Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

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.     

Modulation of protease activity to enhance the recovery of recombinant nucleocapsid protein of Nipah virus

The nucleocapsid (N) protein of Nipah virus (NiV) expressed in Escherichia coli (E. coli) is antigenic and immunogenic. A method to enhance the recovery of recombinant N protein of NiV produced in E. coli is described. A bioinformatics tool, PeptideCutter was used to identify potential protease and cleavage sites from the amino acid sequences deduced from the published DNA sequence of the N protein of NiV. The size of degraded protein was estimated by using the Western blot and PeptideCutter analyse. The identified proteases were serine proteases, hence, a range of serine protease inhibitors were tested to improve the recovery of the N protein. The relative amount of N protein of NiV was 2-fold higher with the addition of PMSF, compared to the control sample (without any protease inhibitor supplementation).     

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.     

Cloning,Expression and Purification of <Emphasis Type="Italic">Microcystis viridis</Emphasis> Lectin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL (R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein.     

Overexpression, purification, and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

Overexpression in Escherichia coli and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 were investigated in this study. PhCpn, the chaperonin gene from the P. horikoshii OT3, was amplified by PCR from the P. horikoshii genomic DNA, subcloned into pET21a vector, and expressed in three E. coli host cells such as BL21, Rosetta, and Codonplus (DE3). Among these host cells, E. coli Rosetta showed the highest expression level of recombinant PhCpn at induction with 1 mM IPTG. The recombinant PhCpn was purified to 91% by heat-shock treatment and anion-exchange chromatography. The ATPase activity of the purified PhCpn increased in a PhCpn concentration-dependent manner. Also, PhCpn protected the inorganic phosphatase from thermal inactivation at 85 and 110°C, speculating that PhCpn is effective in in vitro holding of the protein. The holding efficiency was enhanced by the addition of Mg²⁺ ion. Through the coexpression of pro-carboxypeptidase B (pro-CPB) and PhCpn in E. coli Rosetta, pro-CPB was produced as a soluble and active form with a marked yield. This result indicated that PhCpn facilitated the in vivo correct folding of pro-CPB and could be used as powerful and novel molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.     

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.     

Purification and biochemical characterization of recombinant human H-ferritins from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Recombinant human H-ferritins produced from Saccharomyces cerevisiae were purified and their molecular properties were characterized. Electrophoresis of the recombinant H-ferritins showed revealed bands with mobilities similar to those of the H-ferritins produced by Escherichia coli. The pI of H-ferritins from yeast was more basic than that of H-ferritins produced by E. coli. When the cells were treated with tunicamycin, the pI of H-ferritins was driven to a sharp band with the pI range similar to that of those produced by E. coli, implying that the H-ferritins from yeast are glycosylated. The iron uptake of H-ferritins was rapid in the initial stage, with a slight reduction when compared to ferritins from E. coli. Recombinant ferritins are commonly used during synthesis of inorganic nanoparticles in their cores for chemical and industrial purposes. Transmission electron microscopy revealed that the reconstitution yield and size distribution of the core minerals were affected depending on the protein shells. The H-ferritins with higher reconstitution yields (64.4%) showed slightly larger sizes (mean 6.52 nm) with narrower size distribution.     

High-level Expression of a Synthetic Gene Encoding a Sweet Protein,Monellin, in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE. Revisions requested 13 April 2005 and 26 May 2005; Revisions received 19 May 2005 and 30 August 2005     

Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in <Emphasis Type="BoldItalic">Escherichia coli</Emphasis>

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20°C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.     

Overexpression,Purification, and Characterization of the Recombinant Leucine Aminopeptidase II of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His₆-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-p-nitroanilide, followed by Arg- and Lys-derivatives. The His₆-tagged enzyme was stimulated by Co²⁺ ions, but was strongly inhibited by Cu²⁺ and Hg²⁺ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co²⁺ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme. Received: 16 August 2002 / Accepted: 4 September 2002     

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.     

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.     

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105–115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC₅₀. This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.     

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

Snakin‐1 (SN‐1) is a cysteine‐rich plant antimicrobial peptide and the first purified member of the snakin family. SN‐1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN‐1 in Escherichia coli by a previously developed coexpression method using an aggregation‐prone partner protein. Our goal was to increase the productivity of SN‐1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN‐1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN‐1, the identity of which was verified by MALDI‐TOF MS and NMR studies. The purified recombinant SN‐1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation‐prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN‐1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520–1528, 2017     

High expression and rapid purification of recombinant scorpion anti-insect neurotoxin AaIT

Scorpion long-chain insect neurotoxins are potentially valuable as agricultural pest control agents. Unfortunately, natural insect neurotoxins are limited in quantity and difficult to obtain from scorpion venom. To determine if recombinant insect neurotoxin is active to insects, we expressed and purified an AaIT fusion protein in Escherichia coli and a recombinant AaIT protein in Pichia pastoris. To quantify AaIT expression in P. pichia colonies, we produced highly sensitive antiserum against AaIT in BALB/c mice. P. pastoris transformants that highly expressed AaIT were selected based on immunoassay with the AaIT antiserum. The P. pastoris recombinant AaIT was rapidly purified in a new and efficient two-step method that eliminated all contaminant proteins using ultracentrifugal filters with molecular weight cut-off 10 kDa and 3 kDa. With this new protocol 10 mg of purified recombinant AaIT was harvested from a 1-l P. pastoris culture. Bioactivity tests indicated that the P. pastoris recombinant AaIT was highly toxic to cockroach larvae, but the E. coli AaIT fusion protein was not toxic to cockroaches. The new expression, screening, and purification protocol described here was efficient for quickly producing high concentrations of pure, bioactive protein.     

Cel8H,a novel endoglucanase from the halophilic bacterium <Emphasis Type="Italic">Halomonas</Emphasis> sp. S66-4: Molecular cloning,heterogonous expression,and biochemical characterization

A recombinant Escherichia coli clone expressing an endoglucanase was identified from a genomic library of the halophilic bacterium Halomonas sp. S66-4, and the enzyme was designated Cel8H. The cel8H gene consisted of 1,053 bp and encoded 350 amino acids sharing the highest identity of 48% to other known endoglucanases. The protein was expressed in E. coli BL21 (DE3) and purified to homogeneity. The purified recombinant enzyme had an optimal activity of 4.9 U/mg at pH 5 and 45°C toward the substrate carboxymethylcellulose. It exhibited extraordinary properties which differed from endoglucanases reported previously at the point of high salt tolerance above 5 M, simultaneously with high pH stability at pH 4–12 and high temperature stability at 40–60°C. Various substrate tests indicated that the enzyme hydrolyzes β-1,4-glucosidic bonds specifically.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.