Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.     

High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli

A novel β-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant β-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 β-xylosidase gene from thermophilic fungi.     

High-level expression of human αs1-casein in Escherichia coli

Human _s1-casein was expressed efficiently in Escherichia coli. The overproduced recombinant human a _s1-casein was about 25% of the total cell protein. Two different vectors were constructed to express Met-_s1-casein and Met-_s1-casein with a His-affinity tag at the C-terminus. Recombinant Met-_s1-casein with a His-affinity tag was purified to homogeneity using Ni-nitrilotriacetic acid resin. N-terminal sequence of the first 10 amino acid residues of this purified protein was identical to that of mature human _s1-casein with an extra methionine residue at the N-terminus.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.     

GUS expression in Arabidopsis directed by 5′ regions of the pea metallothionein-like gene PsMT A

Upstream sequences (including the first seven codons) of a metallothionein (MT)-like gene from pea, PsMT A, were fused to GUS and introduced into Arabidopsis. High-level GUS expression was detected in the roots of plants grown on MS medium, except in regions proximal to the root apex. There was precise delineation of the root-shoot boundary. In soil-grown plants there was low GUS expression and this was absent from the more mature regions of the roots. In the aerial tissues of soil-grown plants, GUS expression was restricted to hydathodes, stipules, expanding cotyledons and the following senescent tissues: leaves, cotyledons, petals, sepals, filaments, stigmas, nectaries and siliques. A 298 bp region was shown to be required for GUS expression in roots but not for expression in vegetative aerial tissues of plants grown on MS medium. This region contains predicted ethylene-responsive elements (EREs) but similar patterns of GUS expression were detected in etr1 seedlings. GUS expression was significantly higher in roots exposed to 500 nM copper, but this increase was small in proportion to expression in roots exposed to 50 nM copper.     

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins

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A thermostable β-glucuronidase obtained by directed evolution as a reporter gene in transgenic plants

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.     

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.     

Adaptive Strategies of the Candidate Probiotic E. coli Nissle in the Mammalian Gut

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Pattern of the evolution of HIV-1 enν gene in C?te d?Ivoire

Cête d׳Ivoire continues to have the highest HIV-1 prevalence rate in West Africa, although the infection number is in constant decline. The external envelope protein of the viruses is a likely site of selection, and responsible for receptor binding and entry into host cells, and therefore constitutes an ideal region with which to investigate the evolutionary processes acting on HIV-1. In this study, we analyse 189 envelope glycoprotein V3 loop region sequences of viruse isolates from 1995 to 2009, from HIV-1 untreated patients living in Cête d׳Ivoire, to decipher the temporal relationship between disease diversity, divergence and selection. Our analyses show that the nonsynonymous and synonymous ratio (dN/dS) was lower than 1 for viral populations analysed within 15 years, which showed the sequences did not undergo adequate immune pressure. The phylogenetic tree of the sequences analysed demonstrated distinctly long internal branches and short external branches, suggesting that only a small number of viruses infected the new host cell at each transmission. In addition to identifying sites under purifying selection, we also identified neutral sites that can cause false positive inference of selection. These sites presented form a resource for future studies of selection pressures acting on HIV-1 enν gene in Cête d׳Ivoire and other West African countries.     

Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).     

Molecular identification and properties of a light-insensitive rice catalase-B expressed in E. coli

Catalase plays a central role in plant stress responses but is highly susceptible to photoinhibition. A rice catalase-B protein avoiding photoinhibition was developed by mutagenesis of specific amino acids: Leu-189 to Trp-189 and His-225 to Thr-225 and then recombinantly expressed in E. coli. In addition, the site specific mutation also induced 2–2.5-fold increase in enzyme velocity with high affinity for its substrate and showed nearly a 3-fold lower K _m than the wild protein. These characteristic of mutated rice catalase-B is highly promising in transgenic research to increase plant productivity under stress conditions.     

High level production of bovine angiogenin in E. coli by an efficient refolding procedure

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.     

Production of a fusion protein of sweet potato sporamin from recombinant E. coli XL1 Blue by fed-batch fermentations

Glucose-stat and pH-stat control strategies were employed in order to culture a recombinant E. coli XL1 Blue to produce a fusion protein of sweet potato sporamin (SPA) and glutathione S-transferase (GST) from the recombinant E. coli XL1 Blue. Cell densities up to 25 g l^–1 and 28.9 mg fusion protein (GST-SPA) g^–1 cell dry weight (CDW) was achieved from a fed-batch fermentation controlled by glucose-stat strategy. A pH-stat control fermentation using glycerol as a carbon source gave E. coli up to 27 g l^–1 and 31.5 mg GST-SPA g^–1 CDW. Additionally, a pH-stat control strategy using glucose as a carbon source gave E. coli up to 15 g l^–1 and about 22.7 mg g^–1 CDW of GST-SPA.     

Production of the human immunoglobulin γ1 chain constant region polypeptides in Escherichia coli

We have determined the nucleotide sequence of the constant region exons of the rearranged human immunoglobulin γ₁ chain gene cloned from a human plasma cell leukemia line, ARH-77. The amino acid sequence deduced from the nucleotide sequence revealed that the allotype of the ARH-77 γ₁ chain was Glm (−1, −2, 3).Recombinant plasmids were then constructed in order to express the human γ₁ chain constant region genes (the Fc region gene and the C_H2-C_H3 domains gene) in Escherichia coli. The human γ₁ chain constant region genes without introns were derived from the genomic gene using synthetic DNA fragments. E. coli carrying each expression plasmid produced antigenically active constant region polypeptides as soluble proteins. In the case of the E. coli-derived Fc region polypeptides, they were generated as monomeric forms in the cytoplasm.     

CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.