Expression and purification of soluble B lymphocyte stimulator from recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in a shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30-1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pl) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisified, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 187–192. The text was submitted by the authors in English.     

Expression and purification of soluble recombinant Human Endostatin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

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.     

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.     

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.     

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.     

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.     

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.     

Expression and single-step purification of mercury transporter (merT) from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l⁻¹.     

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.     

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.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Expression and purification of antimicrobial peptide buforin IIb in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel production method in Escherichia coli for an antimicrobial peptide of 21 amino acids, buforin IIb, which is a synthetic analog of buforin II, has been developed. The buforin IIb gene was cloned into the vector pET32a to construct an expression vector pET32a–buforin IIb. The fusion protein Trx-buforin IIb, purified by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant buforin IIb. Purification of recombinant buforin IIb was achieved by HPLC: about 3.1 mg/l active recombinant buforin IIb with purity >99% was obtained. The recombinant buforin IIb showed antimicrobial activities that were similar to the synthetic one.     

Identification and Expression of a <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> Collagenase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.     

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.     

Expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit in transgenic watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. G_M1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to G_M1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.     

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.     

Cloning,Expression, and Characterization of Siamese Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>) Hemoglobin from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.