Preparative Native Polyacrylamide Gel Electrophoresis Research Articles

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.

Optimization of monoclonal antibodies purification expressed in H-192 cells using preparative native-polyacrylamide gel electrophoresis

Monoclonal antibodies (mAbs) are unique and specific drug molecules targeting the treatment of various diseases such as arthritis, immune disorders, infectious diseases, and cancer etc. Different methods such as antibody coupled affinity chromatography, hydrophobic interaction chromatography, etc., can be applied to purify mAbs from various sources. This article provides a simple, cost effective, preparative native-polyacrylamide gel electrophoresis (n-PAGE)technique to purify mAbs expressed in H-192 cells (Hybridoma murine cell lines) against an antigen i.e. 17-alpha-hydroxyprogesterone (17-OHP), which further can have diagnostic application to detect Congenital Adrenal Hyperplasia (CAH). Furthermore, different parameters such as concentration and volume of the feedstock (medium containing antibodies), pore size of gel, height of resolving gel etc. were optimized to obtain the maximum purity and yield of mAbs.

Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera)

Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)2SO4 F3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17μmol/ml, respectively. The optimum conditions for AlTI13 were pH8 and temperatures ranging from 35 to 40°C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that ~28kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.

PURIFICATION AND CHARACTERIZATION OF PHOSPHODIESTERASE I FROM Walterinnesia aegyptia VENOM

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5′-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the Vmax and Km were 1.14 µM/min/mg and 1.9 × 10−3 M, respectively, and the Kcat and Ksp were 7 s−1 and 60 M −1 min−1 respectively. Cysteine was a noncompetitive inhibitor, with Ki = 6.2 × 10−3 M and an IC50 of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with Ki = 0.8 × 10−3 M and an IC50 of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg2+ slightly potentiated the activity. PDE-I hydrolyzed thymidine-5′-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3′-5′-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.

Optimization of a native gel electrophoretic process for the purification of intracellular green fluorescent protein from intact Escherichia coli cells

Intracellular green fluorescent protein (GFP) can be separated and purified from intact Escherichia coli cells by a preparative native polyacrylamide gel electrophoresis (n-PAGE). The effects of operating parameters such as the volume and concentration of feedstock and the pore size and height of resolving gel on the purity and yield of GFP were studied using a 1.7 cm internal diameter gel column. The optimum conditions for this preparative n-PAGE operation were determined to be 100 μl of 15% (w/v) feedstock and a 12% (w/v) resolving gel with a gel height of 2 cm. The purity and yield of the recovered GFP were 98 and 88%, respectively. The results of scalability studies show that the ratio of feedstock volume to cross-sectional area of the column is an important consideration for scaling up the preparative n-PAGE.

Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.

The Effect of Crude and Purified Cerastes Vipera Venom Protein Fractions on Respiratory Chain Function in Cultured Human Fibroblasts

This study was conducted to investigate the effect of crude and venom protein fractions of Cerastes vipera on the major enzymatic components of the respiratory chain in cultured human fibroblasts. Cerastes vipera crude venom was fractionated into seven protein fractions by using 8% preparative native polyacrylamide gel electro-phoresis. The effect of crude venom and purified venom fractions (F1-F7) on respiratory chain function in cultured human fibroblasts was investigated. Confluent fibroblast cultures were incubated with crude venom and venom protein fractions for a fixed period of three hours at 37°C. Crude venom and venom protein fractions 2, 4, 5 and 6 significantly increased the production of lactate as compared to control fibroblasts. The production of pyruvate was markedly decreased in all these cell lines. The ratio of lactate/pyruvate was increased in the cells and in the culture medium by crude venom and venom protein fractions 2, 4, 5 and 6. On the other hand fractions 1,3 and 7 exhibited no effect in relation to lactate and pyruvate production. Similarly the crude venom protein and venom protein fractions 2, 4, 5 and 6 significantly lowered the activity of the major enzymatic component of the respiratory chain in fibroblast mitochondria. In conclusion it is apparent that the crude and venom protein fractions 2, 4, 5 and 6 resulted in a significant lowering of the mitochondrial respiratory chain activity in cultured human fibroblasts.

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1–F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37°C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters. Copyright © 2002 John Wiley & Sons, Ltd.

The effect of Echis carinatus crude venom and purified protein fractions on carbohydrate metabolism in rats

Echis carinatus crude venom was fractionated into 11 protein fractions by preparative native polyacrylamide gel electrophoresis (PAGE). All fractions except fractions 5 and 10 appeared as a single band on analytical native PAGE. Purified venom fractions 1, 4, 8, 10 and 11 appeared as single bands on SDS-PAGE whereas fractions 2, 3 and 7 contained two bands and fraction 6 contained three bands. Fractions 1 and 3 exhibited basic pI (7.3 and 7.6) respectively, while fractions 2, 4, 6, 8, 10 and 11 showed an acidic pI. Amino acid analysis also showed that crude venom is rich in acidic amino acids. A significant hyperglycaemia was produced by i.p. injection of E. carinatus crude venom, after 15 min of envenomation which persisted even after 24 h. Along with hyperglycaemia there was a significant decrease of liver glycogen at 15 min and 1, 12 and 24 h. A significant decrease of plasma [pyr + lac] levels was found from 15 min to 24 h. The liver [pyr + lac] levels increased significantly after 24 h. Skeletal muscle [pyr + lac] level was significantly decreased after 24 h of envenomation. Fractions 2 and 6 produced the highest increase in plasma glucose after 12 h and fraction 7 after 24 h. The plasma insulin level was significantly decreased by these three fractions (2, 6 and 7). So it can be hypothesized that the hyperglycaemia may result from a direct effect of a venom component on plasma insulin. Fractions 7, 8 and 11 caused the highest decrease in plasma [pyr + lac] while fractions 1, 2, 3, 4 and 8 produced the most significant decrease in liver [pyr + lac]. The most significant increase in lactate dehydrogenase level was also produced by fractions 1, 2, 3, 4 and 8. Copyright © 2001 John Wiley & Sons, Ltd.

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins.

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.