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.     

Production of Bioactive γ-Glutamyl Transpeptidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner and Application of the Recombinant Enzyme to <Emphasis Type="SmallCaps">l</Emphasis>-Theanine Synthesis

The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in 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–nitrilotriacetic acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine.     

Recombinant expression of bioactive peptide lunasin in Escherichia coli

Lunasin, a cancer-preventive peptide, was isolated from soybean, barley, and wheat. Previous studies showed that this 43-amino acid peptide has the ability to suppress chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. In this study, we attempted to use the Escherichia coli T7 expression system for expression of lunasin. The lunasin gene was synthesized by overlapping extension polymerase chain reaction and expressed in E. coli BL21(DE3) with the use of vector pET29a. The recombinant lunasin containing his-tag at the C-terminus was expressed in soluble form which could be purified by immobilized metal affinity chromatography. After 4 h, the expression level is above 4.73 mg of recombinant his-tagged lunasin/L of Luria–Bertani broth. It does not affect the bacterial growth and expression levels. This is the first study that successfully uses E. coli as a host to produce valuable bioactive lunasin. The result of in vitro bioassay showed that the purified recombinant lunasin can inhibit histone acetylation. Recombinant lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and nitric oxide production). Compared with other research methods on extraction or chemical synthesis to produce lunasin, our method is very efficient in saving time and cost. In the future, it could be applied in medicine and structure–function determination.     

Over-Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of the Human OCTN2 Transport Protein

The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni²⁺-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni²⁺-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.     

Simple purification ofEscherichia coli-derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8→12→8). Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in <Emphasis Type="Italic">E. coli</Emphasis> with Inhibiting Effect on Endothelial Cells

PEDF34, a functional epitope of pigment epithelium-derived factor (PEDF), obtained by chemical synthesis previously, shows potential anti-angiogenesis activity described before. We perform a novel method in this study for the expression and purification of recombinant PEDF34 in E. coli, and make it convenient, soluble and high yield to obtain this small peptide of PEDF. Human PEDF34 gene was cloned into the fusion-protein expression vector pGEX-4T-1, and the recombinant plasmid was transformed into E. coli strain BL21-DE3. GST-PEDF34 fusion protein was expressed, purified using chromatograph and identified by Western blotting. The purified fusion protein was digested by thrombin, and the small PEDF34 peptide was isolated by ultrafiltration. Circular dichroism (CD) analysis identified that secondary structure of PEDF34 mainly characterizes as α-helix. The 34-AA small peptide could cell-type-specifically inhibit viability of HUVECs in a dose-dependent manner and induce apoptosis of HUVECs. These results suggested that this type of recombinant PEDF34 may have potential in the treatment of angiogenesis-related diseases such as solid tumor.     

An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.     

Signal peptide of <Emphasis Type="Italic">Aureobasidium</Emphasis><Emphasis Type="Italic">pullulans</Emphasis> xylanase: use for extracellular production of a fungal xylanase by <Emphasis Type="Italic">Escherichia coli</Emphasis>

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.     

High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Ubiquitin–peptide fusion protein system enables preparation of stable isotope labeled peptides through the expression of the protein in E. coli cells in labeled media (Kohno et al. (1998) J Biomol NMR 12:109–121). Advantages of the system over others include: very specific cleavage of the bond between ubiquitin and target peptide moieties by yeast ubiquitin hydrolase and low cost for the protease which can also be expressed in E. coli cells. The former point is particularly important since other frequently used proteases, such as factor Xa and thrombin, often show non-specific cleavages at sites unexpected from their nominal specificities. We improved the yield of the peptide by adapting the codon usage of ubiquitin gene for the expression in E. coli cells, by using RNase E-deficient host strains, and by modifying purification procedure. The yield of mastoparan-X was increased threefold by these modifications. We also succeeded in the preparation of labeled magainin 2, an antimicrobial peptide that could not be expressed at all by the previous method, by choosing host strains and culture media. The HSQC signals of the ¹⁵N-labeled magainin 2 in an aqueous solution were completely resolved in spite of the severe overlap of the 1D proton signals, confirming that the stable isotope labeling is quite useful for analysis of peptides.     

Expression and purification of recombinant arginine decarboxylase (speA) from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase’s structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His₁₀-ADC (77.3 kDa) was first purified by Ni–NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His₁₀ tag removed recombinant ADC (74.1 kDa)’s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6–9) and temperature (20–80°C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.     

Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> in batch and repeated batch modes

An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP–glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of Fe₃O₄/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin.     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin on Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Resistant to Conventional Antibiotics with Different Structures

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. To evaluate the effect of S-thanatin (an analog of thanatin, a cationic antimicrobial peptide isolated from the hemipteran insect Podisus maculiventris) against microbial resistant to antibiotics, we studied its bactericidal kinetics, synergistic effect, resistance, and activity on clinical isolates of Klebsiella pneumoniae resistant to conventional antibiotics with different structures. The bactericidal rate of S-thanatin was more than 99% against K. pneumoniae ATCC 700603 when bacterial cultures were monitored for 60 min. The peptide was synergistic with β-lactam cefepime in most of the clinical MDR isolates tested (7/8). An average value of FIC was 0.3708. No synergy was found between the peptide and amoxicillin, gentamycin, tetracycline, or ciprofloxacin in all bacteria tested. A total of 48 isolates of K. pneumoniae with different resistance spectrum tested was susceptible to S-thanatin. The MICs were 6.25–25 μg/ml. No significant difference in the MICs of S-thanatin between the sensitive isolates and the resistant isolates to single antibiotic was observed (P > 0.05). The resistance of K. pneumoniae ATCC 700603 to S-thanatin was slightly higher, when cultured at sub-inhibitory concentration for 5 days. S-thanatin may be an attractive candidate for developing into an antimicrobial agent.     

Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.     

One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.     

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.     

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.