Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

High-level expression and purification of Escherichia coli oligopeptidase B

Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.     

Light producing reporter plasmids for Escherichia coli K-12 that can be integrated into the chromosome

Plasmid vectors using the Photorhabdus luminescenslux operon can be used for real time measurements of promoter activity. We have generated a series of lux vectors that have a conditional origin of replication, different selectable markers and the attP sequence from λ. Single copies of these plasmids can be integrated into the λ attachment site in the Escherichia coli chromosome. We constructed reporter derivatives and compared light production when the plasmids were present in multiple copies and in single copies. We also demonstrated that expression could be induced under the appropriate conditions.     

Isolation of verotoxin-producing Escherichia coli associated with diarrhoea in Malaysia containing plasmids showing homology with biotinylated Shiga-like toxin DNA gene probes

Three strains of verotoxin-producing Escherichia coli isolated from patients with haemorrhagic colitis harboured plasmids ranging in size from 2.7 kb to 91.2 kb. Those plasmids ranging from 2.7 kb to 6.8 kb hybridized to Shiga-like toxin I and Shiga-like toxin II gene probes.R. Son and A. Ansary are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia. G. Rusul and M.I.A. Karim are with the Faculty of Food Science and Biotechnology, University Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

Expression and preparation of recombinant hepcidin in Escherichia coli

Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.     

Dynamics of glucose uptake by single Escherichia coli cells

The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells. When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate. The dependence upon substrate concentration could be described according to Michaelis-Menten kinetics with apparent saturation constant KM = 1.75 microM, and maximum 2-NBDG uptake rate= 197 molecules/cell-second. Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by D-glucose, 3-o-methyl glucose, and D-glucosamine, and lack of inhibition by L-glucose. Inhibition of 2-NBDG uptake by D-glucose was competitive in nature. The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of D-glucose in the culture medium (approximately 0.2 microM) is detectable. The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity. The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics. A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates. The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation.     

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C(8) reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH(2)-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1-C2 and C3-C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.     

Optimization of BLyS production and purification from Escherichia coli

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.     

Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin

A significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, Paraoxon, to non-toxic materials by co-expression of organophosphorus hydrolase (OPH) under trc promoter and Vitreoscilla hemoglobin (VHb) under O₂dependent nar promoter. VHb-expressing whole cells had significant enhancement of OPH activity (48%, 18.7 vs. 27.8 unit l^–1) and bioconversion efficiency V _max/K _m (44%, 0.14 vs. 0.2 min^–1) compared to VHb-free system.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Leakage of recombinant penicillin G acylase from Escherichia coli by adding chloroform under fermentation conditions

A simple method was developed to release periplasmic penicillin G acylase from Escherichia coli BL21(DE3) during the fermentation process. More than 80% of the total penicillin G acylase was released into the broth when 3% (v/v) chloroform was added at 3 h after induction. The activity of extracellular penicillin G acylase reached 20699 U/l. This method was efficient and would facilitate further investigation of penicillin G acylase for industrial applications.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.