Comparison of expression systems for the production of human interferon-a2b.

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform

The performance of a glass bead shaking technique for the disruption of Escherichia coli cells

The efficacy of a simple laboratory method for cell disruption based on the shaking of glass beads on a rotary shaker was assessed in this study, via measurements of the release of total protein and interferon-α2b from E.coli. The optimum conditions for cell disruption were detected after 30 min of shaking in Tris-HCl buffer (pH 8) at 300 rpm with 1.5g of glass beads (diameter: 0.5 mm) per mL of cell suspension volume. Three test runs were conducted under the above conditions and the maximum average protein release values were determined as 3.048, 3.564, and 3.015 mg/mL, respectively. The amount of protein release was comparable to the amount of protein release in ultrasonication and glass bead vortexing procedures. The amount of interferon-α2b release in the ultrasonication, glass bead vortexing, and glass bead shaking trials were 240, 172, and 201 ng/mL, respectively. This method was shown to process between 1 and 10 mL of sample volume in a 50 mL Falcon tube without a great deal of deviation, and was able to handle in excess of 60 samples simultaneously

Improved mannan-degrading enzymes' production by Aspergillus niger through medium optimization.

The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL−1) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL−1), α-cellulose (10.7 nkat mL−1), glucose (8.8 nkat mL−1) and carboxymethylcellulose (4.6 nkat mL−1). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL−1) followed by peptone from meat (1168 nkat mL−1), yeast extract (817 nkat mL−1), ammonium sulphate (241 nkat mL−1), ammonium nitrate (113 nkat mL−1) and ammonium chloride (99 nkat mL−1) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L−1 GG and 57 g L−1 peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL−1) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L)

Determination and optimization of flavonoid and extract yield from brown mango using response surface methodology.

The optimum extraction conditions to obtain the highest flavonoid content and extract yield from Mangifera pajang pericarp (MPP) were analyzed using response surface methodology (RSM). A full factorial central composite design was used to investigate the effects of ethanol concentration (20-80%), temperature (30-65°C) and liquid to solid ratio (20-50 mL/g) on the recovery of extract yield and flavonoids. A second order polynomial model produced a satisfactory fitting of the experimental data with regard to extract yield (R 2 = 0.9890, p < 0.0001) and flavonoids (R 2 = 0.9652, p < 0.0001). The optimum conditions to obtain higher extract yield, were 54%, 50°C, and 42.4 mL/g, while for flavonoids were 68%, 57°C, and 20.2 mL/g, respectively. The experimental values agreed with those predicted with 99% and 96% confidence interval for extract yield and flavonoids respectively. This indicates the suitability of RSM in optimizing the extraction of flavonoids and extract yield from MPP

Sequential optimization of production of a thermostable and organic solvent tolerant lipase by recombinant Escherichia coli

Several medium formulations were screened for the production of a thermostable and organic solvent tolerant lipase by a recombinant Escherichia coli BL21. The highest lipase production (28.9 ± 4.1 IU/mL) was obtained in Luria Bertani medium with the addition of 1% (w/v) glucose. The medium formulation and fermentation conditions were then subjected to sequential optimization. Using a Plackett-Burman design, glucose, NaCl, temperature and induction time were found to be the most significant variables affecting lipase production, and these were then optimized using response surface methodology (RSM). The large value of R 2 (0.979) showed that the quadratic model used for the prediction is highly significant. The optimum levels of these four significant variables (glucose, NaCl, temperature and induction time) as predicted by RSM were 32.4 g/L, 5 g/L, 31.7°C and 2.1 h, respectively. The amount of lipase activity (50.2 ± 4.5 IU/mL) produced under these optimal conditions fitted well to the value (48.9 IU/mL) predicted by RSM. Production of lipase in optimized fermentation was about 2.5-fold higher than in non-optimized fermentation

Molecular docking studies of selected medicinal drugs as dengue virus-2 protease inhibitors

Dengue is a potentially deadly disease with no effective drug. An in silico molecular docking was performed using Autodock 4.2.6 to investigate the molecular interactions between protease inhibitors, comprising antibiotic derivatives namely doxycycline (3), rolitetracycline (5) and a non-steroidal anti-inflammatory drug (NSAID), meclofenamic acid (4), against the NS2B-NS3 protease from dengue virus-2 (DENV-2). The non-competitive inhibitor (3) showed lower binding energy (-5.15 kcal/mol) than the predicted competitive inhibitors 4 and 5 (-3.64 and -3.21 kcal/mol, respectively). Structural analyses showed compound 3 that bound to a specific allosteric site, interacted with Lys74, a significant amino acid residue bonded to one of the catalytic triad, Asp75. Compounds 4 and 5 showed direct binding with two of the catalytic triad, His51 and Ser135, hence, predicted to be competitive inhibitors

Assessment of molecular recognition element for the quantification of human epidermal growth factor using surface plasmon resonance

Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein. Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (KD = 7.39e-10 M) than monoclonal antibody (mAb) (KD = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run. Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment

Optimization of an induction strategy for improving interferon-α2b production in the periplasm of Escherichia coli using response surface methodology

Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively

Response surface optimisation for the extraction of phenolic compounds and antioxidant capacities of underutilised Mangifera pajang Kosterm. peels.

The optimum extraction conditions for highest recovery of total phenolics content (TPC) and antioxidant capacities (AC) were analysed for Mangifera pajang peels (MPP), using response surface methodology. The effects of ethanol concentration (X1: 20-80%), extraction temperature (X2: 30-65°C) and liquid-to-solid ratio (X3: 20-50 mL/g) on the recovery of total phenolics (Y1) and antioxidant capacity (Y 2) were investigated. A second order polynomial model produced a satisfactory fitting of the experimental data with regard to total phenolic content (R2 = 0.9966, p < 0.0001) and antioxidant capacity (R 2 = 0.9953, p < 0.0001). The optimum extraction conditions for TPC were 68%, 55°C and 32.7 mL/g, and for AC were 68%, 56°C and 31.8 mL/g, respectively. Predicted values for extraction of TPC and AC agreed well with the experimental values. Liquid chromatography-mass spectrometry of the optimally obtained extracts from MPP revealed the major phytochemicals as mangiferin, gallic acid, catechin and epicatechin. © 2011 Elsevier Ltd. All rights reserved