IPTG Research Articles

Sequence characteristics, expression and subcellular localization of PtCYP721A57 gene from cytochrome P450 family in Polygala tenuifolia willd.

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins.

Parallel Concentric Wrinkled Polydimethylsiloxane Patterns and Their Potential Biomedical Applications

AbstractThe objective of this study is to examine microbial behavior by fabricating and applying parallel concentric wrinkled patterns to polydimethylsiloxane (PDMS) substrates. The patterns are generated and subsequently relaxed in a rigid silica layer, resulting in the formation of discernible wrinkles that can be analyzed using electron microscopy and light diffraction. The alignment of E. coli and silica microparticles is facilitated by these structures along the creases. Within these creases, E. coli exhibits enhanced attachment and rotational mobility in regions characterized by hydrophobic cracks. Furthermore, the bacteria demonstrate an increase in velocity when propelled by the PDMS creases, suggesting that these structures are capable of guiding microbial motion. In addition, distinct alignment and growth patterns are observed for E. coli proliferation within the PDMS microchannels under confined conditions. The analysis of bacterial morphology and gene expression in response to chemical stimuli, such as isopropyl‐β‐D‐thiogalactopyranoside (IPTG) gradients, demonstrates that the chemical environment has a substantial impact; filamentous cells aligned along the creases. These results illustrate the efficacy of parallel concentric wrinkled PDMS patterns in establishing regulated conditions for investigating microbial behavior and represent significant contributions to the fields of microbiology and biomedicine.

Optimization of fermentation conditions for the production of recombinant feruloyl esterase BpFaeT132C-D143C.

Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAEBpFaeT132C-D143C with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFaeT132C-D143C activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFaeT132C-D143C activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFaeT132C-D143C as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFaeT132C-D143C was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFaeT132C-D143C activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFaeT132C-D143C in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFaeT132C-D143C in degrading agricultural waste transformation applications.

Recombinant Hemagglutinin Protein from H9N2 Avian Influenza Virus Exerts Good Immune Effects in Mice.

The H9N2 subtype of avian influenza virus (AIV) causes enormous economic losses and poses a significant threat to public health; the development of vaccines against avian influenza is ongoing. To study the immunogenicity of hemagglutinin (HA) protein, we constructed a recombinant pET-32a-HA plasmid, induced HA protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG), verified it by SDS-PAGE and Western blotting, and determined the sensitivity of the recombinant protein to acid and heat. Subsequently, mice were immunized with the purified HA protein, and the immunization effect was evaluated according to the hemagglutination inhibition (HI) titer, serum IgG antibody titer, and cytokine secretion level of the mice. The results showed that the molecular weight of the HA protein was approximately 84 kDa, and the protein existed in both soluble and insoluble forms; in addition, the HA protein exhibited good acid and thermal stability, the HI antibody titer reached 6 log2-8 log2, and the IgG-binding antibody titer was 1:1,000,000. Moreover, the levels of IL-2, IL-4, and IL-5 in the immunized mouse spleen cells were significantly increased compared with those in the control group. However, the levels of IL-1β, IL-6, IL-13, IFN-γ, IL-18, TNF-α, and GM-CSF were decreased in the immunized group. The recombinant HA protein utilized in this study exhibited good stability and exerted beneficial immune effects, providing a theoretical basis for further research on influenza vaccines.

OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG.

Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E.coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E.coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett–Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml−1 and a final OD600 of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

Stability and release mechanism of double emulsification (W1/O/W2) for biodegradable pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads loaded with the water-soluble bioactive compound niacinamide

Microbeads of biodegradable polyhydroxybutyrate (PHB) offer environmental benefits and economic competitiveness. The aim of this study was to encapsulate a water-soluble bioactive compound, niacinamide (NIA), in a pH-responsive natural matrix composed of PHB and cellulose acetate phthalate (CAP) by double emulsification (W1/O/W2) to improve the encapsulation efficiency (%EE) and loading capacity (%LC). PHB was produced in-house by Escherichia coli JM109 pUC19-23119phaCABA-04 without the inducing agent isopropyl β-D-1-thiogalactopyranoside (IPTG). The influences of PHB and polyvinyl alcohol (PVA) concentrations, stirring rate, PHB/CAP ratio and initial NIA concentration on the properties of NIA-loaded pH-responsive microbeads were studied. The NIA-loaded pH-responsive PHB/CAP microbeads exhibited a spherical core-shell structure. The average size of the NIA-loaded pH-responsive microbeads was 1243.3 ± 11.5 μm. The EE and LC were 33.3 ± 0.5 % and 28.5 ± 0.4 %, respectively. The release profiles of NIA showed pH-responsive properties, as 94.2 ± 3.5 % of NIA was released at pH 5.5, whereas 99.3 ± 2.4 % of NIA was released at pH 7.0. The NIA-loaded pH-responsive PHB/CAP microbeads were stable for >90 days at 4 °C under darkness, with NIA remaining at 73.65 ± 1.86 %. A cytotoxicity assay in PSVK1 cells confirmed that the NIA-loaded pH-responsive PHB/CAP microbeads were nontoxic at concentrations lower than 31.3 μg/mL, in accordance with ISO 10993-5.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

Phage T4-based colorimetric sensing material and its application to develop a time-dependent bacteria sensing platform for long-term detection of food contamination

Herein, we have developed a new type of bacteria-sensing platform for the naked-eye detection of Escherichia coli (E. coli) via a one-spot sample injection process. The platform comprised chlorophenol red-β-d-galactopyranoside (CPRG), isopropyl β-d-thiogalactopyranoside (IPTG), bacteriophages and two carbohydrates namely, maltodextrin and trehalose. CRPG and IPTG detected the β-galactosidase released from the bacteria and enhance its productivity, respectively. Bacteriophages were used as bioreceptors, and two carbohydrates protected phages during thermal process enabling long-term use of the platform. The developed material was characterized using scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy to determine the presence of the phages in the microcapsules. The detection property of the sensing material was examined using different strains of E. coli. Cold water-soluble guar gum was used as the stable gel matrix to increase the reactivity of platform toward samples stored at low temperature. The final prototype was tested using food samples containing different E. coli strains to demonstrate its food applicability with long-term use. This approach may provide an efficient and feasible phage-based sensing platform for the detection of bacterial contamination in food samples.

Motility-activating mutations upstream of flhDC reduce acid shock survival of Escherichia coli.

Many neutralophilic bacterial species try to evade acid stress with an escape strategy, which is reflected in the increased expression of genes coding for flagellar components. Extremely acid-tolerant bacteria, such as Escherichia coli, survive the strong acid stress, e.g., in the stomach of vertebrates. Recently, we were able to show that the induction of motility genes in E. coli is strictly dependent on the degree of acid stress, i.e., they are induced under mild acid stress but not under severe acid stress. However, it was not known to what extent fine-tuned expression of motility genes is related to fitness and the ability to survive periods of acid shock. In this study, we demonstrate that the expression of FlhDC, the master regulator of flagellation, is inversely correlated with the acid shock survival of E. coli. We encountered this phenomenon when analyzing mutants from the Keio collection, in which the expression of flhDC was altered by an insertion sequence element. These results suggest a fitness trade-off between acid tolerance and motility.IMPORTANCEEscherichia coli is extremely acid-resistant, which is crucial for survival in the gastrointestinal tract of vertebrates. Recently, we systematically studied the response of E. coli to mild and severe acidic conditions using Ribo-Seq and RNA-Seq. We found that motility genes are induced at pH 5.8 but not at pH 4.4, indicating stress-dependent synthesis of flagellar components. In this study, we demonstrate that motility-activating mutations upstream of flhDC, encoding the master regulator of flagella genes, reduce the ability of E. coli to survive periods of acid shock. Furthermore, we show an inverse correlation between motility and acid survival using a chromosomal isopropyl β-D-thio-galactopyranoside (IPTG)-inducible flhDC promoter and by sampling differentially motile subpopulations from swim agar plates. These results reveal a previously undiscovered trade-off between motility and acid tolerance and suggest a differentiation of E. coli into motile and acid-tolerant subpopulations, driven by the integration of insertion sequence elements.

Cloning, expression and application of a novel laccase derived from water buffalo ruminal lignin-degrading bacteria

Water buffalo is the only mammal found to degrade lignin so far, and laccase plays an indispensable role in the degradation of lignin. In this study, multiple laccase genes were amplified based on the water buffalo rumen derived lignin-degrading bacteria Bacillus cereus and Ochrobactrum pseudintermedium. Subsequently, the corresponding recombinant plasmids were transformed into E. coli expression system BL21 (DE3) for induced expression by Isopropyl-β-D-thiogalactopyranoside (IPTG). After preliminary screening, protein purification and enzyme activity assays, Lac3833 with soluble expression and high enzyme activity was selected to test its characteristics, especially the ability of lignin degradation. The results showed that the optimum reaction temperature of Lac3833 was 40 °C for different substrates. The relative activity of Lac3833 reached the highest at pH 4.5 and pH 5.5 when the substrates were ABTS or 2,6-DMP and guaiacol, respectively. Additionally, Lac3833 could maintain high enzyme activity in different temperatures, pH and solutions containing Na+, K+, Mg2+, Ca2+ and Mn2+. Importantly, compared to negative treatment, recombinant laccase Lac3833 treatment showed that it had a significant function in degrading lignin. In conclusion, this is a pioneering study to produce recombinant laccase with lignin-degrading ability by bacteria from water buffalo rumen, which will provide new insights for the exploitation of more lignin-degrading enzymes.

A dual fluorescence protein expression system detects cell cycle dependent protein noise

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, ‘noisy’, gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

LactoSpanks: A Collection of IPTG Inducible Promoters for the Commensal Lactic Acid Bacteria Lactobacillus gasseri.

Lactic acid bacteria (LAB) are important for many biotechnological applications such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides, and little portability to in vivo settings. Using Lactobacillus gasseri, a model commensal bacteria from the human gut, we report the engineering of synthetic LactoSpanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from E.coli and induced by isopropyl β-d-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L.gasseri, albeit with substantial leakage. We then construct and screen a semirational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, LactoSpanks will support many applications in L.gasseri, and potentially other lactic acid and Gram-positive bacteria.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques.Key points• Non-switchable methylases always inhibit the relevant type IIS endonuclease activity• Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site• Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assemblyGraphical

Optimization of culture conditions to express AA9 Polysaccharide monooxygenases AN3860 in Escherichia coli

Lignocellulose biomass is a copious source for second generation biomaterial production. The participant of Polysaccharide monooxygenases enzyme (PMO) in the reactions which convert lignocellulose biomass into monosaccharides enhances the activity and improve the efficiency of hydrolysis of hydrolase enzymes on lignocellulose substrate. Enzyme AN3860, obtained from Aspergillus nidulans strain belonging to AA9 PMO, is expected to catalyze flexibly at C1 and C4 carbon positions of β-glycosidic linkage. As an enzyme with high potential of improving cellulose crystals hydrolysis capacity, AN3860 was successfully cloned into the expression system of E. coli BL21 (DE3) strain. In this study, the culture process of recombinant strain with AN3680 gene is optimized to increase the target proteins yield, thus ensure the outcome of purification process, and save production cost. The results demonstrate that the E. coli recombinant strains grow sufficiently in TB (Terrific Broth) culture media and the highest yield of AN3680 protein achieved when the concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) is 0.05 mM and the temperature of the reaction is 30 0C at 150 rpm. After 6 hours of induction, the biomass reaches 500 mg/L and the yield of AN3860 account for (7-10) % total protein generated. The recombinant AN3860 protein is later harvested on larger scale and purified by Ni-NTA column chromatography method for analysis of bioactivities on lignocellulose substrates in the future. ® 2022 Journal of Science and Technology - NTTU

Synthesis of C-glycoside analogues of isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1-β-D-galactopyranosyl-2-methylpropane. Conformational analysis and evaluation as inhibitors of the lac repressor in E. coli and as galactosidase inhibitors.

Isopropyl 1-thio-β-D-galactopyranoside (IPTG, 1) is used widely as an inducer of protein expression in E. coli and 1-β-D-galactopyranosyl-2-methylpropane (2), a C-glycoside analogue of 1, has also been identified as an inducer. Here, synthesis and study of mimetics of 1 and 2, 1-β-D-galactopyranosyl-2-methylpropan-1-ols and two cyclic acetals derivatives, that constrain the presentation of the iPr group in various geometries is described. Conformational analysis of C-glycosides in protic solvent is performed using (i) Desmond metadynamics simulations (OPLS4) and (ii) use of 3JHH values obtained by 1H-NMR spectroscopy. 1-β-D-Galactopyranosyl-2-methylpropane (2) is an effective protein expression inducer when compared to the new mimetics, which were less effective or did not induce expression. 1-β-D-Galactopyranosyl-2-methylpropane (2) led to significantly reduced proteolysis during protein expression, compared to IPTG suggesting that recombinant protein purification will be easier to achieve with 2, yielding proteins with higher quality and activity. IPTG reduced bacterial growth to a greater degree than 2 compared to the control. IPTG's isopropyl group was observed by molecular dynamics (MD) simulations to be flexible in the binding pocket, deviating from its crystal structure binding mode, without impacting other interactions. The MD simulations predicted that 1-β-D-galactopyranosyl-2-methylpropane (2) was more likely than IPTG to bind the repressor with a conformation favoured in protic solvent, while maintaining interactions observed for IPTG. MD simulations predicted that isobutanol derivatives may disrupt interactions associated with IPTG's binding mode. The compounds were also evaluated as inhibitors of galactosidases, with 2 being the more potent inhibitor of the E. coli β-galactosidase. The constrained cyclic acetals showed similar inhibition constants to IPTG indicating E. coli β-galactosidase can recognize galactopyranoses with varying presentation of the iPr group.

Cloning and Expression of Gonadotropin-releasing Hormone to Develop an Immunological-based Sterilization Vaccine

Background: Gonadotropin hormone-releasing hormone (GnRH) is a peptide involved in mammals’ fertility and may also serve as a candidate target for cancer immunotherapy. Immunonsterilization is known as a proper alternative to surgical castration and has been advocated by European countries in recent years. Immunization with GnRH can effectively inhibit the secretion of gonadotropins and cause infertility in both genders of mammals. In this study, a recombinant trimer of GnRH is designed and expressed in a prokaryotic system. Materials and Methods: A construct containing GnRH trimer was designed and prepared using gene synthesis. A cloning site was embedded and connected to the GnRH using a linker to further clone any protein of interest. The construct was subcloned into a pET-32a+ plasmid vector. The recombinant vector was transferred to BL21, an Escherichia coli strain, and the expression was induced using isopropyl β- d-1-thiogalactopyranoside (IPTG). The cell lysate was prepared using lysis buffer and nickel affinity chromatography purification. The GnRH expression was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after protein purification. Results: The cloning was verified using a polymerase chain reaction (PCR) followed by sequencing the recombinant vector. The result of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis verified the recombinant protein’s expression and the purification process’s function. Conclusion: The GnRH was properly cloned and expressed in BL21. The results also verified the reliability of the purification process. The construct design allows the researchers to express another peptide sequence attached to the GnRH using the embedded linker to improve the stability and antigenicity. A stable recombinant GnRH would be applied in immunocastration and cancer immunotherapies.

Preparation of Alginic Acid/Poly(2‐Oxazoline) Hybrid Gels and Their Use in Cell Preservation

AbstractA hybrid gel is developed aiming to extend the storage period for cell preservation compared with an alginate gel. The introduction of poly(2‐oxazoline)s into alginic acid promises to improve functional materials by changing their functional groups (e.g., poly(2‐isopropyl‐2‐oxazoline) has the lower critical solution temperature behavior). This study demonstrates chemical modification of a carboxylate of sodium alginate by 1,3‐diaminopropane‐terminated poly(2‐oxazoline)s using 3‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxy succinimide (NHS) as the condensation reagents for the aminations. The incorporated ratio of poly(2‐oxazoline)s side chains into the carboxylates estimated by 1H NMR is ca. 5–12% and the remaining pendent carboxylates are used for gelation by adding calcium salts (CaCl2). Gelation is observed within 30 min producing 93–99% gel fractions that swell in water. Similar gelation occurs in the cultivation medium of green‐fluorescence protein (GFP)‐encoded and COVID‐19 spike protein‐encoded DNA recombinant E.coli in both the absence and presence of isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). Even in the presence of IPTG, fluorescence ascribed to GFP is not observed while the cells are kept at 25 °C for 3 weeks. After solvation by ethylenediaminetetraacetic acid (EDTA), fluorescence intensity at 508 nm is clearly observed and cell preservation at room temperature is thereby demonstrated.

Expression and characterization of a lipase EstA from Bacillus subtilis KM-BS for application in bio-hydrolysis of waste cooking oil

A lipase EstA from Bacillus subtilis KM-BS was expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme achieved high activity (49.67 U/mL) with protein concentration of 1.29 mg/mL under optimal conditions at the large-scale expression of 6 h and post-induction time at 30 °C using 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The optimal temperature and pH of the purified enzyme were at 45–55 °C and pH 8.0 − 9.0, respectively. Activity of the purified enzyme was stable in the presence of 1 mM Ca2+; stimulated by 1 mM Mg2+ and Mn2+, and inhibited by Fe3+. A significant amount of fatty acids was released during the hydrolysis of waste cooking oil under the catalysis of purified lipase, indicating that this recombinant lipase showed promise as a suitable candidate in industrial fields, particularly in biodiesel and detergent sector.

Cloning and periplasmic soluble expression of hepatitis B surface antigen gene in Escherichia coli.

The objective of this study was to clone and express the hepatitis B surface antigen gene (HBsAg) in Escherichia coli (E. coli), thereby aiming to develop potential local therapeutics for combating Hepatitis B virus (HBV) infection in the Pakistani community by producing HBsAg in E. coli. Blood serum samples were collected from hepatitis B-infected patients, and their genomic DNA was extracted. Real-time and nested polymerase chain reaction (PCR) was performed to amplify the HBsAg gene. The gene of interest was cloned into the pET20b expression vector and transformed into E. coli BL21 (DE3) using Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The gene's precise size was confirmed with gene-specific external and internal primers (681 bp and 400 bp, respectively). The HBsAg gene was successfully sequenced and submitted to GenBank, exhibiting 98% homology with targeted HBV sequences worldwide. The expression of HBsAg protein was confirmed through silver staining, Coomassie staining, western blot, and dot blot analysis. The expressed protein clones are now available for further development as a local recombinant DNA vaccine to prevent hepatitis B viral infection in the local community.