Filtration Chromatography Research Articles

Manufacture and evaluation of a HER2-positive breast cancer immunotoxin 4D5Fv-PE25

BackgroundHuman epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo.ResultsThe 4D5Fv-PE25 was expressed in high-density Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC50) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation.Conclusionwe succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer.Graphical abstract

ISOLATION AND PURIFICATION OF ANTICANCER PROTEIN EXOTXIN A FROM Pseudomonas aeruginosa

Toxins produced from bacteria constitute promising antitumor agents in treating different cancer types. Exotoxin A from Pseudomonas aeruginosa is the highly toxic virulence component that bind to specific cell receptors. This study aimed to purify Exotoxin A from clinically isolated Pseudomonas aeruginosa. A total of 150 bacterial isolates were taken from clinical samples (burn and wounds) and were screened on selective media and identified as P. aeruginosa using biochemical analysis and molecular test by PCR amplification technique utilizing specific primer 16srRNA. Exotoxin A produced from P. aeruginosa were screened and purified by two steps that include gel filtration and ion-exchange chromatography. In this study, 68 isolates were characterized as P. aeruginosa, furthermore, real-time PCR proved that these isolates revealed 100% specificity, sensitivity and had positive amplified bands with a size of 249bp. The concentration of Exotoxin A extracted from P. aeruginosa using Trypticase soy broth was 18 Mg/ml. The percent of Purification and recovery for Exotoxin A was 21.4 %, 40% respectively. Clinical Isolates of Pseudomonas aeruginosa have the potential to produce Exotoxin A that is responsible for pathogenicity.

Characteristics of anti-Vibrio harveyi compounds produced by Bacillus spp. isolated from shrimp ponds

Abstract. Rusmana I, Isramilda, Akhdiya A. 2021. Characteristics of anti-Vibrio harveyi compounds produced by Bacillus spp. isolated from shrimp ponds. Biodiversitas 22: 4872-4879. Pathogenic Vibrio spp. such as Vibrio vulnificus, V. alginolyticus, V. fluvialis, V. anguillarum, and V. harveyi caused shrimp diseases. The application of bacterial probiotics can control the growth of pathogenic Vibrio in shrimp. Bacillus spp. can produce antimicrobial compounds that inhibit the growth of pathogenic Vibrio spp. Isolation of Bacillus from several pond water samples, pond sediment, and shrimp intestines was successfully collected 175 isolates of Bacillus spp. Three isolates, i.e., Ltw54, Lts36, dan Lts40 had a high inhibitory index and stability in inhibiting the growth of Vibrio harveyi. Verification assay showed that Bacillus sp. Lts40 had the highest antimicrobial activity. The competition assay showed that Bacillus sp Lts40 isolates inhibited the growth of V. harveyi up to 81,8%. The antimicrobial compound produced by Bacillus sp. Lts40 was stable at the pH range of 3-11 and remained stable after heating at 100ºC for 20 minutes. The purification results using the chromatographic filtration technique showed that the protein fraction with a molecular weight of 47,38 kDa effectively inhibited V. harveyi growth. Bacillus sp. Lts40 isolate has potential application as a probiotic agent in shrimp ponds to control the growth of V. harveyi that causes luminous vibriosis diseases and the antimicrobial substance is potentially to be developed and produced as an anti-V. harveyi product that can be applied in a shrimp hatchery.

Лектин, специфічний до N-ацетилнейрамінової кислоти, з червоної водорості Gracilaria canaliculata Sonder та його антибактеріальна активність

A new lectin from the marine red alga Gracilaria canaliculata (GCL) was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion exchange and filtration chromatography. Lectin gave a single band with molecular mass of 22,000 Da in both non-reducing and reducing SDS-PAGE conditions, indicating that GCL is a monomeric protein. The hemagglutination activities of GCL were stable over a wide range of pH from 3 to 10, temperature up 60 oC and not affected by either the presence of EDTA or addition of divalent cations. Lectin GCL had high affinity for N-acetylneuraminic acid through interacting with the acetamido group at equatorial C2 position of these sugar residues, suggesting that GCL is specific for N-acetylneuraminic acid. Furthermore, GCL inhibited the growth of human and shrimp pathogenic bacteria, Staphylococcus aureus and Vibrio alginolyticus, although it did not affect the growth of Escherichia coli, Enterobacter cloace, Vibrio parahaemolyticus and V. harveyi. The red alga G. canaliculata may promise to be a source of valuable lectins for application as antibacterial agents.

N-Acetylneuraminic Acid Specific Lectin and Antibacterial Activity from the Red Alga Gracilaria canaliculata Sonder

A new lectin from the marine red alga Gracilaria canaliculata (GCL) was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion exchange and filtration chromatography. Lectin gave a single band with molecular mass of 22,000 Da in both non-reducing and reducing SDS-PAGE conditions, indicating that GCL is a monomeric protein. The hemagglutination activities of GCL were stable over a wide range of pH from 3 to 10, the temperature rose by 60 °C and it was not affected by either the presence of EDTA or addition of divalent cations. Lectin GCL had high affinity for N-acetylneuraminic acid through interacting with the acetamido group at equatorial C2 position of these sugar residues, suggesting that GCL is specific for N-acetyl-neuraminic acid. Furthermore, GCL inhibited the growth of human and shrimp pathogenic bacteria, Staphylococcus aureus, and Vibrio alginolyticus, although it did not affect the growth of Escherichia coli, Enterobacter cloace, Vibrio parahaemolyticus, and V. harveyi. The red alga G. canaliculata may promise to be a source of valuable lectins for application as antibacterial agents.

β0-Thalassemia Intermedia

Three patients with a relatively mild form of β0-thalassemia who did not require regular blood transfusions are described. Globin synthesis was studied by gel filtration and urea-carboxymethylcellulose chromatography of stroma-free hemolysates prepared from peripheral blood and bone marrow cells labeled in vitro with 14C-leucine. γ/α Synthetic ratios in peripheral blood were in the same range as in patients with the severe clinical form of β0-thalassemia, while γ/α synthetic ratios in bone marrow cells were higher than in that group of patients. The size of the free α-chain pool measured in one case was smaller than in other patients with “classical” Cooley anemia. It is concluded that the severity of the clinical course in β0-thalassemia does not correlate with the imbalance in α versus γ chain synthesis in peripheral blood and is determined by the synthetic ratio in bone marrow cells, where the bulk of hemoglobin synthesis takes place.

Characterization of O-glycan binding lectin from the red alga Hydropuntia eucheumatoides

The red alga, Hydropuntia eucheumatoides is one of the algal genera from which agar is commercially extracted, and is the main source of agar in the world. The lectin HEL from the red alga H. eucheumatoides was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion exchange and filtration chromatography. Lectin gave a single band with molecular mass of 17,000 Da in both non-reducing and reducing SDS-PAGE conditions, therefore lectin exists in monomeric form. The hemagglutination activities of HEL were stable over a wide range of pH from 3 to 10, temperature up 60 oC and not affected by either the presence of EDTA or addition of divalent cations, indicating that lectin requires no metal for biological activity. The hemagglutination activities of HEL were not inhibited by monosaccharides and glycoproteins, D-glucose, D-mannose, D-galactose, D-xylose, N-acety-D-mannosamine, transferin, fetuin and yeast mannan, but strongly inhibited by monosaccharides containing acetamido groups at equatorial C2 position, such as N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetyl-neuraminic acid and glycoprotein porcine stomach mucin bearing O-glycans. Thus, lectin is specific for O-glycans and may recognize the sequences GalNAcαSer/Thr, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)GalNAc- and GluNAc(α1-4)Gal- under interacting with the acetamido groups at equatorial C2 position of the terminal sugar residues in oligosaccharide structures of O-glycans. The red alga H. eucheumatoides could promise to be a source of valuable lectins for application in biochemistry and biomedicine.

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease

The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

Enhanced Production of Shigella flexneri Polysaccharide from a Newly Devised Silicate Method as a Potential Vaccine Conjugate.

Conventional glycoconjugates are prepared with polysaccharides (PS) isolated from bacterial sources by fermentation technique. This approach has some major challenges like lower yield of PS, impurities and usage of hazardous chemicals. Reports on efficient and enhanced production of PS from Shigella flexneri is meager in literature. Hence, in the current study, three different types of media namely Yeast extract medium, Shigella sonnei-defined medium and synthetic medium were utilized for the culture of S. flexneri. Among the selected media it was recognized that the culture of S. flexneri harvested in synthetic media produced significant quantity of PS in less time when compared to the other two media. Different purification techniques such as phenol chloroform extraction, acid precipitation, detergent method, chromatographic purification and a novel silicate method were carried out to refine the harvested PS from impurities. It was observed that large impurities such as bacterial protein, debris and media components were eliminated significantly by using chromatographic and silicate methods. The final yield of purified PS was approximately 20-35% higher in silicate method which is reported for the first time in this study for purification of PS. Further, the characterization of the purified PS was done using high-performance liquid chromatography and high-performance anion-exchange chromatography with pulsed amperometric detection. Hence, the robust process developed in the present study using synthetic media and chromatographic filtration technique along with the novel silicate treatment produced significant quantities of PS from S. flexneri in reduced cost and time, which could be further conjugated to a suitable carrier to generate a potential Shigella conjugate antigen.

Optimization and partial purification of beta-galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue

β-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular β-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal β-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal β-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin’s presence, 80% of activity was preserved after 2 h. These results reveal a A. niger β-galactosidase obtained from residue with favorable characteristics for food industries.

Development of a Stable MGAT1- CHO Cell Line to Produce Clade C gp120 With Improved Binding to Broadly Neutralizing Antibodies.

The high rate of new HIV infections, particularly in Sub-Saharan Africa, emphasizes the need for a safe and effective vaccine to prevent acquired immunodeficiency syndrome (AIDS). To date, the only HIV vaccine trial that has exhibited protective efficacy in humans was the RV144 study completed in Thailand. The finding that protection correlated with antibodies to gp120 suggested that increasing the quality or magnitude of the antibody response that recognize gp120 might improve the modest yet significant protection (31.2%) achieved with this immunization regimen. However, the large-scale production of rgp120 suitable for clinical trials has been challenging due, in part, to low productivity and difficulties in purification. Moreover, the antigens that are currently available were produced largely by the same technology used in the early 1990s and fail to incorporate unique carbohydrates presented on HIV virions required for the binding of several major families of broadly neutralizing antibodies (bNAbs). Here we describe the development of a high-yielding CHO cell line expressing rgp120 from a clade C isolate (TZ97008), representative of the predominant circulating HIV subtype in Southern Africa and Southeast Asia. This cell line, produced using robotic selection, expresses high levels (1.2 g/L) of the TZ97008 rgp120 antigen that incorporates oligomannose glycans required for binding to multiple glycan dependent bNAbs. The resulting rgp120 displays a lower degree of net charge and glycoform heterogeneity as compared to rgp120s produced in normal CHO cells. This homogeneity in net charge facilitates purification by filtration and ion exchange chromatography methods, eliminating the need for expensive custom-made lectin, or immunoaffinity columns. The results described herein document the availability of a novel cell line for the large-scale production of clade C gp120 for clinical trials. Finally, the strategy used to produce a TZ97008 gp120 in the MGAT− CHO cell line can be applied to the production of other candidate HIV vaccines.

Screening, purification and characterization of lipase from Burkholderia pyrrocinia B1213.

A lipase producing strain B1213 isolated from soil was identified as Burkholderia pyrrocinia based on 16S rRNA gene and recA sequeence analysis, making this the first report on the presence of a lipase from B. pyrrocinia. Under an aqueous two-phase purification strategy, which included (ATPE)-ion-exchange chromatography (IEC)-gel and filtration chromatography (GFC), the specific activity of the 35-kDa lipase was determined to be 875.7 U/mg protein. The optimum pH and temperature of this lipase was pH 8.0 and 50°C, respectively. The lipase retained > 85% activity in isopropanol and acetone at 30°C for 10min but the activity was reduced to 10.6% in n-hexane. Mg2+, Al3+, Mn2+, and Fe3+ enhanced lipase activity at both 1mM and 5mM concentrations. p-NPP, a long-chain acyl group 4-NP ester, appeared to be a good substrate candidate.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Recent advances and perspectives on capture and concentration of label-free rare cells for biomedical science and engineering research

Abstract Capture and concentration of the rare cells from a heterogeneous population plays an important role in the development of disease diagnostics, therapeutics, and biomedical research/applications. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer therapy and regenerative medicine. Large-scale supply of the rare cells at high purity and viability is essential, especially on stem cells, cancer stem cells (CSCs), and circulating tumor cells (CTCs). Conventional capture and concentration methods providing efficient and high throughput isolations are introduced; however, most of them require well-defined biochemical markers and immunolabeling procedures which are complicated, uneconomical, and often unreliable. In contrast, the emerging label-free cell capture methods based on biophysical and biomechanical properties of the desired cell population are growing. In this review, we aim to describe the status of label-free rare cell capture and concentration techniques and to highlight exciting new approaches in this field. Label-free rare cell isolation methods using intrinsic biophysical properties such as filtration, size, electrical polarizability, dielectrophoresis, optical dielectrophoresis, and hydrodynamic chromatography are introduced. Moreover, the microfluidic systems adopted to precisely handle cells and interface with other tools have been described earlier; thus, new approaches dealing with merging label-free selection methods and microfluidic systems are merely summarized. Furthermore, the capture and concentration methods using biochemical and materials properties such as cell-materials interaction, spheroid formation, and stem cells/CSCs niche mimicking are highlighted particularly. Advancing cell isolation technique provides the opportunity to avoid the shortcoming of using biochemical labels and presents better flexibility for subsequent characterization, such as on site screening, high throughput screening, and personalized medicine.

Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.

Extraction and purification of alpha- hemolysin which producing by Staphylococcus aureus isolated from different Clinical samples

Four hindered seventy six clinical samples were collected from patients since June 2003 to May 2004 the number of Staphylococcus aureus isolated from the samples were 163 isolates as follows : urine 52 isolates , wound swab 38 isolates , ear swab 21 isolates , tonsillar swab 17 isolates. Boil swab 11 isolates and Nasal swab 24 isolates Alpha hemolysin of Staphylococcus aureus isolates was designated by hemolysin assay it was found that 42% ( 70 ) of isolates were alpha- hemolysin producers. The extraction of alpha- hemolysin from liquid media from producing isolates was done by cooling centrifugation and purified by ammonium sulphate precipitation and gel - filtration chromatography. Activities of purified alpha-hemolysin were detected, it had 2560 HU/ml the optimum temperature of alpha - hemolysin activity was 20 - 40 C and the optimum incubation period was 14hr. The optimum pH of alpha-hemolysin activity was 7. Cytotoxic activity of alpha - hemolysin against human leukocytes were determined and found the survival ratio of human leukocytes was decreased (38%) after treatment with 15 mg/ml for 30 min. while it lost after treatment with 30 mg/ml for 30 min. too.

Structural changes to starch after acid hydrolysis, debranching, autoclaving‐cooling cycles, and heat moisture treatment (HMT): A review

Starch is a carbohydrate polymer consisting of glucose monomers with alpha‐glycosidic bonds comprising amylose (linear chains) and amylopectin (linear and branched chains). Starch modification is generally carried out for two reasons. First, native starch has limited applications. Second, functional properties can be added to starch through the modification to produce resistant starch for use in functional foods. Resistant starch has been revealed to show several health benefits including increasing faecal bulk, lowering colonic pH, and reducing glycaemic response. Acid hydrolysis, debranching, autoclaving‐cooling cycles, and heat moisture treatment (HMT) are the common methods involved in resistant starch production. These modifications can lead to changes in the structure of starch related to its resistance to human digestion. Here, the structural changes observed by gel‐permeation/filtration chromatography (GPC/GFC), Fourier transform infra‐red (FTIR) spectroscopy, X‐ray diffractometry (XRD), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) are discussed.

Purification and characterization of α-amylase in Moroccan locust, Dociostaurus maroccanus Thunberg (Orthoptera: Acrididae) and its inhibition by inhibitors from Phaseolus vulgaris L.

An α-amylase with molecular weight of 73 kDa was purified from midgut of Dociostaurus maroccanus using ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The optimal pH and temperature were 6 and 45 °C, respectively. As calculated using Lineweaver–Burk plots, the Km was about 0.62 mM and the Vmax was 1.113 (μmol/min/mg protein). Mn2+, Hg+, Zn2+ and ethylene diamine tetraacetic acid (EDTA) decreased α-amylase activity of D. maroccanus, whereas the addition of K+, Na+, Fe2+, Ba+, Mg2+, Ca2+ and Co2+ increased enzyme activity. Alpha-amylase inhibitors (AI1, AI2) with molecular weights of 43 kDa and 29 kDa, respectively, were purified from the seeds of Phaseolus vulgaris and its inhibitory effect on purified α-amylase of D. maroccanus was investigated. These inhibitors inhibited the D. maroccanus gut α-amylase activity significantly.

Isolation and characterization of anti-inflammatory peptides derived from whey protein

The present study was conducted to isolate and characterize anti-inflammatory peptides from whey protein hydrolysates using alcalase. Nine subfractions were obtained after sequential purification by ultrafiltration, Sephadex G-25 gel (GE Healthcare, Uppsala, Sweden) filtration chromatography, and preparative HPLC. Among them, subfraction F4e showed the strongest inhibitory activity on interleukin-1β (IL-1β), cyclooxygenase-2, and tumor necrosis factor-α (TNF-α) mRNA expression in lipopolysaccharide-induced RAW 264.7 mouse macrophages. Eight peptides, including 2 new peptides—Asp-Tyr-Lys-Lys-Tyr (DYKKY) and Asp-Gln-Trp-Leu (DQWL)—were identified from subfractions F4c and F4e, respectively, using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Peptide DQWL showed the strongest inhibitory ability on IL-1β, cyclooxygenase-2, and TNF-α mRNA expression and production of IL-1β and TNF-α proteins at concentrations of 10 and 100μg/mL, respectively. Additionally, DQWL treatment significantly inhibited nuclear factor-κB activation by suppressing nuclear translocation of nuclear factor-κB p65 and blocking inhibitor κB kinase phosphorylation and inhibitor κB degradation together with p38 mitogen-activated protein kinase activation. Our study suggests that peptide DQWL has anti-inflammatory potential; further confirmation using an in vivo model is needed.

Extraction, purification and characterisation of dermatan sulphate from bovine collagen waste liquor

The aim of this study was to extract, purify and characterise glycosaminoglycans present in bovine collagen waste liquor. Processing of bovine hides supplies collagen and gelatine to food, cosmetic and pharmaceutical industries generating abundant and unutilised waste streams that contain bioactive polysaccharides known as glycosaminoglycans. Some of these molecules, like heparin and dermatan sulphate, have known anti-thrombotic and anti-coagulant activities and are prescribed therapeutics. The predominant glycosaminoglycan in bovine collagen waste liquor, dermatan sulphate, was extracted and purified to greater than 93% w/w using only serial filtration and anion exchange chromatography. Bioactivity was measured using an in vitro assay revealing anti-thrombotic activity equivalent to commercial dermatan sulphate sourced from porcine mucosa. Fluorophore-assisted carbohydrate electrophoresis revealed a composition rich in the predominant mammalian disaccharide, uronic acid→N-acetyl-D-galactosamine-4-O-sulphate. Depolymerization and fractionation enriched for low molecular weight fragments with significantly improved anti-thrombotic activity. This study identifies an alternative source of anti-thrombotic dermatan sulphate that can be easily extracted without proteolysis or solvent precipitation that can be further processed into low molecular weight fractions with improved anti-thrombotic activity.