Production and purification of a cecropin family antibacterial peptide,hinnavin II,in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly, hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni²⁺-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin II in its biologically active form.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Production of glycoprotein vaccines in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.     

Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

Over-expression,purification and functional characterization of <Emphasis Type="Italic">Celosia</Emphasis> ClpS as a fused protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A ClpS homologue from Celosia cristata was expressed as maltose-binding fusion protein under the control of strong inducible tac promoter of pMALc2X vector in TB1 strain of Escherichia coli. SDS-PAGE analysis showed that fused ClpS is produced as about 63 kDa protein in recombinant bacteria. Expressed product was purified to homogeneity with a yield of about 31 mg/l of bacterial culture. The results indicated that heterologous expression of Celosia ClpS does not affect bacterial growth under different induced conditions. Total cellular antioxidant assessment results revealed that the induction of ClpS activates the bacterial antioxidative system. Since, the purified ClpS did not exhibit antioxidant activity in vitro, we speculated a functional corelation between bacterial proteolytic apparatus and its anti-oxidative system. This prediction may contribute to our better understanding of functional relationship between proteolytic and antioxidative systems in biological worlds in the future investigations.     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the recombinant <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> metalloprotease and its purification and characterization

The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca²⁺, Na⁺ and Mg²⁺ had an activating effect on the enzyme while Zn²⁺ and Cu²⁺ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis.     

Expression and characterization of <Emphasis Type="Italic">Momordica Chanrantia</Emphasis> anti-hyperglycaemic peptide in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS–PAGE and western blot. It revealed that the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent.     

Assessing the impacts of temperature and storage on <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Salmonella</Emphasis>, and <Emphasis Type="Italic">L. monocytogenes</Emphasis> decay in dairy manure

Elevated levels of animal waste-borne pathogen in ambient water is a serious human health issue. Mitigating influx of pathogens from animal waste such as dairy manure to soil and water requires improving our existing knowledge of pathogen reductions in dairy manure treatment methods. This study was conducted to enhance the  understanding of human pathogen decay in liquid dairy manure in anaerobic (AN) and limited aerobic (LA) storage conditions. The decay of three pathogens (Escherichia coli, Salmonella spp., and Listeria monocytogenes) was assessed in bench-scale batch reactors fed with liquid slurry. A series of temperatures (30, 35, 42, and 50 °C) conditions were tested to determine the impacts of temperature on Escherichia coli, Salmonella, and L. monocytogenes decay in AN and LA conditions. Results showed prolonged survival of E. coli compared to Salmonella and L. monocytogenes in both LA and AN environments. Variations in survival among pathogens with temperature and environmental conditions (i.e., LA and AN) indicated the necessity of developing improved dairy manure waste treatment methods for controlling animal waste-borne pathogens. The results of this study will help in improving the current understanding of human pathogen decay in dairy manure for making informed decisions of animal manure treatment by stakeholders.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Production of Bioactive Rockfish (<Emphasis Type="Italic">Sebastes schlegeli</Emphasis>) Myostatin-1 Prodomain in an <Emphasis Type="Italic">Escherichia coli</Emphasis> System

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains of various animal species in E. coli.     

Oxaloacetate decarboxylase of <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: purification,characterization, and expression of the genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The oxaloacetate decarboxylase (OAD) Na⁺ pump consists of subunits , , and , which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin–Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na⁺ dependent, and the activation profile showed strong cooperativity with a Hill coefficient n_H=1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10 M and 200 M, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na⁺ pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570 kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cells.     

Lipoprotein trafficking in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.