Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC₅₀?=?13–15 μg/ml).     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Cloning,expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody

Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni⁺-NTA affinity chromatography (95–98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.     

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.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Secretory Expression and Purification of Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> Heat-Labile Enterotoxin B Subunit and its Applications on Intranasal Vaccination of Hantavirus

In order to further study the B subunit of the Escherichia coli heat-labile enterotoxin (LTB), we obtained the LTB gene from pathogenic E. coli, cloned it into the pET22b (+) prokaryotic expression vector, and expressed it as a fusion protein with His tag in E. coli BL21 (DE3). The recombinant LTB was expressed and purified by Ni²⁺ affinity chromatography. The biological activity of the purified recombinant LTB was assayed in a series of monosialotetrahexosylganglioside (GM1)-ELISA experiments. The recombinant LTB (rLTB) was efficiently expressed under the induction of 10 g/l lactose at 37°C for 6 h and yielded up to 31% of the total bacterial protein. Fused with pelB signal peptide, rLTB was successfully localized to the periplasmic space. GM1-ELISA experiments showed that the rLTB obtained retains strong GM1 ganglioside-binding activity. The ELISA result of hantavirus nucleoprotein-specific secretory immunoglobulin A (sIgA) and IgG showed that intranasal administration of inactivated hantavirus with rLTB significantly increased the levels of hantavirus-specific sIgA (P < 0.01) and IgG (P < 0.01) in comparison with inactivated hatavirus alone. In summary, we have developed a method for the efficient secretory expression and purification of rLTB, and the inactivated hantavirus co-administered intranasally with rLTB could effectively induce both mucosal and humoral immune responses specific to hantavirus. Shouchun Cao and Ying Zhang contributed equally to this work.     

Production and purification of a cecropin family antibacterial peptide,hinnavin II,in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly, hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni²⁺-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin II in its biologically active form.     

Soluble expression and purification of the functional interleukin-30 protein in Escherichia coli

Interleukin-30 (IL-30), or IL-27p28, is the α subunit of IL-27 constructed by Epstein–Barr virus-induced gene 3 (EBI3) and IL-27p28 binding via noncovalent bonds. IL-30 can be independently secreted and function independently of IL-27. Recent studies demonstrated IL-30 could concurrently antagonize T helper 1 (Th1) and Th17 responses and might have therapeutic implications for controlling autoimmune diseases. However, no reports have stated an efficient method to generate a relatively large quantity of IL-30. In this study, an Escherichia coli expression system for the rapid expression of the mouse IL-30 is developed. For the first time, IL-30 was expressed in a form of soluble fusion protein and purified using a method of simple affinity chromatography. In order to avoid the impact of minor codons on expressing eukaryotic protein in E. coli and to improve the expression quantity, the nucleotide sequence of IL-30 was optimized. The optimized gene sequence was then subcloned into the pET-44a(+) vector, which allowed expression of IL-30 with a fusion tag, NusA. The vector was transformed into E. coli and the expressed fusion protein, NusA-IL-30, was purified by Ni chromatography. Then the fusion tag was removed by cleavage with thrombin. The purity of purified IL-30 was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as high-performance liquid chromatography (HPLC) and the purity was up to about 92%. The yield of IL-30 was 8.95 mg from 1 L of bacterial culture. Western blot confirmed the identity of the purified protein. The recombinant IL-30 showed its biological activity by inhibiting Th17 differentiating from naive CD4⁺ T cells. Therefore, this method of express and purifying IL-30 provides novel procedures to facilitate structural and functions studies of IL-30.     

Cloning and Characterization of Extracellular Metal Protease Gene of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.     

Large-scale expression,purification, and glucose uptake activity of recombinant human FGF21 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase–peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

Secretory expression of a β‐xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K_i value of 63 mmol l^?1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.     

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.     

Gene Cloning,Expression, and Characterization of the <Emphasis Type="Italic">Geobacillus Thermoleovorans</Emphasis> CCR11 Thermoalkaliphilic Lipase

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.     

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.