Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
T- and B-lymphocytes are involved in recognition of foreign antigen by the specificity of their surface T-cell receptor and immunoglobulin, generated by gene rearrangement. Each T- and B-lymphocyte carries unique rearranged TCR or immunoglobulin gene, which has been applied to detect clonal from non-clonal T- and B-cell proliferation.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.
OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.
METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.
RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.
CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.
Chikungunya is an acute febrile illness caused by chikungunya virus (CHIKV). In this study, the envelope E1 gene of CHIKV was cloned and expressed in a baculovirus system. The recombinant E1 protein with N-term 6-His residues protein was successfully expressed and purified as confirmed by SDS-PAGE and western blot analysis. The seroreactivity of the recombinant protein was evaluated in immunoassay for anti-CHIKV IgM and IgG antibodies. The recombinant antigen showed 69% sensitivity and 100% specificity for anti-CHIKV IgG by dot blot assay. Detection of anti-CHIKV IgM by dot assay showed 79% sensitivity and 100% specificity. No cross reactivity of the antigen was observed with anti-dengue virus serum samples. The results strongly support that the recombinant E1 protein has potential to be used as diagnostic antigen. The used of the antigen in a dot blot assay gives an advantage for laboratory detection without the need of any specialised equipment.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients' sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients' sera verified the findings of the immunome screening. The patients' humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Purified preparations of Getah virus strains have been analysed by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to reveal their structural proteins. Two envelope proteins (E1 and E2) and core protein (C) were found with the prototype AMM2021 strain both under reducing and nonreducing conditions, while separation of E1 and E2 was observed only under nonreducing conditions for 3 strains isolated in Japan. Limited digestion by Staphylococcus aureus V8 protease revealed difference in the peptide patterns of E1 between AMM2021 and Japanese isolates. Mobility of E1 and E2 was slower for the virus grown in BHK21 cells compared with the virus grown in Aedes albopictus cells, indicating host-controlled modification on the envelope glycoproteins.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Electrophoretically detectable variation in the fungus Neurospora intermedia has been surveyed among isolates from natural populations in Malaya, Papua, Australia and Florida. The principal result is a pattern of genetic variation within and between populations that is qualitatively no different than the well documented patterns for Drosophila and humans. In particular, there is a high level of genetic variation, the majority of which occurs at the level of local populations. Evidence is presented which argues that N. intermedia has a population structure analogous to that of an annual vascular plant with a high level of vegetative reproduction. Sexual reproduction appears to be a regular feature in the biology of the species. Substantial heterokaryon function seems unlikely in natural populations of N. intermedia. Theoretical considerations concerning the mechanisms underlying the observed pattern of variation most likely should be consistent with haploid selection theory. The implications of this constraint upon the theory are discussed in detail, leading to the presentation of a model based upon the concept of environmental heterogenicity. The essence of the model, which is equally applicable to haploid and diploid situations, is a shifting distribution of multiple adaptive niches among local populations such that a given population has a small net selective pressure in favor of one allele or another, depending upon its particular distribution of niches. Gene flow among neighboring populations with differing net selective pressures is postulated as the principal factor underlying intrapopulational allozyme variation.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
An extractive fermentation technique was developed using a thermoseparating reagent to form a two-phase system for simultaneous cell cultivation and downstream processing of extracellular Burkholderia cepacia lipase. A 10% (w/w) solution of ethylene oxide-propylene oxide (EOPO) with a molecular mass of 3900 g/mol and pH 8.5, a 200 rpm speed, and 30 °C were selected as the optimal conditions for lipase production (55 U/ml). Repetitive batch fermentation was performed by continuous replacement of the top phase every 24h, which resulted in an average cell growth mass of 4.7 g/L for 10 extractive batches over 240 h. In scaling-up the process, a bench-scale bioreactor was tested under the conditions that had been optimized in flasks. The production rate and recovery yield were higher in the bioreactor compared to fermentation performed in flasks.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of the PRH1 locus. Gene frequencies of the At productive allele (PRH1(6)) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n = 746). These three proteins were not found in 106 Indian subjects.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The amino-acid composition, 2, 2-Diphenyl-1-picryhydrazyl (DPPH) radical-scavenging activity, and peptide patterns of tilapia protein hydrolysates produced by the enzymatic hydrolysis of Alcalase (AH), Flavourzyme (FH) and Protamex (PH) for 5h using pH-stat method were studied. The ratio of essential amino acids to non-essential amino acids increased after hydrolysis in all samples; however, no significant differences among them were observed. AH had a highest (P < 0.05) DPPH radical-scavenging activity, but no significant difference in the DPPH between FH and PH was observed. SDS-PAGE patterns for all the hydrolysates showed significant (P < 0.05) reduction in the number and the intensity of the bands with increasing time of hydrolysis. Flavourzyme showed the lowest rate of hydrolytic activity towards the tilapia mince.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The present study evaluated the relationship between Bali bull (Bos javanicus) seminal plasma proteins and different semen quality parameters. Semen samples from 10 mature Bali bulls were evaluated for conventional semen parameters (general motility, viability, and normal morphology), sperm functionality (acrosome reaction, sperm penetration rate, sperm penetration index), sperm kinetics (computer-assisted semen analysis parameters such as sperm velocity), and sperm morphology (acrosome and membrane integrity). Frozen-thawed semen with higher sperm motility, viability, acrosome integrity, and membrane integrity (P < 0.05) are consistently higher in acrosome reaction and sperm penetration assay. Three bulls showed the highest, four bulls displayed the medium, and the remaining three bulls showed the lowest for all sperm parameters and SPA. The proteome maps of seminal plasma from high-quality and low-quality Bali bulls were also established. Seminal plasma of both high-quality and low-quality Bali bulls was subjected to two-dimensional SDS-PAGE with isoelectric point ranged from 3 to 10 and molecular weight from 10 to 250 kDa. Approximately 116 spots were detected with Blue Silver stain, and of these spots, 29 were selected and identified by MALDI-TOF/TOF-MS/MS. A majority of the proteins visualized in the seminal plasma two-dimensional maps was successfully identified. An essential group of the identified spots represented binder of sperm 1 (BSP1), clusterin, spermadhesin, tissue inhibitor of metalloproteinases 2 (TIMP-2), and phospholipase A2 (PLA2). Other proteins found in high abundance included seminal ribonuclease, serum albumin, cationic trypsin, and peptide similar to β2 microglobulin. Thus, a reference map of Bali bull seminal plasma proteins has been generated for the very first time and can be used to relate protein pattern changes to physiopathologic events that may influence Bali bull reproductive performance.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Apolipoprotein(a) [apo(a)] exhibits a genetic size polymorphism explaining about 40% of the variability in lipoprotein(a) [Lp(a)] concentration in Tyroleans. Lp(a) concentrations and apo(a) phenotypes were determined in 7 ethnic groups (Tyrolean, Icelandic, Hungarian, Malay, Chinese, Indian, Black Sudanese) and the effects of the apo(a) size polymorphism on Lp(a) levels were estimated in each group. Average Lp(a) concentrations were highly significantly different among these populations, with the Chinese (7.0 mg/dl) having the lowest and the Sudanese (46 mg/dl) the highest levels. Apo(a) phenotype and derived apo(a) allele frequencies were also significantly different among the populations. Apo(a) isoform effects on Lp(a) levels were not significantly different among populations. Lp(a) levels were however roughly twice as high in the same phenotypes in the Indians, and several times as high in the Sudanese, compared with Caucasians. The size variation of apo(a) explains from 0.77 (Malays) to only 0.19 (Sudanese) of the total variability in Lp(a) levels. Together these data show (I) that there is considerable heterogeneity of the Lp(a) polymorphism among populations, (II) that differences in apo(a) allele frequencies alone do not explain the differences in Lp(a) levels among populations and (III) that in some populations, e.g. Sudanese Blacks, Lp(a) levels are mainly determined by factors that are different from the apo(a) size polymorphism.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Traditionally serum and/or CSF specimens have been used for detection of either specific antibodies or antigens as a supportive diagnosis of NCC. However, in recent days, much interest has been shown employing noninvasive specimens such as urine. In our study, we identified and compared a profile of circulating antigenic peptides of parasite origin in three different body fluids (CSF, serum and urine) obtained from confirmed NCC cases and control subjects. The circulating antigenic peptides were resolved by SDS-PAGE and subjected to immunoblotting. For confirmation of their origin as parasite somatic or excretory secretory (ES) material, immunoreactivity was tested employing affinity purified polyclonal Taenia solium metacestode anti-somatic or ES antibodies, respectively. Only lower molecular weight antigenic peptides were found circulating in urine in contrast to serum and CSF specimens. Few somatic peptides were identified to be 100% specific for NCC (19·5 kDa in all three specimens; 131, 70 kDa in CSF and serum only; 128 kDa in CSF only). Similarly, the specific ES peptides detected were 32 kDa (in all three specimens), 16·5 kDa (in serum and CSF only), and 15 kDa (urine only). A test format detecting either one or more of these specific peptides would enhance the sensitivity in diagnosis of NCC.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
BACKGROUND: Prawns and shrimp are a frequent cause of seafood allergy mediated by IgE antibodies. Penaeus monodon and Penaeus latisulcatus, commonly known as black tiger prawn and king prawn, respectively, are among the most frequently consumed prawns in Malaysia. The aim of this study was to identify the IgE-binding proteins of these 2 prawn species.
METHODS: Raw and boiled prawn extracts were prepared and then resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE-immunoblotting was then performed using sera from patients with positive skin prick tests to the raw prawn extracts.
RESULTS: SDS-PAGE analysis of the raw extracts of both prawn species revealed 23 protein bands; the boiled extracts yielded fewer protein bands. The bands in the range of 40 to 100 kDa were sensitive to heat and therefore were not found in the boiled extracts. Immunoblot of raw extracts of black tiger prawns and king prawns yielded 14 and 11 IgE-binding proteins, respectively, with molecular weights of between 15 and 200 kDa. Proteins at 36, 42, and 49 kDa were detected as the major allergens in both species of prawns. A protein of 75 kDa was also identified as a major allergen in black tiger prawns. Other potential allergens were also observed at various molecular masses.
CONCLUSION: Proteins of 36, 42, and 49 kDa were identified as the major allergens of both species of prawns. The 36 and 42 kDa proteins are hypothesised to be tropomyosin and arginine kinase, respectively. A high molecular weight protein of 75 kDa was found to be an additional major allergen in black tiger prawns.
KEYWORDS: Penaeus; allergens; allergy and clinical immunology; hypersensitivity; immunoblotting; tropomyosin
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Adiponectin is an adipocyte-secreting hormone that increases cell sensitivity to insulin. It has been previously demonstrated that this hormone protects against Type II Diabetes and, is found to concurrently promote cell proliferation and differentiation. It is postulated that diabetic patients who suffer from tendinopathy may benefit from using adiponectin, which not only improves the metabolism of diabetic ridden tenocytes but also promotes progenitor cell proliferation and differentiation in tendons. These changes may result in tendon regeneration, which, in diabetic tendinopathy, is difficult to treat. Considering that such findings have yet to be demonstrated, a study was thus conducted using diabetic ridden human tenocyte progenitor cells (TPC) exposed to recombinant adiponectin in vitro. TPC were isolated from tendons of diabetic patients and exposed to 10 μg/ml adiponectin. Cell proliferation rate was investigated at various time points whilst qPCR were used to determine the tenogenic differentiation potential. The results showed that adiponectin significantly reduced blood glucose in animal models. The proliferation rate of adiponectin-treated TPCs was significantly higher at 6, 8 and 10 days as compared to untreated cells (p<0.05). The levels of tenogenic genes expression (collagen I, III, tenomodulin and scleraxis) were also significantly upregulated; whilst the osteogenic (Runx2), chondrogenic (Sox9) and adipogenic (PPARУγ) gene expressions remained unaltered. The results of this study suggest that adiponectin is a potential promoter that not only improves diabetic conditions, but also increases tendon progenitor cell proliferation and differentiation. These features supports the notion that adiponectin may be potentially beneficial in treating diabetic tendinopathy.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The objective of this study was to determine the Immunoglobulin E-binding proteins (IgE) and major allergens of Scomberomorus commerson Lacepede (Narrow-barred Spanish mackerel). Allergen extracts were obtained from uncooked and cooked fish by homogenization in phosphate-buffered saline followed by continuous extraction at 4oC or on ice. Protein profiles and IgEbinding patterns were then detected by means of sodium dodecyl polyacrylamide gel electrophoresis (SDS PAGE) and immunoblotting using sera from patients sensitized to the fish. SDS-PAGE of the uncooked fish extracts revealed 26 protein bands in the range of about 11 to >175 kD, while the cooked extracts produced fewer protein bands. Immunoblotting demonstrated 17 IgE-binding bands, ranging in molecular weight from 11 to 151 kD. Two components with molecular weight of about ~50 and 42 kD showed the highest frequency of IgE-binding (62.2 and 51.4% respectively) and were identified as the major allergens of this fish allergy. Other IgE-binding proteins including a protein at ~12 kD which was equivalent in size to parvalbumin were identified as the minor allergens.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel