Sepharose 4B Column Research Articles

Trypsin from Pyloric Caeca of Asian Seabass: Purification, Characterization, and Its Use in the Hydrolysis of Acid-Soluble Collagen.

The study aimed to purify trypsin from the pyloric caeca of Asian seabass (Lates calcarifer), and investigate its proteolytic capability toward acid-soluble collagen (ASC) in comparison with commercial porcine trypsin (CPT). Trypsin was purified from pyloric caeca, a leftover from the evisceration process, via ammonium sulphate (40-60% saturation) precipitation, and a soybean trypsin inhibitor (SBTI)-Sepharose 4B column. A 18.5-fold purification and a yield of 15.2% were obtained. SDS-PAGE analysis confirmed a single band of trypsin with a molecular weight of 23.5 kDa. Purified trypsin also showed the single band in native-PAGE. The optimal pH and temperature of trypsin for BAPNA (the specific substrate for amidase) hydrolysis were 8.5 and 60 °C, respectively. The trypsin was stable within the pH range of 7.0-9.5 and temperature range of 25-55 °C. Protease inhibition study confirmed that the purified enzyme was trypsin. The purified trypsin had a Michaelis-Menten constant (Km) and catalytic constant (kcat) of 0.078 mM and 5.4 s-1, respectively, when BAPNA was used. For the hydrolysis of TAME (the specific substrate for esterase), the Km and Kcat were 0.09 mM and 4.8 s-1, respectively. Partially purified seabass trypsin (PPST) had a slightly lower hydrolysis capacity toward ASC than CPT, as evidenced by the lower degree of hydrolysis and protein degradation when the former was used. Both the α-chain and β-chain became more degraded as the hydrolysis time increased. Based on MALDI-TOP, peptides with MW of 2992-2970 Da were dominant in the hydrolysates. Therefore, seabass trypsin could be used in the production of hydrolyzed collagen. It could have economic importance to the market, by replacing some commercial proteases, which have religious constraints.

DETECTION OF TERNARY COMPLEX OF FIBRIN DESAB WITH D-DIMER AND D-FRAGMENT OF FIBRIN

The aim of this work is to study the intermolecular interactions of fibrin with D-domain-containing fragments of fibrin(ogen): D-dimer and D-fragment. Materials and methods. Human fibrinogen was obtained from the human blood plasma by salt extraction using 16 % Na2SO4. The content of protein coagulated by thrombin – 96-98%. Analytical size-exclusion chromatography for the detection of molecular complexes was performed on the Sepharose 6B column (30 x 0.5 cm). Components of the analyzed mixture (0.8 ml) were separated by standard chromatography protocol: speed of elution – 0.5 ml/min; collected samples volume – 0.5 ml. Optical density of collected samples was measured by spectrophotometer POP (Optizen, Daejeon, Korea). Composition of each sample was analyzed by SDS-PAGE. Relative amounts of studied compounds in samples were analyzed using densitometry of scanned electropherograms with Totallab TL100 software. Molecular modeling of complexes formed by fibrin desAB and its fragments were performed using UCSF Chimera 1.16 on the basis on earlier developed protofibril structure. The structure of the D-region (PDB ID:1LTJ) was prepared in the same in-protein molecular docking was performed using HDOCK web server. Results. To investigate the complex formation between fibrin desAB. The appearance of D- and DD-fragments in the elution zone of 5.5 mL, which does not overlap with the elution zone of individual fragments (7.5-9.5 mL), was detected, indicating the formation of a ternary complex. Densitometry of electropherograms using TotalLab TL-100 demonstrated that the average densities of pixels in bands of fibrin desAB, D-dimer and D-fragment were equal. It means that the ternary complex of fibrin desAB with D-dimer and D-fragment was composed in the approximate ratio of fibrin desAB, D-dimer and D-fragment 1:1:1. Molecular docking in the HDOCK software was used to establish the spatial arrangement of the D-fragment in relation to the fibrin desAB molecule bound to the D-dimer. Conclusions. We obtained and characterized the ternary complex of fibrin desAB, D-dimer and D-fragment by size-exclusion chromatography followed by SDS-PAGE. Further study of the structure and properties of this complex may clarify certain issues related to fibrin polymerization, namely the process of protofibril formation and their spatial branching.

Overexpression, biochemical characterization, and anticancer activates of L-asparaginase from Bacillus subtilis

L-asparaginases convert L-asparagine into L-aspartate and ammonia. The L-asparaginase from Bacillus subtilis was cloned and expressed in the E. coli strain BL21(DE3)pLysS in the current study. Using glutathione sepharose 4B column chromatography, the L-asparaginase enzyme was uniformly purified 173.34 times, with a final specific activity of 1769.13 IU/mg protein and a yield of 56.14%. The isolated enzyme was identified as a 36 kDa polypeptide chain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immobilized enzyme was placed on top of the Ca alginate beads. The immobilized enzyme is quite stable and retains the majority of its activity at 4 °C (74 percent). The enzymatic and structural characteristics of free and immobilized recombinant L-asparaginase were studied. The activity of the free enzyme peaked after 30 min of incubation at pH 8.0 and 45 °C. After 30 minutes at 50 °C, the immobilized enzyme showed its peak activity at a pH of 8.5. The refined enzyme's amino acid makeup was identified. An enzyme that heals leukemia, Bacillus subtilis L-asparaginase, can be successfully mass-produced using this technique.

Identification of Proteins Responsible for High Activity of Cysteine Proteinase Inhibitor in the Blood of Nile Tilapia Oreochromis niloticus

Cysteine proteinase inhibitors (CPIs) protect tissues and organs against cysteine proteinases in animal blood and have attracted much attention for use in food processing and medical sciences for humans and animals. Several CPI proteins, which include stefins, cystatins, kininogens, histidine-rich glycoproteins (HRG) and fetuins, have been identified and characterized in mammals. Fish blood also contains high CPI activity, but the identity of the major protein responsible for this activity has not been clarified. This study was conducted to screen CPI activity by examining papain inhibitory activity from various different tissues in Nile tilapia Oreochromis niloticus and to identify major proteins for the activity in the blood. CPI activity was highest in the serum among the tissues screened in this study (at least fourfold higher than in other tissues)(P < 0.05). Major proteins for CPI activity in serum were purified using a CNBr-activated sepharose 4B column, gel filtration and an ion exchange FPLC column. From these purifications, two proteins with strong CPI activity were isolated and partially sequenced. Based on their molecular weights and partial amino sequences, the two major proteins with CPI activity from the blood in this species were found to be fetuin B (60 kDa) and kininogen (54 kDa).

Extraction and Purification of Collagen from Marine Squid Uroteuthis Duvauceli

Collagen is a protein, which plays a significant role in the development of tissues and organs. These are used for cosmetic surgery and as wound healing aids in burn patients, remaking of bone and a wide assortment of dental, orthopedic and surgical purposes. These days’ collagen is isolated from calf, porcine tissue and so forth. But it can cause the onset of diseases such as, for example, Bovine spongiform encephalopathy (BSE), Transmissible spongiform encephalopathy (TSE), Foot-and-mouth illness (FMD), and so forth and furthermore cultural and social concerns. As another option, collagen can be separated from marine squid. The aim of the present work is to isolate and partially characterize the collagen from the marine squid, Uroteuthis duvauceli. The confined collagen was evaluated by utilizing Bradford test and partially purified by using gel filtration on a Sepharose 4B column. These filtered collagens were affirmed by SDS PAGE. These days industrially accessible collagen in the market are costly and are not affordable to the common people. The present data on collagens from the marine squid, could help the future endeavors to unwind the therapeutically significant, more secure collagens from marine squid for their utilization in the biomedical field.

Further Analytical Studies on a Mercuri Thiol Adduct Isolated from a Human Prostate Cancer Cell Line (LNCaP)

Thiols play vital roles in cellular metabolism knowledge of which may be important in the design of future anticancer drugs. Previous work on the composition of the thiols present in human cancer cell lines has shown the presence of an unknown low molecular weight species, deemed to be a “Conthiol”, which could be important in this respect. This was prepared and isolated from a human prostate cancer cell line (LNCaP) in the form of an adduct of 2-mercuri-4-nitrophenol; it accounts for 56.5% of the total cellular thiols present in this cell line. Initial LC-MS analysis of this adduct had indicated that the possible molecular weight of the thiol was in the region of 467 daltons. In further analytical studies to identify the thiol, attempts were made to release it from the adduct by passage through a Thiopropyl Sepharose6B column. LC-MS analysis of the column eluate revealed two components yielding negative ion fragments of 427 m/z and 449 m/z. Only the former component contained thiol, indicating that a breakdown and/or possible rearrangement of the Conthiol had occurred. Further investigations of the column thiol eluate using ICP-MS analysis showed that the sulfur content agreed with the spectrophotometric analysis result (Ellman assay) and that the molecule did not contain phosphate. Amino acid analyses of the eluate were negative. In an attempt to prevent the breakdown of the thiol released by the Thiopropyl Sepharose 6B column, the adduct was treated with 5% v/v bromine water prior to applying to the column. In this instance the thiol containing eluate obtained from the column was treated with an equimolar quantity of mercuric chloride forming a fresh adduct, RS-Hg-SR. LC-MS analysis of this mercurial adduct detected a negative ion fragment of 782 m/z which on further ionization gave a ladder like pattern showing loss of mass units of 58 in each rung. This would seem to suggest the presence of a repeat polymer like structure containing 5 monomers, which, plus the thiol atom, gives a possible formula weight of 322; probably revealing only a part of the unknown Conthiol molecule whose properties and formula weight do not correlate with any known cellular thiol. Further analysis of the thiol released from the adduct on the Thiopropyl Sepharose 6B column by Infra-red (FTIR) provided little information except to confirm the presence of the thiol group and C=O stretch bands together with the possibility of a lactam ring at 1651 and 1634 cm·s&#451.

Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.

Characterization of Cytotoxic Lactose Binding Lectin from Sulphur Polypore, Laetiporus sulphureus (Agaricomycetes), from Algeria.

Mushroom lectins have important biological and biomedical applications. Most lectins purified from these organisms exhibit high toxicity in animal cells and toward microbial agents. They are able to induce cell growth inhibition and metabolism by their ability to interact with glyconjugate components (glycoproteins receptors, glycolipids) present in their membrane. After lectins bind to these membrane receptors, they induce cellular signalization chains in which gene expression is regulated and cell death programming (apoptosis) is activated. In this work, a new multimeric lectin was characterized from the rare saprobic edible mushroom, Laetiporus sulphureus strain TMES43, grown in the Algerian forest. Lectin was isolated with ammonium sulfate precipitation followed by affinity chromatography on a Sepharose 4B column, with specific activity of 1204.7 units of hemagglutination activity/mg and 35.55% yield. The protein has a tetrameric structure with a molecular weight of 36 kDa for each subunit, with a total molecular weight of approximately 140 kDa. In addition, a Mascot peptide fingerprint study on a matrix-assisted laser desorption ionization-time of flight tandem fragment showed identity with autophagy-related protein 16 from Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324; Expasy ID: ATG16_PICGU) and no sequence similarity to known mushroom lectins. L. sulphureus hemagglutination activity was reduced by 5 mM of lactose and 10 mM of EDTA incubation and was recovered by metallic cations such as CaCl2, MgCl2, and ZnCl2. L. sulphureus purified lectin had no human ABO group specificity and showed low temperature and alkaline pH stabilities. The MTT preliminary assay showed that L. sulphureus purified lectin induced high cytotoxicity for tumor cells and normal cells.

Structural features and antioxidant activity of a new galactoglucan from edible mushroom Pleurotus djamor

A new water soluble galactoglucan with apparent molecular weight ~1.61 × 105 Da, was isolated from the edible mushroom Pleurotus djamor by hot water extraction followed by purification through dialysis tubing cellulose membrane and sepharose 6B column chromatography. The sugar analysis showed the presence of glucose and galactose in a molar ratio of nearly 3:1 respectively. The structure of the repeating unit in the polysaccharide was determined through chemical and NMR experiments as: [Display omitted] In vitro antioxidant studies showed that the PDPS exhibited hydroxyl radical scavenging activity (EC50 = 1.681 ± 0.034 mg/mL), DPPH radical scavenging activity (EC50 = 3.83 ± 0.427 mg/mL), reducing power (EC50 = 4.258 ± 0.095 mg/mL), and ABTS radical quenching activity (EC50 = 0.816 ± 0.077 mg/mL). So, PDPS should be explored as a natural antioxidant.

Structural studies of immunomodulatory (1 → 3)-, (1 → 4)-α glucan from an edible mushroom Polyporus grammocephalus

A water soluble polysaccharide (PGPS) with molecular weight ~ 1.4 × 105 Da was isolated by alkali treatment from an edible mushroom Polyporus grammocephalus and purified by gel chromatography using sepharose-6B column. Monosaccharide analysis revealed that PGPS was made up of glucose only. PGPS contained (1 → 3)-α-D-Glcp and (1 → 4)-α-D-Glcp moieties in a molar ratio of nearly 1:2. Through a series of chemical and spectroscopic (1D/2D NMR) investigations, the repeating unit of the glucan was established as:→3)-α-D-Glcp(1 → [4)-α-D-Glcp(1]2→This α-glucan was observed to stimulate some prime components of immune system, namely, macrophages, splenocytes, and thymocytes.

Characterization and inhibitory activities on α-amylase and α-glucosidase of the polysaccharide from blue honeysuckle berries

An acidic heteropolysaccharide HEP-2 was obtained from blue honeysuckle berry extracts after purification using Sepharose 6B and Sephadex G-200 column chromatography. The molecular weight of HEP-2, determined with high-performance gel permeation chromatography, was 3.01 × 106 Da. Fourier transform infrared spectroscopy, gas chromatography and nuclear magnetic resonance analyses revealed the presence of α-, β-pyranose ring and six sugar residues in HEP-2. Amorphous aggregates of HEP-2 with irregular shape and lacking the triple helical structure were detected using the Congo red test, scanning electron and atomic force microscopy, and X-ray diffraction studies. Rheological characterization showed typical shear-thinning behavior and viscoelastic property of the polysaccharide. HEP-2 exerted significant inhibitory effects on α-amylase and α-glucosidase and displayed competitive inhibition kinetics. The collective results support the potential utility of HEP-2 as a hypoglycemic agent for enzyme-targeted treatment of diabetes or functional food.

Microwave-assisted extraction of an acidic polysaccharide from Ribes nigrum L.: Structural characteristics and biological activities

Microwave-assisted extraction of the polysaccharide from blackcurrant (Ribes nigrum L.) fruits (MBP) was optimized using response surface methodology (RSM). The yield of MBP was 10.59 % (w/w) under the optimal conditions of power (414 W), liquid to material ratio (31/1 mL/g), and the time (41 min). After purification using D4006 macroporous resin and Sepharose-6B column chromatography, a water-soluble acidic heteropolysaccharide (PMBP) was obtained with a molecular weight of 1.99 × 106 g/mol. PMBP was thermally stable and exhibited excellent rheological properties. Fourier transform infrared spectroscopy, gas chromatography and nuclear magnetic resonance analysis confirmed the presence of α-, β-pyranose and six main glycosidic linkages in PMBP. Scanning electron microscopy, atomic force microscopy, and X-ray diffraction studies demonstrated that PMBP was amorphous aggregates with irregular shape, porous, and smooth surface. Bioactivity assays revealed the notable antioxidant and hypoglycemic activities of PMBP. Moreover, PMBP was kinetically characterized as a competitive inhibitor for α-amylase and α-glucosidase.

Prokaryotic expression, protein purification and functional verification of human homotypic fusion and vacuole protein sorting complex subunit

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant

BackgroundLectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. Methods and resultsHere we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75 μg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. ConclusionLCL can be explored for its clinical potential.

Investigation of the effects of some water-soluble vitamins on glutathionereductase enzyme purified from bovine liver

Glutathione reductase (GR, EC 1.8.1.7) is an antioxidant enzyme and is involved in the reducing reaction of oxidized glutathione. Glutathione and reduced glutathione are very crucial for micro- and macroorganisms because of their barrier function against radicals. Therefore, the aim of this article was to purify GR and evaluate the relationship between purified GR and vitamins. GR was purified from bovine liver using 2',5'-ADP Sepharose 4B column materials. To check the purity of each subunit of the enzyme, SDS-PAGE electrophoresis was performed and each one was 55 kDa. Afterwards, specific activity and purified ratio for the enzyme were calculated as 54 EU/mg $\times $ protein and 2700 times, respectively. Vitamins have a regulatory role in organisms and also some, like vitamin B, are coenzymes due to having cofactor effects. In this project, variable concentrations of some water-soluble vitamins, riboflavin, ascorbic acid, nicotinamide, folic acid, and thiamine, were tested. As a result of these kinetic findings, nicotinamide and folic acid increased the activity of glutathione reductase, while thiamine decreased it. Riboflavin and ascorbic acid did not show a stable effect on enzyme activity. Ki and IC$_{50}$ constants were found as 50.16 $\pm $ 5.63 mM and 21.04 mM, respectively, when Lineweaver--Burk and activity % vs. inhibitor concentration graphs were drawn for thiamine. The results of this article illustrate that the behaviors of vitamins could be complicated and vary from one living organism to other; in other words, they are unpredictable.

Immunological evidence for in\xa0vivo production of novel advanced glycation end-products from 1,5-anhydro-D-fructose, a glycogen metabolite

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

An L-fucose specific lectin from Aspergillus niger isolated from mycotic keratitis patient and its interaction with human pancreatic adenocarcinoma PANC-1 cells

An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30 kDa by SDS–PAGE and 31.6 kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50 °C and over a pH range of 7–10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125 μg for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25 μg/ml at 48 h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85 μg/ml at 48 h revealing promising clinical potential of ANL.

Isolation, Purification, and Characterization of Heparinase from Streptomyces variabilis MTCC 12266

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.

Trypsin inhibitor from duck albumen: Purification and characterization.

Egg albumen is a potential source of trypsin inhibitor (TI), which has been widely used to improve textural property of surimi or surimi-based food products. TI from duck albumen was isolated and purified using ammonium sulfate precipitation at 20%-40% saturation and affinity chromatography using trypsin-CNBr-activated Sepharose 4B column. TI was purified with purity and yield of 111.8-fold and 0.6%, respectively. The purity of inhibitor was confirmed using Native-PAGE as indicated by the presence of single band. Molecular weight of purified TI was 43kDa based on SDS-GAGE and gel filtration. The purified TI remained unchanged at temperatures below 60°C and the pH in the range of 7-9. The inhibitory activity of TI was decreased with the addition of salt higher than 5%. Inhibition kinetic study revealed that purified TI from duck albumen was uncompetitive inhibitor and the inhibition constant (Ki) was 508nM. TI from duck egg albumen could serve as a food grade inhibitor for controlling undesirable proteolysis. PRACTICAL APPLICATIONS: Duck egg albumen has been known to be rich in protease inhibitors, which can be used as a protein additive to enhance gelling properties of surimi or surimi-based products. Therefore, it is of interest to isolate and purify TI from duck egg albumen. Information regarding characteristics of TI from duck albumen could be beneficial for further applications, in which duck albumen is better exploited.

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples.

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line-derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking-in cancer cell-derived EVs to healthy plasma, it is shown that some cancer EV-associated proteins are detectable by nano-LC-MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC-MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient-derived clinical samples.