Size Exclusion Chromatography HPLC Research Articles

Polymorphism of G-quadruplexes formed by short oligonucleotides containing a 3′-3′ inversion of polarity: From G:C:G:C tetrads to π–π stacked G-wires

G-wires are supramolecular DNA structures based on the G-quadruplex (G4) structural motif obtained by the self-assembly of interlocked slipped G-rich oligonucleotide (ON) strands, or by end-to-end stacking of G4 units. Despite the increasing interest towards G-wires due to their potential applications in DNA nanotechnologies, the self-assembly process to obtain G-wires having a predefined length and stability is still neither completely understood nor controlled. In our previous studies, we demonstrated that the d(5′CG2-3′-3′-G2C5′) ON, characterized by the presence of a 3′-3′-inversion of polarity site self-assembles into a G-wire structure when annealed in the presence of K+ ions. Herein, by using CD, PAGE, HPLC size exclusion chromatography, and NMR investigations we studied the propensity of shorter analogues having sequences 5′CGn-3′-3′-GmC5′ (with n = 1 and 1 ≤ m ≤ 3) to form the corresponding G-quadruplexes and stacked G-wires. The results revealed that the formation of G-wires starting from d(5′CGn-3′-3′-GmC5′) ONs is possible only for the sequences having n and m > 1 in which both guanosines flanking the 5′-ending cytosines are not involved into the 3′-3′ phosphodiester bond.

National Control Laboratory Assessment of Quality of Rituximab Biosimilars in India.

In past few years many rituximab (RTX) biosimilars have been launched in India. Biosimilars are products that are similar in terms of quality, safety, and efficacy to its innovator product and are expected to offer improved affordability. The less clinical examination is a significant source of reduction in the cost of development of a biosimilar. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. Therefore, the role of National Control Laboratory become very important to ensure the quality of these drugs by carrying out analytical characterization at the point of drug product release level as when referred by National Regulatory Authority for quality evaluation. To assess the similarity between innovator and biosimilars, different physicochemical and biological quality attributes were assessed. A multitude of state-of-the-art analysis of N = 3 RTX biosimilars marketed in India revealed that the impurity profiles of these biosimilars measured by charge variant analysis (cation exchange chromatography-high performance liquid chromatography [HPLC], capillary zone electrophoresis, and capillary isoelectric focusing), aggregates profiling (size exclusion chromatography-HPLC), fragments analysis (capillary electrophoresis-sodium dodecyl sulfate) were found to be significantly varying as compared with the innovator product. There were significant variations in acidic variants (p = 0.023) and basic variants (p = 0.0005), isoelectric point value (p < 0.0001), aggregates (p = 0.0231), and fragments (p < 0.0001) of biosimilars were found as that of innovator product. However, these differences were not affecting the biological activity in the cell-based potency analysis by complement-dependent cytotoxicity (CDC) assay (p = 0.1026), antibody-dependent cell-mediated cytotoxicity (ADCC) (p = 0.3736), and binding assay by flow cytometer fluorescence-activated cell sorting (p = 0.4005) of these biosimilars as compared with the innovator product.

Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA

i-Motifs, also known as i-tetraplexes, are secondary structures of DNA occurring in cytosine-rich oligonucleotides (CROs) that recall increasing interest in the scientific community for their relevance in various biological processes and DNA nanotechnology. This study reports the design of new structurally modified CROs, named Double-Ended-Linker-CROs (DEL-CROs), capable of forming stable i-motif structures. Here, two C-rich strands having sequences d(AC4A) and d(C6) have been attached, in a parallel fashion, to the two linker's edges by their 3′ or 5′ ends. The resulting DEL-CROs have been investigated for their capability to form i-motif structures by circular dichroism, poly-acrylamide gel electrophoresis, HPLC–size-exclusion chromatography, and NMR studies. This investigation established that DEL-CROs could form more stable i-motif structures than the corresponding unmodified CROs. In particular, the i-motif formed by DEL-5′-d(C6)2 resulted stable enough to be detected even at near physiological conditions (37 °C, pH 7.0). The results open the way to developing pH-switchable nanocarriers and aptamers based on suitably functionalized DEL-CROs.

Physical characteristics comparison between maytansinoid-based and auristatin-based antibody-drug conjugates.

Aim:Direct analytical comparison of two major drug-linkers in the antibody-drug conjugate (ADC) field was conducted.Methods:Four different analytical methods [AlogP calculation, reverse phase (RP) high-performance liquid chromatography (HPLC; RP-HPLC), size exclusion chromatography HPLC (SEC-HPLC), and differential scanning calorimetry (DSC)] were tested for this comparison.Results:Maytansinoid-based ADCs showed less hydrophobicity than auristatin-based ADCs. Regardless of the drug-linker and drug-to-antibody ratios (DARs), the stability detected by DSC was decreased by conjugation.Conclusions:The cost and time-efficient analytical comparison described in this manuscript may be useful information for an initial characterization of ADCs prior to detailed biological studies.

Inhibitory Binding of Angiotensin Converting Enzyme Inhibitors with Carbonic Anhydrase III

Both angiotensin converting enzyme (ACE) and carbonic anhydrase III (CAIII) are zinc-containing enzymes. Interestingly, blocking of the both enzymes is attributed with clinically significant outcomes against hyperlipidemia and obesity. An optimized in-vitro screening approach based on HPLC–size exclusion chromatography was adopted to study the angiotensin converting enzyme inhibitors (ACEIs) affinity against CAIII enzyme. Series of concentrations of the enzyme were injected in the column with a mobile phase containing one of ACEIs in each time. The affinity of the ACEIs toward CAIII was characterized by vacancy (negative) peak whose intensity representing the fraction of the drug bound with CAIII. To explore whether the binding is within the binding site or just a promiscuous binding, exact procedure was repeated with apo-protein part of CAIII. Furthermore, an esterase activity of CAIII was performed to determine if the binding with ACEIs is excitatory or inhibitory. It has been found that ACEIs have real in-vitro inhibitory effects against CAIII at a micro-molar level. The chromatographic study revealed that the ionized carboxylate groups are essential for binding with Zn+2 ion in the enzyme active site. Among the four tested ACEIs, captopril, ramipril, enalapril, and lisinopril, captopril was found to be the most potent inhibitor with Ki = 12.1 μM, while lisinopril is the least potent one with Ki = 25.3 μM. This finding might open the door for further investigation to optimize ACEIs as lead compounds for the discovery and development of selective and potent CAIII inhibitors.

Radical Depolymerization of Alginate Extracted from Moroccan Brown Seaweed Bifurcaria bifurcata

The degradation of alginate extracted from Moroccan Bifurcaria bifurcata has not been fully established to date. In this work, we report the extraction and the characterization of alginate (ASBB) from the brown algae B. bifurcata, as well as the production of oligo-alginates (OGABs) by using a green chemistry process. The depolymerization of ASBB was carried out by controlled radical hydrolysis through our green chemistry process using a hydrogen peroxide (H2O2) catalyst. The molecular weight (Mw) and degree of polymerization (DP) distribution of oligo-alginates (OGABs) obtained were then characterized by HPLC size exclusion chromatography (SEC) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Structural characterization revealed that after 6 h of depolymerization of ASBB, we obtained OGABs with Mw ≤ 5.5 kDa and 2 ≤ DP ≤ 24. These results highlight the effectiveness of the controlled radical hydrolysis of alginate to produce good yields of alginate fractions with controlled Mw with a known polymerization degree (DP) and without altering properties of oligo-alginates. Bifurcaria bifurcata can be a potential source of alginate and oligo-alginates given its abundance on the northwest Atlantic coast. The production and characterization of oligo-alginates promote their exploitation in the cosmetic, pharmaceutic, and agriculture fields.

New G-Quadruplex-Forming Oligodeoxynucleotides Incorporating a Bifunctional Double-Ended Linker (DEL): Effects of DEL Size and ODNs Orientation on the Topology, Stability, and Molecularity of DEL-G-Quadruplexes.

G-quadruplexes (G4s) are unusual secondary structures of DNA occurring in guanosine-rich oligodeoxynucleotide (ODN) strands that are extensively studied for their relevance to the biological processes in which they are involved. In this study, we report the synthesis of a new kind of G4-forming molecule named double-ended-linker ODN (DEL-ODN), in which two TG4T strands are attached to the two ends of symmetric, non-nucleotide linkers. Four DEL-ODNs differing for the incorporation of either a short or long linker and the directionality of the TG4T strands were synthesized, and their ability to form G4 structures and/or multimeric species was investigated by PAGE, HPLC–size-exclusion chromatography (HPLC–SEC), circular dichroism (CD), and NMR studies in comparison with the previously reported monomeric tetra-ended-linker (TEL) analogues and with the corresponding tetramolecular species (TG4T)4. The structural characterization of DEL-ODNs confirmed the formation of stable, bimolecular DEL-G4s for all DEL-ODNs, as well as of additional DEL-G4 multimers with higher molecular weights, thus suggesting a way towards the obtainment of thermally stable DNA nanostructures based on reticulated DEL-G4s.

A Generic Workflow for Bioprocess Analytical Data: Screening Alignment Techniques and Analyzing their Effects on Multivariate Modeling

UV chromatographic data in combination with multivariate data analysis (MVDA) has been extensively used for bioprocess monitoring. However, they are usually attributed to shifts along the retention time and require preprocessing. Misaligned UV chromatographic data result in inconsistent MVDA models. Numerous preprocessing techniques are available, each varying in the number of meta-parameters to optimize, complexity and computational time. Therefore, we aimed at developing a generic workflow to screen for preprocessing techniques. We chose four datasets with increasing complexity containing UV chromatographic data from reverse-phase and size exclusion chromatography HPLC. We aligned all four datasets using three preprocessing techniques, namely icoshift, PAFFT and RAFFT algorithms. We chose several statistical tools to validate the performance of the preprocessing techniques and to screen for meta-parameters. We validated the performance of the preprocessing techniques in terms of data preservation, complexity and computational time, and identified the optimal ranges of meta-parameters for each dataset. Finally, we established principal component analysis (PCA) models to evaluate the chosen alignment technique. Summarizing, in this study a generic workflow has been developed to validate alignment of chromatographic data using statistical tools.

CHARACTERIZATION OF INULIN FROM BLACK SALSIFY (SCORZONERA HISPANICA L.) FOR FOOD AND PHARMACEUTICAL PURPOSES

Objective: The aim of the current study was to evaluate the black salsify (Scorzonera hispanica L.) as a potential source of inulin and to characterize the physicochemical properties of isolated polysaccharide.Methods: The carbohydrate content in its roots and leaves was analyzed by high-performance liquid chromatography with refractive index detection (HPLC-RID) method. Microwave-assisted extraction was performed for isolation of inulin from black salsify roots. The obtained polysaccharide was characterized by HPLC-RID method, HPLC size-exclusion chromatography, and Fourier transformed-infrared spectroscopy. Functional properties as swelling capacity, solubility, and water-holding and oil-holding capacities (OHCs) were also evaluated.Results: Black salsify (S. hispanica L.) roots were evaluated as a rich source of inulin (22% dw) and 1-kestose (6.25 g/100 g dw). The isolated inulin (yield 20%) was characterized with average degree of polymerization 17, with polydispersity index (1.04) that was near to medium-chained inulin. This polysaccharide showed better OHC than water-holding capacity, and it was characterized with swelling capacity 0.5 ml/g sample.Conclusion: For the first time, inulin was isolated from black salsify roots. The chemical characterization of inulin reveals the potential of this plant to be used as a valuable source of this polysaccharide for future application in food technology and pharmaceutical industry for dietary fibers, stabilizer, and coating agent.

Polyphenylsulfone (PPSU) for baby bottles: a comprehensive assessment on polymer-related non-intentionally added substances (NIAS)

ABSTRACTPolyphenylsulfone (PPSU) is a new material for the production of baby bottles. PPSU is a polyether plastic formally composed of bisphenol S (BPS) and 4,4ʹ-dihydroxybiphenyl (DHBP), which both have slight endocrine activities in in vitro tests. So far, little is known about the presence and the release of potentially hazardous substances from PPSU baby bottles. In this study, we present a three-step approach for the analysis of PPSU starting with polymer characterisation in terms of chemical structure, total oligomer content and hydrolytic stability. Second is the determination of extractables focussing on monomers, monomer derivatives, linear and cyclic oligomers below 1000 Da and residual solvent. Third is a risk assessment on migration-related substances in accordance to European Union plastics regulation no. 10/2011 based on triplicate consecutive migration experiments using official milk simulant 50% ethanol. We analysed five types of PPSU baby bottles from different brands as well as corresponding raw materials from different manufacturers by various analytical techniques (high-performance liquid chromatography (HPLC)-diode array detector /fluorescence detector/Corona/electrospray ionisation-MS, HPLC-size exclusion chromatography, gas chromatography-mass spectrometry (GC-MS), 1H-NMR). We found significant variations of PPSU materials from different producers with regard to polymer and oligomer chain end groups (methoxylation, chlorination), while total oligomer content below 1000 Da was similar (mean about 0.48%). BPS was not detected above 0.3 mg/kg polymer in any PPSU sample. Residual DHBP content ranged between 1.7 and 15.5 mg/kg polymer. The most common oligomer in all PPSU samples was the cyclic tetramer (about 1200 mg/kg polymer), which is the only cyclic compound below 1000 Da. Residual solvent, sulfolane, was determined to a maximum of 1300 mg/kg polymer. In migration tests, we detected exceedances of neither specific migration limits for listed substances nor of thresholds of toxicological concern for non-listed substances (monomer derivatives, oligomers). Based on our analytical results, no concerns exist regarding migration of polymer-related substances from PPSU baby bottles.

In vitro digestion comparison from fish and poultry by-product meals from simulated digestive process at different times of the Pacific Bluefin tuna, Thunnus orientalis

The objective of this study was to compare the hydrolysates of fish meal (FM) and poultry by-product meal (PBM) upon in vitro simulation of the digestion process of the Pacific Bluefin tuna, Thunnus orientalis. Two grams of crude protein from each meal were hydrolyzed in triplicate for 60min using pepsin followed by alkaline hydrolysis for 180min using a pancreatic crude extract. Aliquots were collected during the alkaline hydrolysis (0, 30, 60, 120 and 180min). Protein digestibility was estimated; aliquots collected at 0 and 180min of incubation were analyzed for amino acid composition; and peptide molecular size distribution from all aliquots was characterized using size exclusion chromatography-HPLC. Protein digestibility for FM was greater (P<0.01) compared to PBM with estimates of 68.4 and 57.2 (±0.87% SEM) for FM and PBM, respectively. In hydrolyzed precipitates, protein source did not affect (P≥0.29) molecular size distribution; however, time had an effect. From 0 to 180min incubation, estimates for peptides >150kDa decreased (P<0.05) from 0.53 to 0.23±0.070g, and estimates for peptides <6.5kDa increased (P<0.01) from 0.03 to 0.17±0.050g. In hydrolyzed supernatants, protein source did not affect (P≥0.31) molecular size distribution; however, time had an effect. From 0 to 180min incubation, peptides >150kDa decreased (P<0.01) from 0.87 to 0.38±0.059g, peptides 150–29kDa increased (P<0.01) from 0.12 to 0.17±0.016g, and peptides 12.4–6.5kDa increased (P<0.01) from 0.08 to 0.32±0.090g; furthermore peptides <6.5kDa increased (P<0.01) from 0.07 to 0.32±0.060g. Overall, protein digestibility was greater for FM compared to PBM; the generation of low molecular weight peptides increased towards the end of the in vitro incubation. There was no difference in the molecular weight distribution between hydrolysates of FM and PBM; however, based on analysis of the slope for the release of peptides during incubation, there was a tendency for slower production of peptides <6.5kDa from PBM. Statement of relevanceEvaluation of feed ingredients in aquaculture should include analysis for the metabolism through the gastrointestinal tract. We simulated the digestion of the Pacific Bluefin tuna to evaluate the degradation of fish and poultry by-product meals. Low molecular size peptides increased towards the end of the incubation period. Degradation patterns for fish and poultry by-product meals were similar.

Synthesis, optimization and structural characterization of a chitosan–glucose derivative obtained by the Maillard reaction

Chitosan (Chit) was submitted to the Maillard reaction (MR) by co-heating a solution with glucose (Glc). Different reaction conditions as temperature (40, 60 and 80°C), Glc concentration (0.5%, 1%, and 2%, w/v), and reaction time (72, 52 and 24h) were evaluated. Assessment of the reaction extent was monitored by measuring changes in UV absorbance, browning and fluorescence. Under the best conditions, 2% (w/v) of Chit, 2% (w/v) of Glc at 60°C and 32h of reaction time, a chitosan–glucose (Chit–Glc) derivative was purified and submitted to structural characterization to confirm its formation. Analysis of its molecular weight (MW) and the degree of substitution (DS) was carried out by HPLC–Size Exclusion Chromatography (SEC) and a colloid titration method, respectively. FT-IR and 1H NMR were also used to analyze the functional groups and evaluate the introduction of Glc into the Chit molecule. According to our objectives, the results obtained in this work allowed to better understand the key parameters influencing the MR with Chit as well as to confirm the successful introduction of Glc into the Chit molecule obtaining a Chit–Glc derivative with a DS of 64.76±4.40% and a MW of 210.37kDa.

Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks

Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed 3H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation.

Correlation of sensory bitterness in dairy protein hydrolysates: Comparison of prediction models built using sensory, chromatographic and electronic tongue data

Sensory evaluation can be problematic for ingredients with a bitter taste during research and development phase of new food products. In this study, 19 dairy protein hydrolysates (DPH) were analysed by an electronic tongue and their physicochemical characteristics, the data obtained from these methods were correlated with their bitterness intensity as scored by a trained sensory panel and each model was also assessed by its predictive capabilities. The physiochemical characteristics of the DPHs investigated were degree of hydrolysis (DH%), and data relating to peptide size and relative hydrophobicity from size exclusion chromatography (SEC) and reverse phase (RP) HPLC. Partial least square regression (PLS) was used to construct the prediction models. All PLS regressions had good correlations (0.78 to 0.93) with the strongest being the combination of data obtained from SEC and RP HPLC. However, the PLS with the strongest predictive power was based on the e-tongue which had the PLS regression with the lowest root mean predicted residual error sum of squares (PRESS) in the study. The results show that the PLS models constructed with the e-tongue and the combination of SEC and RP-HPLC has potential to be used for prediction of bitterness and thus reducing the reliance on sensory analysis in DPHs for future food research.

Stability of Monoclonal Antibodies at High-Concentration: Head-to-Head Comparison of the IgG1 and IgG4 Subclass

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [sizeexclusion chromatography (SEC)–HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC–HPLC and capillary isoelectric focusing). The impact of pH (4–9) and ionic strength (10 and 150mm) was investigated using highlyconcentrated (150mg/mL) mAb formulations. Lower conformational stability was identified for the IgG4 resulting in increased levels of soluble aggregates. The IgG1 was chemically less stable as compared with the IgG4, presumably because of the higher flexibility in the IgG1 hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:115–127, 2014

Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. CAs are present in many pathogenic species and are involved in the bicarbonate metabolism/biosynthetic reactions involving this ion. Ubiquity of these enzymes suggests a pivotal role in microbial virulence and pathogenicity. Porphyromonas gingivalis is an anaerobic bacterium, which colonizes the oral cavity, being involved in the pathogenesis of periodontitis, an inflammatory disease leading to tooth loss. Recently, we reported an anion inhibitory study on the γ-CA (denominated PgiCA) identified in the genome of this Gram-negative bacterium. In this paper we continue our research on PgiCA, and describe the biochemical characterization of the recombinant protein, its thermal stability, the oligomeric state and the enzyme kinetics. PgiCA is a polypeptide chain formed of 192 amino acids and displays an identity of 30–33% when compared with the prototypical γ-CAs, CAM or CAMH (from Methanosarcina thermophila) or CcmM (from Thermosynechococcus elongatus). A subunit molecular mass of 21 kDa was estimated by SDS-PAGE, while HPLC size exclusion chromatography under native conditions gave an estimated molecular mass of 65 kDa suggesting that the recombinant enzyme self-associate in a homotrimer, as all other γ-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other γ-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.

Effects of Divalent Metal Ions on the Chaperone Activity and Structure of Rat Lens H18G Mutant αB‐Crystallin

AbstractαB‐crystalin, a small heat shock protein and a component of α‐crystalin, is a molecular chaperone playing an important role in preventing the formation of cataracts. It has been reported that His18 is an important site for Cu2+ to bind with to form a stable metal complex and thus to enhance this chaperone‐like activity of human αB‐crystalin. In this work, we used site‐directed mutagenesis to clone and express H18G rat lens αB‐crystalin in order to investigate the role of His18 in chaperoning activity. We found that 1 mM of Cu2+, or Zn2+, rather than Mg2+, significantly enhanced the chaperone‐like activity of wild type αB‐crystalin. Whereas, it is Zn2+ and Mg2+, not Cu2+, that significantly reduced this activity of H18G αB‐crystalin. In the absence of cation, H18G showed better activity compared to the wild type αB‐crystalin. ANS fluorescence measurement showed there was no linear relationship between chaperone‐like activity and surface hydrophobicity, indicating that surface hydrophobicity is not a prerequisite for chaperone‐like activity. An HPLC size‐exclusion chromatography study showed that in the presence of metal ions, wild type αB‐crystalin tended to aggregate via dissociation and re‐association into a high molecular aggregate with a molecular weight higher than 1400 kDa and then precipitated, suggesting that the presence of metal ions is a factor leading to the formation of cataracts. Both the near and far UV‐CD spectra suggested that the wild type αB‐crystalin reflected more β‐sheet structural characteristics; whereas the H18G reflected more random coil characteristics. The H18G induced structural alterations as to develop more random coil characteristics and more micro‐environmental changes around the tryptophan residues. This work suggested that His18 may not be a crucial binding site for Cu2+, but rather that it may be an important binding site for Zn2+ in terms of chaperone‐like activity and the process of metal induced self‐aggregation is prerequisite for chaperone‐like activity to occur.

Partial characterization of an exopolysaccharide secreted by a marine bacterium,Vibrio neocaledonicussp. nov., from New Caledonia

Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS. Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA-DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC-size exclusion chromatography. This EPS exhibits a high N-acetyl-hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672×10(3) Da. These data are relevant for possible technological exploitation. We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer. This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.

Alsophinase, a new P-III metalloproteinase with α-fibrinogenolytic and hemorrhagic activity from the venom of the rear-fanged Puerto Rican Racer Alsophis portoricensis (Serpentes: Dipsadidae)

Metalloproteinases from snake venoms are often multi-domain enzymes involved in degradation of a variety of structural proteins. Hemorrhage and tissue necrosis are common manifestations of viperid envenomations in humans, largely due to the actions of prominent metalloproteinases, and envenomation by rear-fanged snakes may also cause hemorrhage. We purified the major metalloproteinase in Alsophis portoricensis (Puerto Rican Racer) venom through HPLC size exclusion and ion exchange chromatography. Named alsophinase, it is the first protein purified and characterized from the venom of Alsophis. Alsophinase is a single polypeptide chain protein, and based on mass, activity and complete inhibition by 1,10-phenanthroline, it is a class P-III snake venom member of the M12 ADAM family of metalloproteinases. Alsophinase has a molecular mass of 56.003kDa and an N-terminal sequence of QDTYLNAKKYIEFYLVVDNGMFxKYSxxFTV, with 67% sequence identity to a metalloproteinase isolated from venom of Philodryas olfersii (another rear-fanged species). Alsophinase rapidly catalyzed cleavage of only the Ala14–Leu15 bond of oxidized insulin B chain, had potent hemorrhagic activity in mice, and degraded only the α-subunit of human fibrinogen in vitro. Alsophinase is responsible for hemorrhagic and fibrinogenolytic activity of crude venom, and it may contribute to localized edema and ecchymosis associated with human envenomations by A. portoricensis. It may be more specific in peptide bond recognition than many well-characterized viperid P-III metalloproteinases, and it could have utility as a new protein fragmentation enzyme for mass spectrometry studies.

Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.