQ group are able to replicate in A. baumannii and can stably co-reside with derivatives of pWH1277. Furthermore, we have constructed a series of four plasmids that allow inducible expression of Flag-tagged proteins in A. baumannii by arabinose or isopropyl β-d-1-thiogalactopyranoside. Together with constructs previously developed, these plasmids will accommodate the need in genetic analysis of this increasingly important pathogen.
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Ahn, Yeh-Jin y Na-Hyun Song. "A Cytosolic Heat Shock Protein Expressed in Carrot (Daucus carota L.) Enhances Cell Viability under Oxidative and Osmotic Stress Conditions". HortScience 47, n.º 1 (enero de 2012): 143–48. http://dx.doi.org/10.21273/hortsci.47.1.143.
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The expression and function of DcHsp17.7, a small heat shock protein expressed in carrot (Daucus carota L.), was examined under oxidative and osmotic stress conditions. Comparative analysis revealed that DcHsp17.7 is a cytosolic Class I protein. Sequence alignment showed that DcHsp17.7 has the characteristic α-crystalline domain-containing consensus regions I and II. Under oxidative [hydrogen peroxide (H2O2)] and osmotic (polyethylene glycol) stress conditions, DcHsp17.7 accumulated in carrot leaf tissue. To examine its function under these abiotic stress conditions, the coding sequence of DcHsp17.7 was introduced into Escherichia coli and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Under both oxidative and osmotic stress conditions, heterologously expressed DcHsp17.7 enhanced bacterial cell viability. The expression level of soluble proteins was higher in transgenic cells expressing DcHsp17.7 when compared with controls under these stress conditions. These results suggest that DcHsp17.7 confers tolerance to both oxidative and osmotic stresses and thereby functions as a molecular chaperone during the stresses examined.
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Wyatt, Morgan A. y Nathan A. Magarvey. "Optimizing dimodular nonribosomal peptide synthetases and natural dipeptides in an Escherichia coli heterologous host". Biochemistry and Cell Biology 91, n.º 4 (agosto de 2013): 203–8. http://dx.doi.org/10.1139/bcb-2012-0097.
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Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.
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Gomes, Luciana, Gabriel Monteiro y Filipe Mergulhão. "The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms". International Journal of Molecular Sciences 21, n.º 2 (16 de enero de 2020): 576. http://dx.doi.org/10.3390/ijms21020576.
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This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.
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Tan, Boon Hooi, Thean Chor Leow, Hooi Ling Foo y Raha Abdul Rahim. "Molecular Characterization of a Recombinant Manganese Superoxide Dismutase fromLactococcus lactisM4". BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/469298.
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A superoxide dismutase (SOD) gene ofLactococcus lactisM4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression ofsodAunder T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD ofL. lactisIL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).
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Takagi, Motoki, Tomohisa Kuzuyama, Shunji Takahashi y Haruo Seto. "A Gene Cluster for the Mevalonate Pathway from Streptomyces sp. Strain CL190". Journal of Bacteriology 182, n.º 15 (1 de agosto de 2000): 4153–57. http://dx.doi.org/10.1128/jb.182.15.4153-4157.2000.
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ABSTRACT A biosynthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC1.1.1.34 ), the rate-limiting enzyme of the mevalonate pathway for isopentenyl diphosphate biosynthesis, had previously been purified fromStreptomyces sp. strain CL190 and its corresponding gene (hmgr) had been cloned (S. Takahashi, T. Kuzuyama, and H. Seto, J. Bacteriol. 181:1256–1263, 1999). Sequence analysis of the flanking regions of the hmgr gene revealed five new open reading frames, orfA to -E, which showed similarity to those encoding eucaryotic and archaebacterial enzymes for the mevalonate pathway. Feeding experiments with [1-13C]acetate demonstrated that Escherichia coli JM109 harboring the hmgr gene and these open reading frames used the mevalonate pathway under induction with isopropyl β-d-thiogalactopyranoside. This transformant could grow in the presence of fosmidomycin, a potent and specific inhibitor of the nonmevalonate pathway, indicating that the mevalonate pathway, intrinsically absent in E. coli, is operating in the E. coli transformant. The hmgr gene andorfABCDE are thus unambiguously shown to be responsible for the mevalonate pathway and to form a gene cluster in the genome ofStreptomyces sp. strain CL190.
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Rucká, Lenka, Natalia Kulik, Petr Novotný, Anastasia Sedova, Lucie Petrásková, Romana Příhodová, Barbora Křístková, Petr Halada, Miroslav Pátek y Ludmila Martínková. "Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus". Molecules 25, n.º 17 (25 de agosto de 2020): 3861. http://dx.doi.org/10.3390/molecules25173861.
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Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl β-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed β-cyano-L-alanine (β-CA) with the highest specific activities (ca. 132 and 40 U mg−1, respectively) similar to plant NLase NIT4. β-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5–9 vs. pH 5–7). These NLases may participate in plant–fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.
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Ishuk, Sergey A., Elena G. Bogomolova, Olga A. Dobrovolskaya, Alyona O. Akhmetshina, Daria S. Krasnoshchek, Anna A. Lukovenko, Ekaterina A. Fedorova et al. "Production of recombinant IGF1 and its action on neuroblastoma cells in vitro". Medical academic journal 18, n.º 4 (15 de diciembre de 2018): 34–41. http://dx.doi.org/10.17816/maj18434-41.
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This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein.
To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography.
The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons.
Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.
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Zhao-Ju, Tian, Zheng Yu-Shu, Liu Cui-Yan, Hu Jing-Dong y Zhao Hong-Kun. "Expression and biological activity assay of bovine interleukin-18 fusion protein". Chinese Journal of Agricultural Biotechnology 5, n.º 1 (abril de 2008): 7–11. http://dx.doi.org/10.1017/s1479236207001830.
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AbstractThe cDNA of bovine interleukin-18 (BoIL-18) was subcloned into pGEX6P-1 vector and transformed into Escherichia coli BL21(DE3). The recombinant protein was successfully expressed in E. coli by induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 0.3 mmol/l for 8 h. SDS-PAGE indicated that the BoIL-18 fusion protein, 44 kDa, was highly expressed. Densitometric scanning showed that the fusion protein expression was about 31.8% of the total bacterial protein. The biological activity of the chromatographically purified protein was assayed. The peripheral blood mononuclear cells (PBMC) proliferation test indicated that the BoIL-18 fusion protein could enhance PBMC proliferation when its concentration was more than 0.10 mg/l. Enzyme-linked immunosorbent assay (ELISA) showed that the BoIL-18 fusion protein could induce interferon (IFN)-γ production from spleen lymphocytes when it was at a concentration of more than 0.20 mg/l, and that the inducing effect of BoIL-18 fusion protein on IFN-γ was directly proportional to its concentration. This verified that the purified BoIL-18 fusion protein possessed a functional activity and could be applied in further studies.
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Hwang, Hyun-Ju, Jin-Woo Han, Hancheol Jeon y Jong Han. "Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin". Biomolecules 8, n.º 4 (14 de noviembre de 2018): 146. http://dx.doi.org/10.3390/biom8040146.
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Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)—i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl β-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.
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Cheng, Chih-Yu, Chia-Huang Tsai, Pei-Jyun Liou y Chi-Hang Wang. "Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach". Polymers 13, n.º 2 (18 de enero de 2021): 290. http://dx.doi.org/10.3390/polym13020290.
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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.
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Shang, Fei, Hui Wang, Dan Zhang, Wenhui Wang, Jiangliu Yu y Ting Xue. "Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli". PeerJ 9 (16 de noviembre de 2021): e12497. http://dx.doi.org/10.7717/peerj.12497.
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Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.
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Varman, Arul M., Yi Xiao, Himadri B. Pakrasi y Yinjie J. Tang. "Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production". Applied and Environmental Microbiology 79, n.º 3 (26 de noviembre de 2012): 908–14. http://dx.doi.org/10.1128/aem.02827-12.
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ABSTRACTGlobal warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacteriumSynechocystissp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employedin situremoval of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions.
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Granicka, L. H., M. Wdowiak, A. Kosek, S. Świezewski, D. Wasilewska, E. Jankowska, A. Weryński y J. Kawiak. "Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report". Cell Transplantation 14, n.º 5 (mayo de 2005): 323–30. http://dx.doi.org/10.3727/000000005783983043.
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The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.
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Fang-Fang, Gao, Wu Zhong-Yi y Zeng Shen-Ming. "Bovine interferon-tau expression in Escherichia coli and identification of its biological activities". Chinese Journal of Agricultural Biotechnology 5, n.º 3 (diciembre de 2008): 189–95. http://dx.doi.org/10.1017/s147923620800226x.
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AbstractThe bovine interferon-tau (bIFN-τ) gene, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and subcloned into a pGEM-T vector. After being verified, the fragments, with or without signal sequence, were inserted into the expression vector pET-30a(+). Two recombinant plasmids were induced to express the recombinant proteins by isopropyl β-d-1-thiogalactopyranoside. The results showed that the bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. It had 99% homology with nucleotides and 97% with amino acids in the GenBank sequence (accession number: XM_593584). The products of recombinant bIFN-τ, minus signal sequence, expressed in pET-30a(+) were analysed by SDS-PAGE. A new 20 kDa protein was detected and its molecular weight was as expected. The antiviral activity of recombinant bIFN-τ was 1×104 IU/mg using a standard cytopathic reduction assay. Marked morphological changes were induced by recombinant bIFN-τ in bovine endometrial epithelial cells. The cell volume was larger than that of controls and a lot of vesicles appeared in the cytoplasm after 24 h culture in the presence of 2.9 μg/ml recombinant bIFN-τ. In conclusion, a purified recombinant, biologically active bIFN-τ was obtained in this experiment.
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Yang, Junbao, Terence Moyana, Samuel MacKenzie, Qun Xia y Jim Xiang. "One Hundred Seventy-Fold Increase in Excretion of an FV Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-α) fromEscherichia coli Caused by the Synergistic Effects of Glycine and Triton X-100". Applied and Environmental Microbiology 64, n.º 8 (1 de agosto de 1998): 2869–74. http://dx.doi.org/10.1128/aem.64.8.2869-2874.1998.
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ABSTRACT To target tumor necrosis factor alpha (TNF-α) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH–TNF-α, which encodes the secreted form of single-chain fusion protein sFV/TNF-α in Escherichia coli. sFV/TNF-α was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30°C and 37 μmol of isopropyl-β-d-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37°C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-α was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-α in E. coli by using glycine and Triton X-100.
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Phan, Hanh Thi-Thu, Nguyen Ngoc Yen Nhi, Le Thuy Tien, Chu Thi Bich Phuong, Le Thi Phuong Ngan, Phan Thi Phuong Trang y Hoang Duc Nguyen. "Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter". Science and Technology Development Journal 22, n.º 2 (19 de junio de 2019): 239–46. http://dx.doi.org/10.32508/stdj.v22i2.1284.
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Introduction: In basic research, it is essential to use an inducible promoter which can be controlled to express a small amount of protein for studying their roles in the cell. Pspac, a well-known weak promoter for Bacillus subtilis, uses isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. However, plasmids carrying this promoter such as pHCMC05 still have a disadvantage which harbors a repetitive DNA fragment of about 200 bp, resulting in structural instability in Escherichia coli, causing difficulty during cloning.
Methods: In this study, we constructed a plasmid that does not carry the repetitive sequences and investigated plasmid structural stability in E. coli, then measured the β-galactosidase reporter gene (bgaB) expression in B. subtilis.
Results: The constructed plasmid pHT2002 was stable over 56 generations while pHCMC05-bgaB was structurally instable and ultimately lost after 42 generations. BgaB activities and Western-blot indicated that BgaB-coding gene under control of IPTG-inducible promoter Pspac could be expressed at low levels.
Conclusion: The study demonstrated that the new expression plasmid without the repeated sequences retained its structural stability in E. coli facilitating the cloning step. The expression plasmid with Pspac promoter for B. subtilis could be used to express a modest amount of the heterologous protein in the presence of IPTG.
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PERITO, Brunella, Nerino ALLOCATI, Enrico CASALONE, Michele MASULLI, Beatrice DRAGANI, Mario POLSINELLI, Antonio ACETO y Carmine DI ILIO. "Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis". Biochemical Journal 318, n.º 1 (15 de agosto de 1996): 157–62. http://dx.doi.org/10.1042/bj3180157.
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The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421–425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli σ70 consensus promoter and to promoters of P. mirabiliscat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside and growth at 37 °C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.
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Kim, Young Kyeung, Cyril F. Bourgeois, Catherine Isel, Mark J. Churcher y Jonathan Karn. "Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation". Molecular and Cellular Biology 22, n.º 13 (1 de julio de 2002): 4622–37. http://dx.doi.org/10.1128/mcb.22.13.4622-4637.2002.
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ABSTRACT Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-β-d-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.
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Jin, Zhuo, Jeonghwan Seo, Backki Kim, Seung Young Lee y Hee-Jong Koh. "Identification of a Candidate Gene for the Novel Cytoplasmic Male Sterility Derived from Inter-Subspecific Crosses in Rice (Oryza sativa L.)". Genes 12, n.º 4 (17 de abril de 2021): 590. http://dx.doi.org/10.3390/genes12040590.
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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.
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Nishimura, Atsuhisa, Hiroshi Oyama, Takatoshi Hamada, Katsunori Nobuoka, Takashi Shin, Sawao Murao y Kohei Oda. "Molecular Cloning, Sequencing, and Expression inEscherichia coli of the Gene Encoding a Novel 5-Oxoprolinase without ATP-Hydrolyzing Activity from Alcaligenes faecalis N-38A". Applied and Environmental Microbiology 66, n.º 8 (1 de agosto de 2000): 3201–5. http://dx.doi.org/10.1128/aem.66.8.3201-3205.2000.
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ABSTRACT The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-β-d-thiogalactopyranoside-inducibletac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited byp-chloromercuribenzoic acid, EDTA,o-phenanthroline, HgCl2, and CuSO4. The EDTA-inactivated enzyme was completely restored by the addition of Zn2+ or Co2+. In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.
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Watanabe, Shouji, Miyuki Hamano, Hiroshi Kakeshita, Keigo Bunai, Shigeo Tojo, Hirotake Yamaguchi, Yasutaro Fujita, Sui-Lam Wong y Kunio Yamane. "Mannitol-1-Phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in Bacillus subtilis: Possible Cooperation of mtl and gut Operons". Journal of Bacteriology 185, n.º 16 (15 de agosto de 2003): 4816–24. http://dx.doi.org/10.1128/jb.185.16.4816-4824.2003.
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ABSTRACT We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBAmt1) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-β-d-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.
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Nie, Yingbin, Songmei Ma y Wanquan Ji. "Cloning, prokaryotic expression, and subcellular localisation of the TaMAPK10-like gene in common wheat". Canadian Journal of Plant Science 99, n.º 4 (1 de agosto de 2019): 460–66. http://dx.doi.org/10.1139/cjps-2018-0195.
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Mitogen-activated protein kinase (MAPK/MPK) is a group of serine-threonine protein kinases that are activated by different extracellular stimuli. To explore the function of MAPK in wheat infected with powdery mildew, a new wheat germplasm N9134 was used to obtain the full-length MAPK gene and the MAPK sequence was used to identify its prokaryotic expression and subcellular localisation. Wheat MAPK was obtained by homologous gene cloning and designated as TaMAPK10-like. The open reading frame of TaMAPK10-like was 1638 bp, which coded a deduced protein of 545 amino acids. Phylogenetic analysis revealed that TaMAPK10-like was most closely related to MAPK10-like of Aegilops tauschii at the protein level. It had high nucleotide similarity with the reported MAPK gene in A. tauschii, Sorghum bicolor, and Setaria italica, and had features typical of MAPK family genes. Subcellular localisation showed that TaMAPK10-like was mainly located in the cytoplasm along the microtubules, and a small number were located in the cell membrane and the nucleus. A pMD-MAPK10-like fusion expression vector was constructed and the TaMAPK10-like fusion protein was 70 kDa. The expressions of protein in bacteria were best obtained using 0.5 mmol L−1 isopropyl β-d-1-thiogalactopyranoside at 37 °C for 12 h. These results provide the basic data for further understanding the biological function of the TaMAPK10-like gene.
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Hanh, Vu Thi. "EXPRESSION OF GLUTARYL7-AMINOCEPHALOSPORANIC ACID ACYLASE IN ESCHERICHIA COLI BL21(DE3) AND IMMOBILIZATION OF RECOMBINANT ENZYME ON NANOPOROUS MATERIALS". Vietnam Journal of Science and Technology 54, n.º 4A (21 de marzo de 2018): 123. http://dx.doi.org/10.15625/2525-2518/54/4a/12012.
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The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 oC and induced by 0.4 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 °C, pH7.0 and immobilization time –60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials.
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AL-Jabri, Zaaima, Roxana Zamudio, Eva Horvath-Papp, Joseph Ralph, Zakariya AL-Muharrami, Kumar Rajakumar y Marco Oggioni. "Integrase-Controlled Excision of Metal-Resistance Genomic Islands in Acinetobacter baumannii". Genes 9, n.º 7 (20 de julio de 2018): 366. http://dx.doi.org/10.3390/genes9070366.
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Genomic islands (GIs) are discrete gene clusters encoding for a variety of functions including antibiotic and heavy metal resistance, some of which are tightly associated to lineages of the core genome phylogenetic tree. We have investigated the functions of two distinct integrase genes in the mobilization of two metal resistant GIs, G08 and G62, of Acinetobacter baumannii. Real-time PCR demonstrated integrase-dependent GI excision, utilizing isopropyl β-d-1-thiogalactopyranoside IPTG-inducible integrase genes in plasmid-based mini-GIs in Escherichia coli. In A. baumannii, integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli plasmids and A. baumannii chromosome, the rate of excision and circularization was found to be dependent on the expression level of the integrases. Susceptibility testing in A. baumannii strain ATCC 17978, A424, and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility. In summary, the data evidenced the functionality of two integrases in the excision and circularization of the two Acinetobacter heavy-metal resistance GIs, G08 and G62, in E. coli, as well as when chromosomally located in their natural host. These recombination events occur at different frequencies resulting in genome plasticity and may participate in the spread of resistance determinants in A. baumannii.
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Pérez-Ortega, Jesús, Ria van Boxtel, Eline F. de Jonge y Jan Tommassen. "Regulated Expression of lpxC Allows for Reduction of Endotoxicity in Bordetella pertussis". International Journal of Molecular Sciences 23, n.º 14 (21 de julio de 2022): 8027. http://dx.doi.org/10.3390/ijms23148027.
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The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria.
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Kang, Christina S. y Han Seung Kim. "Function Analysis of Chlorophenol Monooxygenase for Chlorophenol Degradation". Advanced Materials Research 1073-1076 (diciembre de 2014): 700–703. http://dx.doi.org/10.4028/www.scientific.net/amr.1073-1076.700.
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4-Chlorophenol is well-known as an extensively used antiseptic, and it may cause severe damage to the environment and human health. 4-Chlorophenol can be biologically degraded by Arthrobacter chlorophenolicus A6, which can be attributed to the cphC-I and cphB genes of the microorganism that encode for a two-component flavin-diffusible monooxygenase (TC-FDM), composed of oxygenase and reductase components. This study reports the cloning, overexpression, purification, and function analysis of the oxygenase and reductase components from the genes cphC-I and cphB. The genes were cloned into vector pET-24a, and 4 different strains of Escherichia coli were transformed with these recombinant genes. The optimization of expression conditions indicated that cphC-I is best overexpressed in E. coli BL21(DE3) incubated for 24 hours at 15°C with 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside. However, cphB was not expressed into soluble form enzyme in any of the conditions, and therefore fre of E. coli was used instead to analyze the function of CphC-I. CphC-I was able to degrade approximately 13.86% of 4-chlorophenol, indicating that it is indeed a reduced flavin-dependent monooxygenase and utilizes 4-chlorophenol as a substrate. The results of this study are expected to establish the basic understanding of TC-FDM for its application to enzymatic bioremediation of phenol-contaminated environment.
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Boneca, Ivo G., Chantal Ecobichon, Catherine Chaput, Aurélie Mathieu, Stéphanie Guadagnini, Marie-Christine Prévost, Frédéric Colland, Agnès Labigne y Hilde de Reuse. "Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori". Applied and Environmental Microbiology 74, n.º 7 (1 de febrero de 2008): 2095–102. http://dx.doi.org/10.1128/aem.01348-07.
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ABSTRACT The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.
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Rui, Huan y Liqun Wang. "Prokaryotic Expression, Purification and Identification of Human PDE9A Protein". Journal of Biology and Life Science 10, n.º 1 (15 de noviembre de 2018): 22. http://dx.doi.org/10.5296/jbls.v10i1.13667.
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Cerebral aggregation of beta amyloid plaques (Aβ) and neurofibrillary tangles is responsible for the onset of Alzheimer’s disease (AD), and PDE9A inhibition rescues Aβ-induced deficits in synaptic plasticity and cognition. This study was aimed to express active PDE9A protein for subsequent inhibitor screening. The PDE9A gene was cloned from human cDNA by real-time polymerase chain reaction, and then the gene sequence and its amino acid sequence were analyzed on Lasergene. An inducible expression vector was constructed by enzyme digestion-seamless cloning and transformed into Escherichia coli BL21 (DE3) for PDE9A expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The recombinant protein was purified by Ni-NTA affinity chromatography and its activity was determined by a phosphodiesterase assay kit. It was found the open reading frame of PED9A was 1035 bp long, the deduced protein was composed of 345 amino acids, and its predicted isoelectric point was about 4.84. The E. coli vector ST6-PDE9A successfully expressed the recombinant PDE9A protein in the supernatant of bacterial lysate. The optimal culture conditions were that the bacterium ST6-PDE9A was grown first in a lysogeny broth at 37 ºC to an OD600 of 0.6-0.8 and then at 16 ºC for 40 h with the addition of 1 M IPTG. Activity test showed PDE9A significantly hydrolyzed the substrate cyclic guanosine monophosphate. In conclusion, we constructed a prokaryotic expression vector and expressed active proteins, laying a solid foundation for screening PDE9 inhibitors.
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Kadiyala, Venkateswarlu, Lloyd J. Nadeau y Jim C. Spain. "Construction of Escherichia coli Strains for Conversion of Nitroacetophenones to ortho-Aminophenols". Applied and Environmental Microbiology 69, n.º 11 (noviembre de 2003): 6520–26. http://dx.doi.org/10.1128/aem.69.11.6520-6526.2003.
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ABSTRACT The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.
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Nakamura, Takashi, Emi Takeda, Tomoko Kiryu, Kentaro Mori, Miyu Ohori, Eiki Kikugawa y Kazuhiko Ishikawa. "Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli". Current Biotechnology 8, n.º 1 (25 de septiembre de 2019): 15–23. http://dx.doi.org/10.2174/2211550108666190418125138.
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Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.
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Mega, Olfa, Cece Sumantri, Irma Isnafia Arief y Cahyo Budiman. "Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)". Jurnal Agripet 19, n.º 2 (1 de octubre de 2019): 149–58. http://dx.doi.org/10.17969/agripet.v19i2.14904.
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Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico. pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively. Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C. As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.
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Latif, Kunza, Shagufta Naz, Imran Altaf, Jian dong Huang, Rasheeda Bashir, Farheen Aslam y Shaozhen Xing. "Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi". Bioscience Journal 38 (23 de septiembre de 2022): e38084. http://dx.doi.org/10.14393/bj-v38n0a2022-61149.
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We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.
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Al-Obaidi, R. M., G. F. Salih y B. F. Nore. "BIOACTIVITY CHARACTERIZATION OF PURIFIED RECOMBINANT HYPOTHETICAL PROTEIN CODED BY OPEN READING FRAME-112 OF STREPTOMYCES." IRAQI JOURNAL OF AGRICULTURAL SCIENCES 52, n.º 2 (20 de abril de 2021): 502–11. http://dx.doi.org/10.36103/ijas.v52i2.1314.
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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.
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Werner, Erin, Frank Roe, Amandine Bugnicourt, Michael J. Franklin, Arne Heydorn, Søren Molin, Betsey Pitts y Philip S. Stewart. "Stratified Growth in Pseudomonas aeruginosa Biofilms". Applied and Environmental Microbiology 70, n.º 10 (octubre de 2004): 6188–96. http://dx.doi.org/10.1128/aem.70.10.6188-6196.2004.
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ABSTRACT In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp 1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 μm wide in colony biofilms and 30 μm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 μm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.
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Eugster, Marcel R. y Martin J. Loessner. "Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan". Journal of Bacteriology 194, n.º 23 (21 de septiembre de 2012): 6498–506. http://dx.doi.org/10.1128/jb.00808-12.
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ABSTRACTThe C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. TheListeria monocytogenesPly118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure ofListeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding usingL. monocytogenesserovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundanttagOhomologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of eithertagO1(EGDelmo0959) ortagO2(EGDelmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficientListeriacells. Substitution oftagOfrom an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from otherListeriaphage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.
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Liška, Vladimír, Jan Evangelista Dyr, Jiří Suttnar, Ivan Hirsch y Vladimír Vonka. "Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-β-d-thiogalactopyranoside". Journal of Chromatography B: Biomedical Sciences and Applications 656, n.º 1 (junio de 1994): 127–33. http://dx.doi.org/10.1016/0378-4347(94)00079-4.
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Nooh, Hisham, Malihe Masomian, Abu Salleh, Rosfarizan Mohamad, Mohd Ali y Raja Rahman. "Production of Thermostable T1 Lipase Using Agroindustrial Waste Medium Formulation". Catalysts 8, n.º 11 (23 de octubre de 2018): 485. http://dx.doi.org/10.3390/catal8110485.
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Large-scale production of T1 lipase using conventional culture media is costly. To reduce the cost of production, an alternative growth medium using local resources has been developed. In this study, the growth of recombinant Escherichia coli and expression of T1 lipase were tested using different agroindustrial wastes as carbon and nitrogen sources by conventional method. Subsequently, by using central composite rotatable design (CCRD), a set of 30 experiments was generated to evaluate the effect of different parameters, including the amount of molasses (as carbon source), fish waste (as nitrogen source), NaCl, and inducer concentration on production of T1 lipase. Response surface methodology (RSM) analysis indicated that all factors had significant effects on T1 lipase production. This statistical analysis was utilised to develop a quadratic model to correlate various important variables for the growth of the recombinant strain and regulation of gene expression to the response (T1 lipase activity). Optimum conditions for T1 lipase production were observed to be 1.0 g/L of molasses, 2.29 g/L of fish waste, 3.46 g/L of NaCl, and 0.03 mM of IPTG (Isopropyl β-d-1-thiogalactopyranoside). Based on these conditions, the actual lipase activity was found to be 164.37 U/mL, which fitted well with the maximum predicted value of 172.89 U/mL. Therefore, the results demonstrated that, the statistical analysis, performed using RSM, was efficient in optimising T1 lipase production. Moreover, the optimum conditions obtained can be applied to scale up the process and minimise the cost of enzyme production.
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Ratnaningsih, Enny, Rachmad Ade, Rindia Maharani Putri y Idris Idris. "Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1". International Journal of Renewable Energy Development 10, n.º 4 (8 de julio de 2021): 857–63. http://dx.doi.org/10.14710/ijred.2021.38887.
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In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37ºC with shaking, 2 hours IPTG induction at 30ºC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation
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Rukmana, Andriansjah, Burhanuddin Rasyid y Fitriyah Sjatha. "Gluthathione S-transferase-resuscitation-promoting factor B recombinant protein of Mycobacterium tuberculosis induces the production of interferon-γ and interleukin-12 in mice splenocytes". Medical Journal of Indonesia 28, n.º 3 (4 de octubre de 2019): 234–40. http://dx.doi.org/10.13181/mji.v28i3.2444.
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BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guérin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro.
METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg/ ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays.
RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10.
CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.
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Wu, Weihua, Fang Liu y Seema Singh. "Toward engineeringE. coliwith an autoregulatory system for lignin valorization". Proceedings of the National Academy of Sciences 115, n.º 12 (2 de marzo de 2018): 2970–75. http://dx.doi.org/10.1073/pnas.1720129115.
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Efficient lignin valorization could add more than 10-fold the value gained from burning it for energy and is critical for economic viability of future biorefineries. However, lignin-derived aromatics from biomass pretreatment are known to be potent fermentation inhibitors in microbial production of fuels and other value-added chemicals. In addition, isopropyl-β-d-1-thiogalactopyranoside and other inducers are routinely added into fermentation broth to induce the expression of pathway enzymes, which further adds to the overall process cost. An autoregulatory system that can diminish the aromatics’ toxicity as well as be substrate-inducible can be the key for successful integration of lignin valorization into future lignocellulosic biorefineries. Toward that goal, in this study an autoregulatory system is demonstrated that alleviates the toxicity issue and eliminates the cost of an external inducer. Specifically, this system is composed of a catechol biosynthesis pathway coexpressed with an active aromatic transporter CouP under induction by a vanillin self-inducible promoter, ADH7, to effectively convert the lignin-derived aromatics into value-added chemicals usingEscherichia colias a host. The constructed autoregulatory system can efficiently transport vanillin across the cell membrane and convert it to catechol. Compared with the system without CouP expression, the expression of catechol biosynthesis pathway with transporter CouP significantly improved the catechol yields about 30% and 40% under promoter pTrc and ADH7, respectively. This study demonstrated an aromatic-induced autoregulatory system that enabled conversion of lignin-derived aromatics into catechol without the addition of any costly, external inducers, providing a promising and economically viable route for lignin valorization.