Marine-Derived Cytosine Arabinoside (Ara-C) Inhibits Biofilm Formation by Inhibiting PEL Operon Proteins (Pel A and Pel B) of Pseudomonas aeruginosa: An In Silico Approach.

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative biofilm-forming opportunistic human pathogen whose vital mechanism is biofilm formation for better survival. PelA and PelB proteins of the PEL operon are essential for bacterial-synthesized pellicle polysaccharide (PEL), which is a vital structural component of the biofilm. It helps in adherence of biofilm on the surface and maintenance of cell-to-cell interactions and with other matrix components. Here, in-silico molecular docking and simulation studies were performed against PelA and PelB using ten natural bioactive compounds, individually [podocarpic acids, ferruginol, scopadulcic acid B, pisiferic acid, metachromin A, Cytarabine (cytosine arabinoside; Ara-C), ursolic acid, oleanolic acid, maslinic acid, and betulinic acid], those have already been established as anti-infectious compounds. The results obtained from AutoDock and Glide-Schordinger stated that a marine-derived cytosine arabinoside (Ara-C) among the ten compounds binds active sites of PelA and PelB, exhibiting strong binding affinity [Trp224 (hydrogen), Ser219 (polar), Val234 (hydrophobic) for PelA; Leu365 and Glu389 (hydrogen), Gln366 (polar) for PelB] with high negative binding energy - 5.518 kcal/mol and - 6.056 kcal/mol, respectively. The molecular dynamic and simulation studies for 100 ns showed the MMGBSA binding energy scores are - 16.4 kcal/mol (Ara-C with PelA), and - 22.25 kcal/mol (Ara-C with PelB). Further, ADME/T studies indicate the IC50 values of AraC are 6.10 mM for PelA and 18.78 mM for PelB, which is a comparatively very low dose. The zero violation of Lipinski's Rule of Five further established that Ara-C is a good candidate for drug development. Thus, Ara-C could be considered a potent anti-biofilm compound against PEL operon-dependent biofilm formation of P. aeruginosa.

Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80.

pelB has been known as a successful signal peptide to translocate the protein target extracellularly in the Escherichia coli system. However, in our previous study, the yield of MPT64 protein extracellular recovery was still low and plenty of this protein was remain trapped in cytoplasm and periplasm. Recently, nonionic surfactants were efficiently reported to secrete recombinant protein extracellularly. Nonetheless, it must be clarified whether the surfactant supplementation can improve the yield of MPT64 extracellular protein significantly without giving impact on the structure of isolated MPT64 protein and can minimized the cell lysis effect. MPT64 protein secretion was carried out by comparing the effects of surfactants Tween 80 and Triton × 100 at various concentrations. Triton × 100 was able to increase the extracellular MPT64 protein gain up to 3 times higher than Tween 80 and it was in line with the greater level ratio of cell leakage of Triton × 100 compared to that of Tween 80. Similarly, the viable cell of the cultures decreased dramatically. However, both surfactants did not interfere the structure of MPT64 protein. In conclusion, Triton × 100 can be chosen as the supporting surfactant to assist the act of peptide signal in improving the resulting of MPT64 extracellular protein.

Introduction of a Hexalysine (6 K) Tag Can Protect from N-Terminal Cleavage and Increase Yield of Recombinant Proteins Expressed in the Periplasm of E. coli.

Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and activity, as the cytosol of E. coli constitutes a reducing environment. The periplasm in contrast is an oxidative environment which supports proper protein folding. However, yields can be limited compared with cytoplasmic expression, and protocols must be adjusted to avoid overloading the periplasmic transportation machinery. Another less-appreciated issue with periplasmic expression is the potential generation of unwanted N-terminal cleavage products, a persistent issue which we encountered when expressing the disulfide bond containing extracellular regions of several Helicobacter pylori adhesins (BabA, BabB, BabC, and LabA) in the periplasm of E. coli XL10 GOLD, a strain traditionally not used for proteins expression. Here, we describe how introducing a C-terminal hexa-lysine (6 K) tag enhanced solubility and protected BabA from N-terminal proteolytic degradation (BabA), enabling crystallization and subsequent X-ray structural analysis. However. the same strategy had no advantageous effect for LabA, which using this protocol could be retrieved from the periplasm in relatively high yields (20-40 mg/L).

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli.

BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.

Enhanced Extracellular Production of IsPETase in Escherichia coli via Engineering of the pelB Signal Peptide.

Poly(ethylene terephthalate) (PET) is one of the most commonly used plastics worldwide and its accumulation in the environment is a global problem. PETase from Ideonella sakaiensis 201-F6 was reported to exhibit higher hydrolytic activity and specificity for PET than other enzymes at ambient temperature. Enzymatic degradation of PET using PETase provides an attractive approach for plastic degradation and recycling. In this work, extracellular PETase was achieved by Escherichia coli BL21 using a Sec-dependent translocation signal peptide, pelB, for secretion. Furthermore, engineering of the pelB through random mutagenesis and screening was performed to improve the secretion efficiency of PETase. Evolved pelB enabled higher PETase secretion by up to 1.7-fold. The improved secretion of PETase led to more efficient hydrolysis of the PET model compound, bis (2-hydroxyethyl) terephthalic acid (BHET), PET powder, and PET film. Our study presents the first example of the increasing secretion of PETase by an engineered signal peptide, providing a promising approach to obtain extracellular PETase for efficient enzymatic degradation of PET.

Real-time monitoring of rhamnose induction effect on the expression of mpt64 gene fused with pelB signal peptide in Escherichia coli BL21 (DE3).

In this research, Escherichia coli BL21 (DE3) harboring an expression vector constructed with a rhamnose-inducible promoter and a pelB signal peptide was used as a host cell to produce MPT64 protein. The objective of this research was to figure out the optimum time of mpt64 gene expression through real-time monitoring of MPT64 protein production and distribution in host compartments. The mpt64 expression was regulated by the rhamnose presence at a concentration of 4 mM. The real-time isolated protein was monitored using polyacrylamide gel electrophoresis in denaturation condition. Based on real-time monitoring, the MPT64 protein (24 kDa) in the cytoplasm was optimum detected at 24 h after induction. For periplasmic fraction, the protein was detected at 4 h after induction but thinning at 15 h after induction. At 16 h after induction, the MPT64 protein band was found in the medium with increasing concentrations until 24 h. Thus, it can be concluded that the mpt64 gene expression was regulated in the presence of rhamnose as an inducer, and the proteins were shown to be translocated throughout the host cell compartment with different levels of protein accumulation at different times, according to the role of pelB as a signal peptide.

Engineered small metal-binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C-terminal his-tag added for subsequent purification. Our research group has proposed the small metal-binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB-SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2-11 cells, was equivalent to commercial growth hormone at 50 ng·mL-1 . Therefore, we strongly recommend the use of PelB-SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.

Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-ß structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0-11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries.

BACKGROUND: Antibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it's also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed. RESULTS: In the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well. CONCLUSIONS: In conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Effect of PelB signal sequences on Pfe1 expression and ω-hydroxyundec-9-enoic acid biotransformation in recombinant Escherichia coli.

ω-Hydroxyundec-9-enoic acid (ω-HUA) was reported as a valuable medium-chain fatty acid with industrial potentials. For bioconversion of ricinoleic acid to ω-HUA, in this study, an alcohol dehydrogenase (Adh) from Micrococcus luteus, a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 and an esterase (Pfe1) from Pseudomonas fluorescens SIK WI were overexpressed in Escherichia coli BL21(DE3). In order to enhance accessibility of Pfe1 to the (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA) substrate, a truncated PelB signal sequence without the recognition site of signal peptidase (tPelB) was attached to the N-terminus of Pfe1, resulting in the construction of E. coli AB-tPE strain expressing Adh, BVMO and the tPelB-Pfe1 fusion protein. A batch-type biotransformation of ricinoleic acid by E. coli AB-tPE resulted in 1.8- and 2.2-fold increases in ω-HUA conversion yield and productivity, respectively, relative to those for the control strain without the PelB sequence (AB-E). By fed-batch-type biotransformation with glycerol feeding, the AB-tPE strain produced 23.7 mM (equivalent to 4.7 g/L) of ω-HUA with 60.8%(mol/mol) of conversion yield and 1.2 mM/h of productivity, which were 13.2, 2.9, and 2.6 times higher than those in a batch-type biotransformation using the AB-E strain. In conclusion, combination of the truncated PelB-Pfe1 fusion and fed-batch process with glycerol feeding provided the highest efficiency of ω-HUA biotransformation, of which strategies might be applicable for biotransformation of hydrophobic substances.

Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli.

In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.

Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.

Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl ß-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.

Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli.

An Escherichia coli expression system was established to produce recombinant extracellular Pseudozyma (Candida) antarctica lipase B (CALB). With the aim of producing the genuine CALB without additional amino acid residues, the mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3) using the pET system. Inducing gene expression at low temperature (20°C) was crucial for the production of active CALB; higher temperatures caused inclusion body formation. Prolonged induction for 48 h at 20°C allowed for the enzyme to be released into the culture medium, with more than half of the activity detected in the culture supernatant. A catalytically inactive CALB mutant (S105A) protein was similarly released, suggesting that the lipid-hydrolyzing activity of the enzyme was not the reason for the release. The CALB production level was further improved by optimizing the culture medium. Under the optimized conditions, the CALB in the culture supernatant amounted to 550 mg/L. The recombinant CALB was purified from the culture supernatant, yielding 5.67 mg of purified CALB from 50 mL of culture. N-terminal sequencing and ESI-MS analyses showed proper removal of the pelB signal sequence and the correct molecular weight of the protein, respectively, confirming the structural integrity of the recombinant CALB. The kinetic parameters towards p-nitrophenylbutyrate and the enantiomeric selectivity on rac-1-phenylethylacetate of the recombinant CALB were consistent with those of the authentic CALB. This is the first example of E. coli-based extracellular production of a CALB enzyme without extra amino acid residues.

Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.

Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli.

Our previous studies demonstrated that Thermobifida fusca cutinase is released into culture medium when expressed without a signal peptide in Escherichia coli, and this extracellular expression results from an enhanced membrane permeability caused by cutinase's phospholipid hydrolase activity. The present study investigated whether this phenomenon would also occur during the expression of cutinase fused to pelB signal peptide (pelB-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pelB-cutinase was expressed in a secB knockout strain, which has a defective type II secretion pathway, there was still a large amount of cutinase in the culture medium. Additional experiments confirmed that the periplasmic and cytoplasmic fractions of the expressing cells had hydrolytic activity toward phosphatidyl ethanolamine, and the recombinant cells showed correspondingly improved membrane permeability. All these phenomena were also observed in the parent E. coli strain. Moreover, the secretion efficiency of the inactive cutinase mutant was found to be significantly lower than that of pelB-cutinase in the parent E. coli. Based on these results, the phospholipid hydrolase activity of pelB-cutinase must play a larger role in its extracellular production than does type II secretion pathway.

Improved extracellular expression and purification of recombinant Staphylococcus aureus protein A.

Protein A from Staphylococcus aureus plays one key role as an immobilized affinity ligand for the purification of antibodies. A simple method for its extracellular expression in Escherichia coli and subsequent purification is reported herein. The N-terminus of the gene coding for the five IgG binding domains was fused to a pelB signal peptide which is responsible for periplasmic localization and which is removed after translocation into the periplasmic space of E.coli. Different additives, which were added at the same time with the induction of the protein expression by IPTG, were tested in order to facilitate the release of the target protein. With help of this optimized release protocol, more than 380mgL(-1) of protein A were obtained when Tris-HCl pH 8.5 was added up to a final concentration of 180mM in shaking flask experiments. Based on these observations, a protocol was developed for the extracellular production of SpA in a stirred tank bioreactor yielding 5.5gL(-1) of the secreted target protein. After cell removal by centrifugation, the protein A-containing supernatant was concentrated and dialyzed by tangential flow filtration. The target protein was subsequently purified by anion exchange chromatography with a total process yield of 90% and a final purity of ⩾95% (RP HPLC) was achieved.

Expression and secretion of functional recombinant µO-conotoxin MrVIB-His-tag in Escherichia coli.

µO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.