SDS-polyacrylamide Gel Electrophoresis Analysis Research Articles

ChemInform Abstract: Purification and Characterization of Laccase from Coriolopsis floccosa MTCC‐1177 and Its Use in the Selective Oxidation of Aromatic Methyl Group to Aldehyde Without Mediators.

A laccase from the culture filtrate of white rot fungus Coriolopsis floccosa MTCC-1177 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as well as native polyacrylamide gel electrophoresis (native-PAGE) produced single protein bands indicating that the enzyme preparation was pure. Molecular mass of the enzyme determined from SDS-PAGE analysis was 64 kDa. Using 2,6-dimethoxyphenol (DMP), 2,2′-[Azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid (ABTS) diammonium salt and 4-hydroxy-3,5-dimethoxy benzaldehyde azine (syringaldazine) as the substrates, the K m, k cat and k cat/K m values of the laccase were found to be 112.5 μM, 5.16 s−1, 4.60 × 104 M − 1 s−1, 58 μM, 5.16 s−1, 8.90 × 104 M − 1 s−1 and 100 μM, 5.16 s−1, 5.16 × 104 M − 1 s−1, respectively. The pH and temperature optima were 5.0°C and 40°C, respectively. Activation energy for thermal denaturation of the enzyme was 36.6 kJ/mol/K. The enzyme was most stable at pH 4.0 when exposed for 1 h. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. The enzyme transforms toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively, in the absence of electron transfer mediators.

Influence of processing conditions on the physicochemical and sensory properties of sesame milk: A novel nutritional beverage

Production of sesame milk is one of the methods for increasing consumption of sesame as an excellent nutritional resource. The aim of this study was to examine the effects of sodium bicarbonate concentration in soaking water (0, 0.5 and 1 g/100 mL NaHCO3), roasting temperature (0 and 145 °C) and blanching time (0, 15 and 30 min) on physicochemical and sensory properties of sesame milk. Changes promoted by these processing conditions were also evaluated via color analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The processing parameters mainly affected pH, total solids, protein, fat, ash, lipoxygenase activity, stability, specific gravity, viscosity, color features and sensory properties. Sensory evaluation revealed that overall acceptability was higher in all treatments than the control. SDS-PAGE analysis showed that 7S globulins decreased by roasting and soaking while 11S globulins of sesame milk proteins increased by roasting. The optimum processing conditions were found to be soaking in water containing 0.5 g/100 mL NaHCO3, blanching for 15 min and without any roasting when desirability function method was applied.

Purification and characterization of laccase from Coriolopsis floccosa MTCC-1177 and its use in the selective oxidation of aromatic methyl group to aldehyde without mediators

A laccase from the culture filtrate of white rot fungus Coriolopsis floccosa MTCC-1177 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as well as native polyacrylamide gel electrophoresis (native-PAGE) produced single protein bands indicating that the enzyme preparation was pure. Molecular mass of the enzyme determined from SDS-PAGE analysis was 64 kDa. Using 2,6-dimethoxyphenol (DMP), 2,2′-[Azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid (ABTS) diammonium salt and 4-hydroxy-3,5-dimethoxy benzaldehyde azine (syringaldazine) as the substrates, the K m, k cat and k cat/K m values of the laccase were found to be 112.5 μM, 5.16 s−1, 4.60 × 104 M − 1 s−1, 58 μM, 5.16 s−1, 8.90 × 104 M − 1 s−1 and 100 μM, 5.16 s−1, 5.16 × 104 M − 1 s−1, respectively. The pH and temperature optima were 5.0°C and 40°C, respectively. Activation energy for thermal denaturation of the enzyme was 36.6 kJ/mol/K. The enzyme was most stable at pH 4.0 when exposed for 1 h. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. The enzyme transforms toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively, in the absence of electron transfer mediators. A yellow laccase has been purified and characterized from the culture filtrate of white rot fungus Coriolopsis floccosa MTCC-1177. This laccase selectively transforms the aromatic methyl group to aromatic aldehyde group in the absence of mediator molecules. The average yield of the products was 90%.

Defensive Responses of Rice Genotypes for Resistance Against Rice Leaffolder Cnaphalocrocis medinalis

The experiment was carried out to assess the reaction of different categories of rice genotypes viz., resistant, susceptible, hybrid, scented, popular and wild in response to the infestation by rice leaffolder (RLF), Cnaphalocrocis medinalis (Guenee) and to explore the possible use of these genotypes in developing RLF-resistant rice varieties. The changes of various biochemical constituents such as leaf soluble protein, phenol, ortho-dihydroxy phenol, tannin and enzymes viz., peroxidase, phenyl alanine ammonia lyase (PAL) were assessed spectrophotometrically in all the rice genotypes before and after RLF infestation. The protein profile was analyzed using sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) method. A significant constituent of biochemical content such as tannin, phenol and ortho-dihydroxy phenol has been increased along with enzyme activities of peroxidase and PAL in the infested resistant (Ptb 33, TKM6 and LFR831311) and wild rice genotypes (Oryza minuta and O. rhizomatis). A decrease in leaf protein content was evident invariably in all the infested rice genotypes. It is also evident that the contents of biochemicals such as phenol, ortho-dihydroxy phenol and tannin were negatively correlated with leaffolder damage. However, leaf protein content was positively correlated with the damage by rice leaffolder. SDS-PAGE analysis for total protein profiling of healthy and C. medinalis-infested genotypes revealed the enhanced expression of a high molecular weight (> 97 kDa) protein in all the genotypes. Besides, there was also an increased induction of a 38 kDa protein in C. medinalis infested resistant genotypes, which was absent in uninfested plants. The present investigation proved that the elevated levels of biochemicals and enzymes may play a vital role in rice plants resistance to RLF.

English

Genetic relationship between some species of Zea mays and Sorghum was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of seed protein and random amplification of polymorphic DNA (RAPD-PCR) markers. According to SDS-PAGE analysis, 78 band were identified across the studied species. The number of bands varies from 17 bands in sample number 5 to 6 in sample number 6. Analysis of RAPD-PCR of DNA provided more precise information concerning relationships between Zea mays and Sorghum species than SDS-PAGE analysis. A remarkable result from this study was identifying a close relationship between Zea mays spp mays and Zea mays spp Mexicana. Further support comes from the molecular data of RAPD, which indicate that close relationship between Sorghum valgare and Sorghum bicolor. Key words: Zea mays, Sorgum volgare pres, Punciu milia ceaum L. protein, random amplification of polymorphic DNA.

Production of extracellular proteins, cellulases and antifungal metabolites by Chaetomium globosum Kunze ex. Fr.

Chaetomium globosum has been well-known potential antagonist of several seed and soilborne fungus. Eight isolates of C. globosum were obtained from different sources and were identified by morphological characters. C. globosum isolates examined for the presence of extra cellular proteins, cellulases and antifungal metabolites in culture filtrate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), thin-layer chromatography and high-performance liquid chromatography. Variation in the mycelial protein of C. globosum isolates was noted in the SDS-PAGE analysis. Different C. globosum isolates that showed more number of bands in protein profile was further screened for the production of cellulases in culture filtrate. Cellulase activity of C. globosum isolates revealed that maximum activity was observed in the isolate Cg-6 after 11 days of incubation, while Cg-2 had least activity. C. globosum isolates were tested for antibiotic production, among which three isolates viz. Cg-6, Cg-7 and Cg-5 were found to produce the antibiotic Chaetoglobosin A in the culture filtrate. The antibiotic Chaetoglobosin A appeared blue colour under UV spectrum with a wavelength of 250 nm.

Biochemical and biological analysis of Philodryas baroni (Baron’s Green Racer; Dipsadidae) venom

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bβ- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 μg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Separation of Recombinant Geranylgeranyl Diphosphate Synthase of Deinococcus radiodurans from Expressed Strain Cell Homogenate by Immobilized Metal Affinity Chromatography on a Characterized Monolithic Cryogel Column

Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative carotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experimental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeability is 5.04×10−12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10−4 m·−1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and effective approach to isolate GGPPS from cell homogenate of engineering strains.

An acellular aortic matrix of buffalo origin crosslinked with 1‐ethyl‐3‐3‐dimethylaminopropylcarbodiimide hydrochloride for the repair of inguinal hernia in horses

SummaryAn acellular aortic matrix (AAM) crosslinked with 1‐ethyl‐3‐3‐dimethylaminopropylcarbodiimide hydrochloride (EDC) was evaluated for the repair of inguinal hernias in 5 horses. The aorta from buffalo was acellularised using 1% sodium dodecyl sulfate (SDS) and 0.25% trypsin. The AAM was crosslinked with 1% EDC. Under anaesthesia, inguinal hernias were repaired with EDC‐crosslinked AAM graft using an inlay graft technique. Blood samples collected on Days 0, 15 and 30 post implantation, were used for SDS polyacrylamide gel electrophoresis (PAGE) analysis to assess the animals' serum protein concentration, and gelatin zymography for the identification of matrix metalloproteinases. All animals that underwent hernia repair demonstrated successful healing without clinical signs of wound dehiscence, infection or recurrence during a 6‐month follow‐up period. SDS‐PAGE analysis of serum protein concentrations revealed that, this was increased at Day 15 and had decreased again at Day 30. Gelatin zymography of serum of implanted horses expressed a band of 92 kDa, corresponding to MMP‐9 activity. The relative amount of the 92 kDa band was higher at Day 15 as compared to Days 0 and 30. It may be concluded that EDC‐crosslinked AAM of buffalo origin can be used safely in horses for the repair of inguinal hernia with adequate strength and minimal foreign body reaction.

Increased prevalence of low oligomeric state surfactant protein D with restricted lectin activity in bronchoalveolar lavage fluid from preterm infants

BackgroundSurfactant protein D (SP-D) is a soluble oligomeric C-type lectin known to protect against lipopolysaccharide and ventilator-induced lung injury in preterm lambs. Here we assess the expression and functional status...

Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restricted fragment length polymorphism among fenugreek accessions

Protein and DNA polymorphismswere surveyed among seven accessions of wild fenugreek (Trigonellafoenum-graecum L.) to estimate their genetic diversity and relationships. Samples were obtained from diverse ecogeographical areas in Saudi Arabia and Yemen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of seed storage protein showed genetic variations among fenugreek germplasms, both quantitatively and qualitatively, generating a total of 168 polypeptide bands with different molecular weights ranging from 4.5 to 300 kDa. Twenty-six of these bands were polymorphic, with a considerable polymorphism value (80.00%). Furthermore, restriction fragment length polymorphism (RFLP) analysis was also employed, which was based on the ability of four restriction enzymes (EagI, EcoRI, FspI, and HindIII) to cleave genomic DNA of the plant materials at specific target nucleotide sequences into different numbers of DNA fragments. RFLP analysis revealed 166 fragments with known sequences and variable lengths ranging from 80 to 4000 bp with a highly degree of polymorphism (88.71%). Data derived from SDS-PAGE or RFLP analyses were used to produce dendrograms, which clustered the studied fenugreek accessions into different groups based on the unweighted pair group method with arithmetic mean (UPGMA). The resulting relationships indicated that these two marker techniques were nearly equivalent, but not identical, with respect to phylogenetic information. In conclusion, SDS-PAGE analysis of seed proteins should be augmented with RFLP analysis of DNA for reliable estimates of genetic diversity among fenugreek germplasms.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS

Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. STATISTICAL METHOD USED: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.

Salivary gland proteome of the human malaria vector, Anopheles campestris-like (Diptera: Culicidae)

Anopheles campestris-like is proven to be a high-potential vector of Plasmodium vivax in Thailand. In this study, A. campestris-like salivary gland proteins were determined and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and nano-liquid chromatography-mass spectrometry. The total amount of salivary gland proteins in the mosquitoes aged 3-5days was approximately 0.1 ± 0.05μg/male and 1.38 ± 0.01μg/female. SDS-PAGE analysis revealed at least 12 major proteins found in the female salivary glands and each morphological region of the female glands contained different major proteins. Two-dimensional gel electrophoresis showed approximately 20 major and several minor protein spots displaying relative molecular masses from 10 to 72kDa with electric points ranging from 3.9 to 10. At least 15 glycoproteins were detected in the female glands. Similar electrophoretic protein profiles were detected comparing the male and proximal-lateral lobes of the female glands, suggesting that these lobes are responsible for sugar feeding. Blood-feeding proteins, i.e., putative 5'-nucleotidase/apyrase, anti-platelet protein, long-form D7 salivary protein, D7-related 1 protein, and gSG6, were detected in the distal-lateral lobes (DL) and/or medial lobes (ML) of the female glands. The major spots related to housekeeping proteins from other arthropod species including Culex quinquefasciatus serine/threonine-protein kinase rio3 expressed in both male and female glands, Ixodes scapularis putative sil1 expressed in DL and ML, and I. scapularis putative cyclophilin A expressed in DL. These results provide information for further study on the salivary gland proteins of A. campestris-like that are involved in hematophagy and disease transmission.

Decomposition of proteins by photocatalytic Ti(IV)-doped calcium hydroxyapatite particles

The decomposition of protein molecules on the surface of Ti(IV)-doped calcium hydroxyapatite (TiHap) particles with a Ti/(Ca+Ti) atomic ratio among 0–0.20 under UV irradiation of 365nm in wavelength was disclosed. The acidic bovine serum albumin (BSA), neutral myoglobin (MGB) and basic lysozyme (LSZ) were employed as a model of pathogenic proteins. The photocatalytic activities of TiHap particles were estimated from the decomposition of each protein under 1mW/cm2 UV irradiation dispersed in 10mL quartz tube. The concentrations of each protein in the supernatant after centrifugation during the UV irradiation were determined both by a HPLC and a SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis methods. No change in BSA concentration ([BSA]) by UV irradiation was observed for all the unheated original TiHap particles with low photocatalytic activity. The similar results were observed for the systems employed heat treated particles endowed a high photocatalytic activity by heat treatment at 650°C for 1h. These results indicated that the decomposition of BSA molecules is hard to take place. The heme structured MGB molecules are decomposed by UV irradiation irrespective of the presence of TiHap particles. In the case of heat treated particles, MGB molecules were further decomposed by the UV irradiation. The strongest photocatalytic activity was observed for the decomposition systems of LSZ by using heat treated particles. In this system, all the TiHap particles completely decomposed LSZ molecules after started the UV irradiation. It was concluded that the ability of decomposition of proteins is strongly related to the molecular weight and rigidity of proteins molecules. The LSZ molecule with low molecular weight and rigid structure was easily decomposed on the surface of heat treated TiHap particles under UV irradiation.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

In vitro digestibility and rheological properties of caseinates treated by an oxidative system containing horseradish peroxidase, glucose oxidase and glucose

Sodium caseinate was modified by an oxidative system comprising horseradish peroxidase (HRP), glucose oxidase (GO) and glucose in either a one-step or a two-step protocol. In the former, a sodium caseinate suspension was treated simultaneously with HRP, GO and glucose, whereas in the latter the suspension was treated first with GO and glucose, then subsequently with HRP. Fluorescence spectroscopy or sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis confirmed dityrosine or polypeptide formation in the treated caseinates, respectively. The treated caseinates had higher surface hydrophobicity and lower in vitro digestibility than the original caseinate, as well as a higher apparent viscosity, elastic modulus and viscous modulus. Caseinate treated with the one-step method contained more cross-linked polypeptides and higher surface hydrophobicity, apparent viscosity, elastic and viscous modulus higher and lower in vitro digestibility than caseinate treated with the two-step method. The oxidative system showed its potential to modify functional properties of food proteins.

Expression, purification and antigenic evaluation of toxin-coregulated pilus B protein of Vibrio cholera

The toxin co-regulated pilus B (TcpB) as well as tcpA has been verified as a critical colonization factor for Vibrio cholera O1. TcpB is a candidate for making subunit vaccine against cholera; this study aims to produce an oral vaccine by expressing recombinant toxin co-regulated pilus B in Escherichia coli (E. coli). The toxin co-regulated pilus B (tcpB) gene was amplified by polymerase chain reaction (PCR) method. PCR product was sub-cloned to prokaryotic expression vector PET32a, was transformed in E. coliBL21 (DE3) and was induced by Isopropyl_β-D-1-thiogalactopyranoside (IPTG). Recombinant protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by Ni-NTA resin. The immune response to TcpB in animal model was studied. PCR product of tcpB gene has 1297 bp and it was confirmed by sequencing. In SDS-PAGE analysis a band with 65 kDa was seen. In patient recovering from cholera and animal model such as Mice, rabbit with oral inoculation, high titer of antibody in serum was detected. These results demonstrated that the recombinant TcpA is antigenic and can be used in a carrier host as an oral vaccine against cholera. Key words: Antigenicity, Escherichia coli, recombinant protein, toxin co-regulated pilus B.

Characterization of Protein-like Fluorophores Released from Lake Phytoplankton on the Basis of Fractionation and Electrophoresis

Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.

Molecular determination of genotoxic effects of cobalt and nickel on maize (Zea mays L.) by RAPD and protein analyses

Assessment of DNA damages stemming from toxic chemicals is an important issue in terms of genotoxicology. In this study, maize (Zea mays L.) seedlings were used for screening the genotoxic effects of cobalt (Co) and nickel (Ni) treatments at various concentrations (5 mM, 10 mM, 20 mM and 40 mM). For this purpose, randomly amplified polymorphic DNA (RAPD) technique was applied to genomic DNA extracted from metal-exposed and unexposed plant materials. Besides, changes in total protein contents were screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. For RAPD analysis, 16 RAPD primers were found to produce unique polymorphic band profiles on different concentrations of Co-/Ni-treated maize seedlings. Increased polymorphism resulting from the appearance of new bands or disappearance of normal bands was observed with increasing concentration of Co and Ni treatments. Genomic template stability, a qualitative measurement of changes in RAPD patterns of genomic DNA, decreased with increasing metal concentration. In SDS-PAGE analysis, it was observed that the total soluble protein content decreased by Co treatment, while it increased by Ni treatment. The results obtained from this study revealed that RAPD profiles and total soluble protein levels can be applied to detect genotoxicity, and these analyses can offer useful biomarker assays for the evaluation of genotoxic effects on Co- and Ni-polluted plants.