Size-exclusion Fast Protein Liquid Chromatography Research Articles

Protein decomplexation and proteomics: A complementary assessment method of the physicochemical purity of antivenom

The quality of antivenom is governed by its safety and efficacy profiles. These quality characteristics are much influenced by the purity of antivenom content. Rigorous assessment and meticulous monitoring of antivenom purity at the preclinical setting is hence crucial. This study aimed to explore an integrative proteomic method to assess the physicochemical purity of four commercially available antivenoms in the region. The antivenoms were subjected to Superdex 200 HR 10/30 size-exclusion fast-protein liquid chromatography (SE-FPLC). The proteins in each fraction were trypsin-digested and analyzed by nano-ESI-liquid chromatography-tandem mass spectrometry (LC-MS/MS). SE-FPLC resolved the antivenom proteins into three major protein components of very high (>200 kDa), high (100–120 kDa) and medium (<60 kDa) molecular weights. The major components (80–95% of total proteins) in the antivenoms were proteins of 100–120 kDa consisting of mainly the light and partially digested heavy immunoglobulin chains, consistent with F(ab’)2 as the active principle of the antivenoms. However, LC-MS/MS also detected substantial quantity of large proteins (e.g. alpha-2-macroglobulins), immunoglobulin aggregates and impurities e.g. albumins in some products. The method is practical and able to unveil the quantitative and qualitative aspects of antivenom protein compositions. It is therefore a potentially useful preclinical assessment tool of antivenom purity.

Australian Scorpion Hormurus waigiensis Venom Fractions Show Broad Bioactivity Through Modulation of Bio-Impedance and Cytosolic Calcium.

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan

Objective:The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three majorThaisspecies (T. bufo,T. hippocastanumandT. rudolphi) from the Sindh Coast, Pakistan.Methods:Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA &amp; Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis.Results:Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the threeThais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmedviaSDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined.Conclusion:Our findings suggest thatThais sp.are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

Formation and characterization of self-assembled bovine serum albumin nanoparticles as chrysin delivery systems.

Chrysin (5,7-dihydroxyflavone) (Chrys) is a natural flavone extracted from many plants, and it has been proposed as a bioactive agent for cancer therapy. Nevertheless, its use is limited mainly due to its poor water solubility. Bovine serum albumin (BSA) is a water soluble, biocompatible and non-toxic protein with a promising application in lipophilic bioactive compound delivery. Moreover, BSA is heat sensitive, feature that could be used for producing self-assembled nanoparticle with tailor-made properties. In this contribution, we studied the formation of BSA nanoparticles (BSAnp) by thermal treatment at different conditions of temperature (70 °C/5 min and 85 °C/5 min), protein concentration (1.0-4.0%wt.) and aqueous medium pH values (9.0 and 11.0) in which it is known that BSA is found in different unfolded conformations. Binding of Chrys dissolved in dimethyl sulfoxide (DMSO) was studied by fluorescence titration experiments. Characterization of Chrys-loaded and unloaded BSAnp was performed in phosphate buffered saline (PBS) pH 7.4 by applying a set of complementary techniques: dynamic light scattering (DLS), size exclusion fast protein liquid chromatography (SEC-FPLC) and transmission electron microscopy (TEM). Different populations of BSAnp were obtained, which showed different diameters in the range of 1328 nm, ζ potentials around -10.0 mV, molecular weight in the range of 400-1000 kDa and spherical shape. Chrys encapsulation efficiency (EE. %) was also determined, and values between 44-84% were obtained, which mainly depended on the mode of Chrys binding and physicochemical BSAnp properties. Results highlight the ability of self-assembled BSAnp for Chrys vehiculization in an aqueous medium which could found potential application in antitumor therapies.

Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system.

The γ-aminobutyric acid (GABA) transporter 1 (GAT1) belongs to a family of Na+ and Cl−-coupled transport proteins and possesses 12 putative transmembrane domains. To perform structural analyses of the GAT1 protein, the GAT1/green fluorescent protein (GFP) fusion protein was functionally expressed in insect Sf9 cells by the BAC-TO-BAC® baculovirus expression system. A two-step procedure to purify the GAT1/GFP fusion protein from insect Sf9 cells has been established and involves immunoaffinity chromatography using self-prepared anti-GFP antibodies and size-exclusion fast protein liquid chromatography (SE-FPLC). A yield of 200–300 μg of the GAT1/GFP protein could be purified from 400–600 mL of infected Sf9 cells. The purified protein was analyzed by transmission electron microscopy (TEM), which revealed that the GAT1/GFP fusion protein was isolated in its monomeric form.

Fractionation of unreduced gluten proteins on SEC and their relationship with cookie quality characteristics.

The size distribution assessment of unreduced wheat gluten proteins of commercial Indian wheat varieties was examined using Size-Exclusion Fast Protein Liquid Chromatography. Elution profiles were fractionated into five peaks, with the molecular weights of eluting peaks as 130-30, 55-20, 28-10 and <10kDa (IV and V), respectively. Peak I (glutenins) and II (gliadins) exhibited contrary results with AWRC (r=0.928** and r=-0.831**), R/E ratio (r=0.745** and r=-0.869**), gluten index (r=0.959** and r=-0.994**), gliadin/glutenin ratio (r=-0.952** and r=0.948**), dough development time (r=0.830** and r=-0.930**), dough stability (r=0.901** and r=-0.979**) and dough weakening (r=-0.969** and r=0.986**). Significant statistical correlation was also observed for peak I and II with cookie hardness (r=0.948** and r=-0.924**) and cookie spread (r=-0.837** and r=-0.743**) respectively. Peak III, IV and V occupied a minor fraction of whole and did not exhibit a statistically significant correlation with any of the quality parameters.

Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP–HPLC Methodology

Uricase (Uc), a therapeutic enzyme, is widely used in its PEGylated form to treat hyperuricemia and is largely manufactured by means of random/first generation PEGylation approach. Currently available randomly PEGylated uricase conjugates exhibit inadequacies like reduced uricolytic activity, risk of inducing immunogenic reactions, lack of selectivity, and molecular heterogeneity. In the present study, site-specific/second generation PEGylation strategy involving modification of specific and rare amino acids by means of terminally functionalized PEG polymers was applied. Uricase was conjugated with methoxypolyethyelenglycol-maleimide (mPEG-mal) by means of thiol PEGylation to synthesize monoPEGylated uricase conjugates. For enhancing the yield of monoPEGylated uricase conjugates, response surface methodology was employed to determine the yield of monoPEGylated conjugates using reverse phase high performance liquid chromatography. Using the optimized conditions, the developed method was validated for the production of monoPEGylated uricase conjugates which were further purified by size exclusion fast protein liquid chromatography (SE-FPLC). The molecular weights of the purified conjugates were determined by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE). The optimum values of reaction conditions were determined as 1:12 concentration ratio of Uc to mPEG-mal, 2.76 kDa as mPEG-mal molecular weight and 3.55 mM EDTA concentration which resulted in a very high conjugate yield of 95.16 %. The conjugate synthesized using the optimized method retained a residual uricolytic activity of 84 % and a thiol group modification extent of 68.3 %. The PEGylation reaction was optimized using OVAT and statistical methods. Using the optimized conditions very high yield of conjugates were obtained and RP–HPLC method was used to quantify the PEGylated uricase.

Differential antioxidative and hypocholesterolemic responses to two fish protein hydrolysates (Sardina pilchardus and Boops boops) in cholesterol-fed rats

Purpose– The purpose of this study was to evaluate the antioxidant and hypocholesterolemic activities of sardine and bogue protein hydrolysates in cholesterol-fed rats.Design/methodology/approach– In total, 18 male Wistar rats (220 ± 10 g) fed 20 per cent casein, 1 per cent cholesterol and 0.5 per cent cholic acid were divided into three groups and received a daily gavage of 250 mg of sardine (SPH) or bogue (BPH) protein hydrolysates for 30 days. The third group, named control group (CG), received in the same conditions water. Lipoproteins were fractionated by size-exclusion fast protein liquid chromatography, and serum lipids, apolipoproteins and lipoproteins were assayed.Findings– In SPH and BPH groups, serum total cholesterol concentrations were −66 per cent lower than in CG. This corresponded to the decreased very low-density lipoprotein-C in the former groups. Moreover, BPH treatment reduced low-density lipoprotein-C compared with CG and SPH groups. Compared with CG, serum phospholipids were reduced by SPH and BPH. Furthermore, BPH increased significantly APOA4 and sphingomyelin but lowered phosphatidylcholine. In the latter group, serum lecithin cholesterol acyltransferase activity was +23 per cent higher, but with SPH, this activity was −35 per cent reduced compared with CG. Apolipoprotein A-I contents were similar in the three groups. Compared with CG, hydroperoxide and lipid peroxidation contents in serum and lipoprotein fractions were reduced by SPH and BPH. Compared with CG, serum superoxide dismutase and glutathione peroxidase activities were increased in the treated groups, particularly in the BPH group.Originality/value– These results suggest that sardine protein hydrolysates and particularly those of bogue could be a very useful natural compound to prevent hypercholesterolemia by both improving the lipid profile and modulating oxidative stress in cholesterol-fed rats.

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and 1H{15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

Surface modification of cowpea chlorotic mottle virus capsids via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and their adhesion behavior with HeLa cells

A copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was exploited for the surface modification of cowpea chlorotic mottle virus (CCMV). The exposed carboxyl residues of the CCMV capsids were modified with an alkyne and then further modified with an azide, using a triazole connection in the presence of CuSO4, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and a bathocuproin disulfonic acid disodium salt (BCDS). Fluorogenic coumarin was successfully grafted onto the CCMV capsids and monitored by fast protein liquid chromatography (FPLC) and UV-irradiated SDS-PAGE. An oligo-ethylene glycol (OEG) short chain and an Arg-Gly-Asp (RGD) peptide were also connected to the CCMV capsids via the CuAAC reaction. Size-exclusion FPLC, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analyses confirmed the modification and integrity of the viral capsids. Interestingly, OEG-CCMV displayed a unique phenomenon of connected bridges with the intact capsids crosslinked to each other. Coumarin-CCMV, OEG-CCMV, and RGD-CCMV were absorbed onto APTES slides for cell binding with HeLa cells. The opposite adhesion behavior of OEG-CCMV and RGD-CCMV indicated the inhibition effect of OEG and the promotion effect of RGD for cell attachment. This provides a generalized method for chemical modification of the surface of virus capsids with multivalent ligands, which demonstrates the potential applications in bioimaging, tissue engineering, and drug delivery.

A simple and sensitive method for lipoprotein and lipids profiles analysis of individual micro-liter scale serum samples

A simple and sensitive method to determine lipoprotein and lipids profiles in micro-liter scale individual serum sample is not presently available. Traditional lipoprotein separation techniques either by ultra-centrifugation or by liquid chromatography methods have their disadvantages in both lipoprotein separation and lipids component quantification. In this study we used small volume needing size-exclusion fast protein liquid chromatography to separate different lipoprotein subclasses in 50μL serum. And lipids contents, such as cholesterol, cholesterol ester and triacylglycerol, were measured by using two different fluorescence-based lipid detection methods. With this method, very low density lipoprotein, low density lipoprotein and high density lipoprotein could be easily separated, and follow-up lipid detection was completed by simple kinds of reactions. Serum lipoprotein and lipids profiling from C57BL/6 mice (n=5) and human (n=5) were analyzed. The elution profiles of five individuals were highly reproducible, and there were lipoprotein and lipids distribution variations between C57BL/6 mice and human beings. In conclusion, this method which combined small volume needing size-exclusion fast protein liquid chromatography and fluorescence-based lipids measurement, provided a simple, efficient, integrity and reproducible procedure for determining serum lipoprotein and lipids profiles in micro-liter scale levels. It becomes possible that determination of lipoprotein profiles and gaining information of lipids in different lipoproteins can be accomplished simultaneously.

On the Relevance of the Met-turn Methionine in Metzincins

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.

A superior drug carrier – aponeocarzinostatin in partially unfolded state fully protects the labile antitumor enediyne

BackgroundNeocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier.MethodsWe characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy.ResultsThe partially unfolded protein is in molten globule-like state, in which ~60% and ~20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state.ConclusionThese findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.

Tg-SwDI Transgenic Mice Exhibit Novel Alterations in AβPP Processing, Aβ Degradation, and Resilient Amyloid Angiopathy

Alzheimer's disease (AD) is characterized by the accumulation of extracellular insoluble amyloid, primarily derived from polymerized amyloid-beta (Abeta) peptides. We characterized the chemical composition of the Abeta peptides deposited in the brain parenchyma and cerebrovascular walls of triple transgenic Tg-SwDI mice that produce a rapid and profuse Abeta accumulation. The processing of the N- and C-terminal regions of mutant AbetaPP differs substantially from humans because the brain parenchyma accumulates numerous, diffuse, nonfibrillar plaques, whereas the thalamic microvessels harbor overwhelming amounts of compact, fibrillar, thioflavine-S- and apolipoprotein E-positive amyloid deposits. The abundant accretion of vascular amyloid, despite low AbetaPP transgene expression levels, suggests that inefficient Abeta proteolysis because of conformational changes and dimerization may be key pathogenic factors in this animal model. The disruption of amyloid plaque cores by immunotherapy is accompanied by increased perivascular deposition in both humans and transgenic mice. This analogous susceptibility and response to the disruption of amyloid deposits suggests that Tg-SwDI mice provide an excellent model in which to study the functional aftermath of immunotherapeutic interventions. These mice might also reveal new avenues to promote amyloidogenic AbetaPP processing and fundamental insights into the faulty degradation and clearance of Abeta in AD, pivotal issues in understanding AD pathophysiology and the assessment of new therapeutic agents.

Apolipoprotein B and triacylglycerol secretion in human triacylglycerol hydrolase transgenic mice

Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.

Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases

The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1-15) and with the majority (KLK4-15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs (i.e. the KLK "activome") are unknown, to a significant extent, but have been postulated to involve "activation cascades" with other KLKs and endopeptidases. To characterize more completely the KLK activome, we have expressed from Escherichia coli individual KLK propeptides fused to the amino terminus of a soluble carrier protein. The ability of 12 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross-activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system.

Identification of Ghrelin in Human Saliva: Production by the Salivary Glands and Potential Role in Proliferation of Oral Keratinocytes

We investigated whether ghrelin is present in human saliva, is produced by salivary glands, and physiologic consequences of these findings. Expression of ghrelin and specific receptor mRNA was determined by PCR. Proteins were identified by immunoblotting and size-exclusion fast protein liquid chromatography (FPLC) with consecutive RIA. Specific RIAs were used for quantification of salivary total and bioactive ghrelin. Distribution of ghrelin was investigated by immunohistochemistry in cryosections of the salivary glands. The effect of ghrelin on incorporation of 5-bromo-2'-deoxyuridine as a measure of cell proliferation was investigated in primary oral keratinocytes. Ghrelin is produced by the salivary glands. The hormone was identified in saliva and glands by immunoblotting and by FPLC fractionation of saliva. Immunohistochemistry demonstrated ghrelin distribution in the salivary glands. The receptor was also produced by the glands and by oral keratinocytes and was shown to be functional. Comparison of total ghrelin values for healthy individuals (body mass index, 18-27 kg/m2) showed significantly lower concentrations in saliva than in serum (P < 0.01). The correlation between both matrices was r2 = 0.56 (P < 0.001) with a negative correlation to body mass index (r2 = 0.314; P < 0.01). Bioactive acylated ghrelin was also present in saliva. Incubation of keratinocytes with ghrelin led to significantly increased cell proliferation (P < 0.001). This effect could be completely suppressed by co-incubation with NOX-B11 (50 nmol/L), a novel specific inhibitor of acylated ghrelin. Ghrelin in saliva is produced and released by salivary glands. The effect of ghrelin on oral cell proliferation adds to the pro-proliferative action of other salivary growth factors.

Isolation and Characterization of Copolymers of β-Lactoglobulin, α-Lactalbumin, κ-Casein, and αs1-Casein Generated by Pressurization and Thermal Treatment of Raw Milk

Raw skim milk was submitted to high pressure (300 to 600MPa) and temperature (4 to 70°C) treatments for 2 or 5min. The combined effects of pressure and temperature on milk proteins induced structural changes and polymer and copolymer formation characterized by anion-exchange and size-exclusion fast protein liquid chromatography and electrophoretic techniques. Approximately half of the β-lactoglobulin formed polymers, and the other half formed large copolymers, mainly with κ-casein, α-lactalbumin via intermolecular disulfide bond exchange, and αs1-casein via physicochemical interactions, in proportions of 1.0:0.7:0.3:0.1, respectively. Minor whey proteins (serum albumin, immunoglobulins, and lactoferrin) also participated in the formation of the copolymers but to a lesser extent. Two populations of the copolymers were found with apparent molecular masses ranging from 440 to 2000 kDa for the first and more than 2000 kDa for the second. On the contrary, for heated milks the aggregation kinetics obtained by combination of high pressure and thermal treatment were very fast, as no intermediates such as dimers and small size oligomers were observed after pressurization, whatever the temperature studied. Lactosylation of proteins as well as proteolysis were very limited. A β-casein amino-terminal peptide of 22 kDa was specifically recovered in milk samples treated under the more drastic conditions (500 MPa/55°C per 5min and 600 MPa/70°C per 5min) and might have been generated by neutral proteases such as elastase released from somatic cells present in milk. No casein was released from the micelle whatever the combination of high pressure and temperature studied.

Echinococcus granulosus coproantigens: chromatographic fractionation and characterization.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights > 670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at > 670 kDa corresponding to fraction F1 from a culture derived excretory-secretory (E-S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-beta-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Target cell-restricted and -enhanced apoptosis induction by a scFv:sTRAIL fusion protein with specificity for the pancarcinoma-associated antigen EGP2.

The apparent tumor selective apoptosis-inducing activity of recombinant soluble TNF-related apoptosis-inducing ligand (TRAIL) has aroused much interest for use in clinical application. However, to exploit fully its therapeutic potential, the characteristics of both the TRAIL receptor system and soluble TRAIL (sTRAIL) should be taken into account: first, the widespread expression of the various TRAIL receptors throughout the human body; second, the differential binding affinities and crosslinking requirements of the agonistic receptors TRAIL-R1 and TRAIL-R2; and third, the solution behavior of particular sTRAIL preparations. Therefore, we constructed a novel TRAIL fusion protein, designated scFvC54:sTRAIL, comprising the human scFv antibody fragment C54 genetically linked to the N-terminus of human sTRAIL. The scFvC54:sTRAIL fusion protein was designed to induce apoptosis by crosslinking of agonistic TRAIL receptors only after specific binding of scFvC54:sTRAIL to the abundantly expressed carcinoma-associated cell surface antigen EGP2 (alias EpCAM). Target antigen-restricted apoptosis induction was demonstrated for various EGP2-positive tumor cells and could be inhibited by an EGP2 competing antibody. Target antigen binding converted soluble scFvC54:sTRAIL into a membrane-bound form of TRAIL that was capable of signaling apoptosis not only through TRAIL-R1 but also through TRAIL-R2. Size-exclusion fast protein liquid chromatography (FPLC) indicated that scFvC54:sTRAIL was produced as stable and homogeneous trimers in the absence of detectable TRAIL aggregates. The favorable characteristics of the scFvC54:sTRAIL fusion protein potentially reduce the amount of sTRAIL required for antitumor activity and may be of value for the treatment of various human carcinomas.