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 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.     

Extracellular recombinant protein production from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stable expression and secretion of polyhydroxybutyrate depolymerase of <Emphasis Type="Italic">Paucimonas lemoignei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.     

Characterization of a novel <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> xanthine dehydrogenase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications.

Results

The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40–45 °C, was stable under alkaline conditions (pH 7–11) and the half-inactivation temperature was 60 °C. The K _m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s^?1 and 2.7 μM^?1 s^?1, respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized.

Conclusion

The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

     

Peroxidase activity of cytochrome <Emphasis Type="Italic">bd</Emphasis> from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H₂O₂ concentration is linear up to the concentration of 8 mM. At higher concentrations of H₂O₂, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b ₅₅₈, and H₂O₂ is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.     

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.     

Expression of <Emphasis Type="Italic">Treponema denticola</Emphasis> Oligopeptidase B in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases. This bacterium possesses a particular arginine peptidase activity (previously called BANA-peptidase or trypsin-like enzyme) that is common to the three cultivable bacterial species most highly associated with severe periodontal disease. We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T. denticola through DNA sequencing and mutagenesis studies. In the present study, we report expression of T. denticola OpdB peptidase in Escherichia coli. The opdB PCR product was cloned into pET30b and then transformed into the E. coli BL21 (DE3)/pLysS expression strain. Assays of enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola. Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.     

Biosynthesis of two quercetin <Emphasis Type="Italic">O</Emphasis>-diglycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Various flavonoid glycosides are found in nature, and their biological activities are as variable as their number. In some cases, the sugar moiety attached to the flavonoid modulates its biological activities. Flavonoid glycones are not easily synthesized chemically. Therefore, in this study, we attempted to synthesize quercetin 3-O-glucosyl (1→2) xyloside and quercetin 3-O-glucosyl (1→6) rhamnoside (also called rutin) using two uridine diphosphate-dependent glycosyltransferases (UGTs) in Escherichia coli. To synthesize quercetin 3-O-glucosyl (1→2) xyloside, sequential glycosylation was carried out by regulating the expression time of the two UGTs. AtUGT78D2 was subcloned into a vector controlled by a Tac promoter without a lacI operator, while AtUGT79B1 was subcloned into a vector controlled by a T7 promoter. UDP-xyloside was supplied by concomitantly expressing UDP-glucose dehydrogenase (ugd) and UDP-xyloside synthase (UXS) in the E. coli. Using these strategies, 65.0 mg/L of quercetin 3-O-glucosyl (1→2) xyloside was produced. For the synthesis of rutin, one UGT (BcGT1) was integrated into the E. coli chromosome and the other UGT (Fg2) was expressed in a plasmid along with RHM2 (rhamnose synthase gene 2). After optimization of the initial cell concentration and incubation temperature, 119.8 mg/L of rutin was produced. The strategies used in this study thus show promise for the synthesis of flavonoid diglucosides in E. coli.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Cyclohexanone-induced stress metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Solvent stress occurs during whole-cell biocatalysis of organic chemicals. Organic substrates and/or products may accumulate in the cellular membranes of whole cells, causing structural destabilization of the membranes, which leads to disturbances in cellular carbon and energy metabolism. Here, we investigate the effect of cyclohexanone on carbon metabolism in Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032. Adding cyclohexanone to the culture medium (i.e., glucose mineral medium) resulted in a decreased specific growth rate and increased cellular maintenance energy in both strains of bacteria. Notably, carbon metabolism, which is mainly involved to increase cellular maintenance energy, was very different between the bacteria. Carbon flux into the acetic acid fermentation pathway was dominantly enhanced in E. coli, whereas the TCA cycle appeared to be activated in C. glutamicum. In fact, carbon flux into the TCA cycle in E. coli appeared to be reduced with increasing amounts of cyclohexanone in the culture medium. Metabolic engineering of E. coli cells to maintain or improve TCA cycle activity and, presumably, that of the electron transport chain, which are involved in regeneration of cofactors (e.g., NAD(P)H and ATP) and formation of toxic metabolites (e.g., acetic acid), may be useful in increasing solvent tolerance and biotransformation of organic chemicals (e.g., cyclohexanone).     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Purification of dibenzothiophene monooxygenase from a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dibenzothiophene monooxygenase is the first enzyme involved in the degradation of dibenzothiophene. This gene was expressed via the pET28a vector in E. coli and was purified in a single step using affinity chromatography. The protein was purified 39-fold with a specific activity of 38 U/mg.     

Oligomeric organization of recombinant human neurotrophins expressed in Escherichia coli cells

Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3lambda) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure.     

Enhancing Functional Expression of Heterologous <Emphasis Type="Italic">Burkholderia</Emphasis> Lipase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip–HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.     

Magnetic-dependent ATP pool in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The ATP pool in Escherichia coli is a magnetic-dependent characteristic of microorganism vital activity. It depends on the values of the external static magnetic field and the existence of magnetic moment of magnesium isotopes nuclei added to the growth nutrient medium. The combined effects of the magnetic field 70–95 mT and magnesium magnetic isotope ²⁵Mg on E. coli bacteria leads to increase intracellular concentration of ATP. Magnetic-field effects in the range of 0.8–16 mT, registered for all bacteria regardless of the magnesium-isotopic enrichment of nutrient medium, evidence about the sensitivity of intracellular processes to weak magnetic fields.