High Efficient Expression of Bioactive Human BMP-14 in <Emphasis Type="Italic">E. coli</Emphasis> Using SUMO Fusion Partner

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-β superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of human BMP-14. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography. After the SUMO-BMP14 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 45 mg recombinant hBMP-14 was obtained from 1 litre bacterial culture with no less than 95% purity. The purified hBMP-14 dimer was over 90% purity and could induce the expression of alkaline phosphatase activity in C2C12 cells in a dose-dependent manner. Thus the SUMO-mediated peptide expression and purification system potentially could be employed for the production of other homodimeric proteins.     

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon?+?and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~?25.5% and ~?6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.     

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.     

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.     

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.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

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.     

SUMO Mediating Fusion Expression of Antimicrobial Peptide CM4 from two Joined Genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. To improve the expression level of CM4 in Escherichia coli, two tandem repeats of CM4 genes were cloned into the vector pSUMO to construct an expression vector pSUMO–2CM4. The fusion protein SUMO–2CM4, purified by Ni²⁺-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. After the cleaved sample was re-applied to a Ni-IDA column, finally, about 48 mg recombinant CM4 was obtained from 1 L bacterial culture with no less than 96% purity, which was the highest yield of CM4 reported so far.     

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.     

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.     

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.     

Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

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.     

Production of Vanillin by Metabolically Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l⁻¹ was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

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).     

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.     

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.