Post-induction Temperature Research Articles

Optimizing proinsulin production in E. coli BL21 (DE3) using taguchi method and efficient one-step insulin purification by on-column enzymatic cleavage.

Diabetes is a critical worldwide health problem. Numerous studies have focused on producing recombinant human insulin to address this issue. In this research, the process factors of production of recombinant His-tagged proinsulin in E. coli BL21 (DE3) strain were studied. Bacterial culture factors with significant effects on the amount of produced recombinant proinsulin were screened using a Taguchi L8 orthogonal array. Proinsulin expression was conducted under predicted optimal conditions. The folded impure His-tagged proinsulin was purified using immobilized metal ion affinity chromatography (IMAC). A novel IMAC sequence order combined with the use of non-His-tagged C-peptide cleavage enzymes followed by His- tagged enterokinase enzyme enabled simultaneous protein purification and elimination of C-peptide and His-tag in just one step. Statistical analysis revealed that the amount of produced proinsulin was significantly affected by several factors including the post-induction incubation temperature, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration, pre-induction incubation temperature, the glucose concentration, bacterial cell population at induction step, and the time of harvesting. The optimized model resulted in an empirical maximum proinsulin concentration of 254.5 ± 11.7 µg/ml. The high purity of the purified insulin (> 96% by SDS-PAGE) indicated that applied IMAC sequence order could be considered an efficient technique for on-column cleavage and insulin purification.

Efficient biosynthesis of bilirubin by overexpressing heme oxygenase, biliverdin reductase and 5-aminolevulinic acid dehydratase in Escherichia coli

Bilirubin IXα (BR) offers health benefits in cardiovascular health, stroke, diabetes and metabolic syndrome. BR is mainly extracted from pig bile, bringing potential risks due to infectious viruses. In this study, an efficient pathway for BR biosynthesis was constructed by overexpression of heme oxygenase (HO1) and biliverdin reductase (BvdR) in Escherichia coli. Co-expression of 5-aminolevulinic acid (ALA) dehydratase improved BR production by the recombinant E. coli. In addition, glutamate supplementation in culture media enhanced BR production. Interestingly, it was discovered that ALA exhibited inhibitory effects on HO1 activity and reduced BR production. Culture conditions including post-induction temperature, IPTG concentration, pH of TB media and induction start time were optimized. Finally, fed-batch fermentation was carried out for the strain SH2, producing a BR titer of 62.1 mg/L in a 20 L bioreactor. This study lays foundation for future scalable production of BR in recombinant E. coli.

Integrated bioprocessing and genetic strategies to enhance soluble expression of anti-HER2 immunotoxin in E. Coli

Immunotoxins are widely applied for cancer therapy. However, bacterial expression of immunotoxins usually leads to the formation of insoluble and non-functional recombinant proteins. This study was aimed to improve soluble expression of a novel anti-HER2 immunotoxin under the regulation of the trc promoter in Escherichia coli by optimization of the cultivation conditions using response surface methodology (RSM). To conduct RSM, four cultivation variables (i.e., inducer concentration, post-induction time, post-induction temperature, and medium recipe), were selected for statistical characterization and optimization using the Box-Behnken design and Design Expert software. Based on the developed model using the Box-Behnken design, the optimal cultivation conditions for soluble expression of anti-HER2 immunotoxin were determined to be 0.1 mM IPTG for induction in the LB medium at 33 °C for 18 h. The expressed immunotoxin was successfully purified using affinity chromatography with more than 90% purity and its bioactivity was confirmed using cell-based ELISA. Technical approach developed in this study can be generally applied to enhance the production yield and quality of recombinant proteins using E. coli as the gene expression system.

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.

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048vs. 0.064). Induction at the cell density of 0.7 and an isopropyl β-D-1-thiogalactopyranoside concentration of 0.6mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83mg/L) were more in line with the experimental data (259.51mg/L) than the RSM (276.13mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBankaccession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35°C post induction temperature with overall lipase production of 50.50IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.

Optimizing recombinant production of L-asparaginase 1 from Saccharomyces cerevisiae using response surface methodology.

L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7mM IPTG for 4h at 30°C) resulted in a significant amount of L-asparaginase with a titer of 93.52μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Response Surface Methodology Approach to Optimize the Expression of Thioredoxin-MOG Fusion Protein

Background: The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) has been shown to generate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A considerable amount of MOG must be accessible for EAE induction. Here, for the first time, Response Surface Methodology-Box-Behnken (RSM-BBD) was employed to identify the ideal culture conditions for causing Escherichia coli (E. coli) BL21 to overproduce the Thioredoxin-MOG (Trx-MOG) fusion protein. The RSM method is a powerful, efficient, and reliable alternative to the One-Factor-At-A-Time (OFAT) method in optimizing process variables, allowing for a smaller number of experimental runs, investigating variable interaction, and being cheaper and less time-consuming. Methods: Here, using the 29 experimental assays, the direct and indirect effects of factors including post-induction time, IPTGinducer concentration, pre-induction optical density, and post-induction temperature on the protein expression level content were evaluated. Results: The proposed quadratic model demonstrated a significant effect of the two variables A (time) and C (temperature) on protein synthesis. An inducer concentration of 0.491 mM, the pre-induction optical density (OD600) of 0.8, and a temperature of 23 °C for 23.878 hours were found to be the best growth conditions for high yield Trx-MOG synthesis. The optimum protein concentration was attained (163.96 µg/mL) and was within the range of (200.04 µg/mL), which was the value predicted. Conclusion: The study concluded that RSM optimization effectively increased the production of Trx-MOG in E. coli, which could have the potential for large-scale fermentation.

Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia

BackgroundAlthough E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection.MethodsThe core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab−) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD450) and S/Co values were estimated.ResultsThe integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab−sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection.ConclusionThe applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.

Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology.

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1g/L) in shake flasks was achieved using the following predicted optimal conditions: OD600nm before induction of 0.4 and the induction at 32°C for 7.7h, with 0.6mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/μl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale‑up studies to enhance the biomass for the production of other recombinant proteins.

SCREENING THE EFFECT OF THE EXPRESSION MEDIUM AND GROWTH CONDITIONS ON THE PERFORMANCE OF ENGINEERED XYLANASE PRODUCED BY IMMOBILIZED RECOMBINANT E. COLI

Escherichia coli is the most prevalent host organism for the production of recombinant en-zymes. This was feasible due to the possibility of genetic modification and the availability of multiple E. coli strains as recombinant systems. The primary disadvantage of using E. coli as a host, however, is bacterial cell lysis due to tension build-up in the periplasmic space caused by the overexpression of the recombinant enzyme. Therefore, immobilization is preferable to cytoplasmic excretion for directing the expression of recombinant enzymes into the culture medium. This research investigated the effect of graphene oxide (GO) on the xylanase and β-galactosidase activity of immobilized recombinant E. coli. The effect of culture conditions (expression medium, IPTG, post induction temperature, post induction duration, agitation rate, and pH) on xylanase excretion and cell survival of an immobilized cell was studied using the one factor at a time (OFAT) method. After 24 hours of induction, using terrific broth (TB) as a medium increased xylanase excretion to 0.060 U/ml and resulted in decreased β-galactosidase activity (1.218 U/ml). Apart from that, a lower concentration of isopropyl -D-1-thiogalactopyranoside (IPTG) at 0.01 mM, a lower post-induction temperature (25°C), a 5-hour post-induction time, neutral pH, and 150 rpm significantly increased the xylanase excretion of immobilized cells with low β-galactosidase activity. This study established that immobilizing recombinant E. coli on GO may be advantageous for the excretion of recombinant proteins with a high cell viability.

A study on the effect of preoperative warming on post-induction core temperature and incidence of postoperative shivering in patients under general anesthesia

BackgroundInadvertent perioperative hypothermia (IPH) defined as core temperature below 36.0 °C is a common complication of general anesthesia with prevalence up to 70%. Warming of peripheral tissues prior to induction of anesthesia reduces the central to peripheral temperature gradient, thereby minimizing central heat loss due to heat redistribution, after induction of anesthesia. This study aimed to evaluate the effect of prewarming on post-induction core temperature and incidence of perioperative inadvertent hypothermia leading to postanesthetic shivering (PAS) in patients undergoing general anesthesia.This is a single-arm study performed after authorization from the scientific review committee (IRB no.:10/2015/05) in a cohort of patients between the ages of 18 and 65 years in ASA I and II physical status, undergoing GA for elective surgeries lasting less than 3 h. Rates of IPH and PAS in 60 patients who were warmed before anesthesia over a 30-min period with a forced-air warmer set at 38.0 °C were compared with existing data from an equal number of cohorts who received only intraoperative warming, during similar surgical procedures according to routine GA.Comparisons between the two groups were made using the Student’s t-test and chi-square test. A paired t-test or Wilcoxon’s signed rank test was applied for pairwise comparisons. The results were considered statistically significant when the P-value was < 0.05.ResultsThe mean decrease in core temperature in the unwarmed group was 0.7 °C (+ /- 0.23) compared with a 0.2 °C decrease (+ /- 0.06) in the prewarmed group of patients. A total of 31.70% of patients in the unwarmed group developed IPH compared with one patient (1.7%) in the prewarmed group shortly after onset. Twenty-six patients (43.30%) in the unwarmed group had hypothermia at the end of surgery, compared with one patient (1.7%) in the prewarmed group. Shivering was observed in 46% of patients in the unwarmed group, while no shivering was observed in the prewarmed group.ConclusionsPreoperative warming is an effective intervention to reduce the frequency of core temperature drops after induction of anesthesia, thereby preventing inadvertent perioperative hypothermia and the incidence of postoperative shivering.

Effects of peripheral active warming and passive insulation on core body temperature during feline ovariohysterectomy: a multi-arm randomized clinical trial.

Hypothermia is a common complication of anesthesia, particularly in cats. Some veterinarians insulate the extremities of cats as a preventive measure, and there is evidence that heating the extremities of dogs decreases the rate of heat loss from the core. This study investigated whether active warming or passive insulation of the extremities of cats resulted in a slower decrease in rectal temperature during anesthesia. Female cats were assigned via block randomization to passive (cotton toddler socks), active (heated toddler socks) or control group (uncovered extremities). Rectal temperature was monitored every 5 mins from induction through return to trap/carrier (final temperature). Multivariable linear regression models were used to compare temperature (rate change and final) between groups. There were 164 cats with 1757 temperature readings. Mean total duration of anesthesia was 53 ± 13 mins. The temperature of all groups decreased linearly over time (all P <0.0001), with the rates of temperature decrease being -0.039°F/min (95% confidence interval [CI] -0.043 to -0.035)/-0.022°C (95% CI -0.024 to -0.019), -0.039°F/min (95% CI -0.042 to -0.035)/-0.022°C (95% CI -0.023 to -0.019) and -0.029°F/min (95% CI -0.032 to -0.025)/-0.016°C (95% CI -0.018 to -0.014) for the control, passive and active groups, respectively. The control, passive and active groups had median final temperatures of 98.4°F (interquartile range [IQR] 97.6-99.4)/36.9°C (IQR 36.4-37.4), 98.0°F (IQR 97.2-98.7)/36.7°C (IQR 36.2-37.1) and 99.1°F (IQR 97.7-100.0)/37.3°C (IQR 36.5-37.8), respectively. After controlling for weight, postinduction temperature and duration of anesthesia, and as compared with controls, the final temperature of the active group was predicted to be 0.54°F (95% CI 0.03-1.01)/0.3°C (95% CI 0.02-0.56) greater (P = 0.023), while the passive group was not significantly different (P = 0.130). The rate of rectal temperature decrease was significantly slower for the active group compared with the other groups. Although the cumulative difference in final temperature reading was modest, superior materials might enhance performance. Cotton toddler socks alone did not slow the rate of temperature decrease.

Computational and Experimental Evaluation of Linker Peptides and Thioredoxin Fusion Tag in CD20-rituximab Specific Interactions.

Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs). The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins. By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G4S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G4S molecule. Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.

Maximizing the recovery of the native p28 bacterial peptide with improved activity and maintained solubility and stability in Escherichia coli BL21 (DE3)

p28 is a natural bacterial product, which recently has attracted much attention as an efficient cell penetrating peptide (CPP) and a promising anticancer agent. Considering the interesting biological qualities of p28, maximizing its expression appears to be a prominent priority. The optimization of such bioprocesses might be facilitated by utilizing statistical approaches such as Design of Experiment (DoE). In this study, we aimed to maximize the expression of “biologically active” p28 in Escherichia coli BL21 (DE3) host by harnessing statistical tools and experimental methods. Using Minitab, Plackett-Burman and Box-Behnken Response Surface Methodology (RSM) designs were generated to optimize the conditions for the expression of p28. Each condition was experimentally investigated by assessing the biological activity of the purified p28 in the MCF-7 breast cancer cell line. Seven independent variables were investigated, and three of them including ethanol concentration, OD600 of the culture at the time of induction, and the post-induction temperature were demonstrated to significantly affect the p28 expression in E. coli. The cytotoxicity, penetration efficiency, and total process time were measured as dependent variables. The optimized expression conditions were validated experimentally, and the final products were investigated in terms of expression yield, solubility, and stability in vitro. Following the optimization, an 8-fold increase of the concentration of p28 expression was observed. In this study, we suggest an optimized combination of effective factors to produce soluble p28 in the E. coli host, a protocol that results in the production of a significantly high amount of the biologically active peptide with retained solubility and stability.

Optimization of the heterologous expression and purification of Plasmodium falciparum generative cell specific 1 in Escherichia coli

Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.

Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17

Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis–trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis–trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.

Machine learning modeling for solubility prediction of recombinant antibody fragment in four different E. coli strains

The solubility of proteins is usually a necessity for their functioning. Recently an emergence of machine learning approaches as trained alternatives to statistical models has been evidenced for empirical modeling and optimization. Here, soluble production of anti-EpCAM extracellular domain (EpEx) single chain variable fragment (scFv) antibody was modeled and optimized as a function of four literature based numerical factors (post-induction temperature, post-induction time, cell density of induction time, and inducer concentration) and one categorical variable using artificial neural network (ANN) and response surface methodology (RSM). Models were established by the CCD experimental data derived from 232 separate experiments. The concentration of soluble scFv reached 112.4 mg/L at the optimum condition and strain (induction at cell density 0.6 with 0.4 mM IPTG for 24 h at 23 °C in Origami). The predicted value obtained by ANN for the response (106.1 mg/L) was closer to the experimental result than that obtained by RSM (97.9 mg/L), which again confirmed a higher accuracy of ANN model. To the author’s knowledge this is the first report on comparison of ANN and RSM in statistical optimization of fermentation conditions of E.coli for the soluble production of recombinant scFv.

A statistical approach to boost soluble expression of E. coli-derived virus-like particles in shake-flask cultivation

Hepatitis B core virus-like particles (HBc-VLP) have been widely used as carrier platforms to present an epitope of interest. Escherichia coli expression system is cost effective and produces high yields of recombinant protein. However major drawbacks include difficulties in obtaining soluble expression and tendency to form inclusion bodies. To boost solubility of proteins during expression of E. coli-derived HBc-VLPs carrying EBNA1 epitope, a statistical approach involving fractional factorial design and response surface methodology was used. For the first time, this approach was applied to quantitatively determine the impact of key parameters in shake-flask cultivation. Expression conditions including post-induction temperature and shaker-speed, and cell density at induction were optimized. Based on native agarose gel electrophoresis, optimized soluble protein cellular yield was 210.5 mg g−1 dry cell mass and volumetric yield was 272 mg L−1 of culture media. Findings highlight: 1) the significant interaction between post-induction temperature and shaker-speed on production, and; 2) sufficient oxygen level is required during induction. It is concluded that this statistical approach can be practically applied to optimize expression of HBc-VLP in shake-flask cultivation, and to determine key parameters for large-scale productions.