Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Hb S-Sao Paulo: A new sickling hemoglobin with stable polymers and decreased oxygen affinity

Hb S-Sao Paulo (SP) [HBB:c.20A > T p.Glu6Val: c.196A > G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations. (C) 2012 Elsevier Inc. All rights reserved.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50 mu M of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circulardichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized. (C) 2012 Elsevier Inc. All rights reserved.

A novel xylan degrading beta-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular beta-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. beta-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 A degrees C and 3.0-5.5, respectively. beta-xylosidase was stable in acidic pH (3.0-6.0) and 70 A degrees C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The beta-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-beta-d-xylopyranoside, exhibiting apparent K-m and V-max values of 0.66 mM and 39 U (mg protein)(-1) respectively, and to a lesser extent p-nitrophenyl-beta-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel beta-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by beta-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring

The affinity of the d-galactose-binding lectin from Artocarpus heterophyllus lectin, known as jacalin, with immonuglobulins (Igs) was determined by biofunctionalization of a piezoelectric transducer. This piezoelectric biofunctionalized transducer was used as a mass-sensitive analytical tool, allowing the real-time binding analysis of jacalin-human immunoglobulin A1 (IgA(1)) and jacalin-bovine IgG(1) interactions from which the apparent affinity constant was calculated. The strategy was centered in immobilizing jacalin on the gold electrode's surface of the piezoelectric crystal resonator using appropriate procedures based on self-assembling of 11-mercaptoundecanoic acid and 2-mercaptoethanol thiol's mixture, a particular immobilization strategy by which it was possible to avoid cross-interaction between the proteins over electrode's surface. The apparent affinity constants obtained between jacalin-human IgA(1) and jacalin-bovine IgG(1) differed by 1 order of magnitude [(8.0 +/- 0.9) x 10(5) vs (8.3 +/- 0.1) x 10(6) L mol(-1)]. On the other hand, the difference found between human IgA(1) and human IgA(2) interaction with jacalin, eight times higher for IgA(1), was attributed to the presence of O-linked glycans in the IgA(1) hinge region, which is absent in IgA(2). Specific interaction of jacalin with O-glycans, proved to be present in the human IgA(1) and hypothetically present in bovine IgG(1) structures, is discussed as responsible for the obtained affinity values.

A fragment-based approach for ligand binding affinity and selectivity for the liver X receptor beta

Selective modulation of liver X receptor beta (LXR beta) has been recognized as an important approach to prevent or reverse the atherosclerotic process. In the present work, we have developed robust conformation-independent fragment-based quantitative structure-activity and structure-selectivity relationship models for a series of quinolines and cinnolines as potent modulators of the two LXR sub-types. The generated models were then used to predict the potency of an external test set and the predicted values were in good agreement with the experimental results, indicating the potential of the models for untested compounds. The final 2D molecular recognition patterns obtained were integrated to 3D structure-based molecular modeling studies to provide useful insights into the chemical and structural determinants for increased LXR beta binding affinity and selectivity. (C) 2011 Elsevier Inc. All rights reserved.

Purification of bromelain from pineapple wastes by ethanol precipitation

Bromelain is an aqueous extract of pineapple that contains a complex mixture of proteases and non-protease components. These enzymes perform an important role in proteolytic modulation of the cellular matrix in numerous physiologic processes, including anti-inflammatory, anti-thrombotic and fibrinolytic functions. Due to the scale of global production of pineapple (Ananas comosus L.), and the high percentage of waste generated in their cultivation and processing, several studies have been conducted on the recovery of bromelain. The aim of this study was to purify bromelain from pineapple wastes using an easy-to-scale-up process of precipitation by ethanol. The results showed that bromelain was recovered by using ethanol at concentrations of 30% and 70%, in which a purification factor of 2.28 fold was achieved, and yielded more than 98% of the total enzymatic activity. This enzyme proved to be susceptible to denaturation after the lyophilization process. However, by using 10% (w/v) glucose as a cryoprotector, it was possible to preserve 90% of the original enzymatic activity. The efficiency of the purification process was confirmed by SDS-PAGE, and native-PAGE electrophoresis, fluorimetry, circular dichroism and FTIR analyzes, showing that this method could be used to obtain highly purified and structurally stable bromelain. (C) 2012 Elsevier B.V. All rights reserved.

A comparative study of the purification of betanin

Betanin is a natural pigment with antioxidant properties used as a food colourant. This work describes the spectrophotometric and chromatographic quantification of betanin (2S/15S) and its epimer isobetanin (2S/15R) in fresh beetroot juice, food-grade beetroot powder and betanin standard diluted in dextrin. Absorption spectra of all three samples were deconvoluted using a mixed three-function model. Food-grade beetroot powder has the largest amount of violet-red impurities, probably formed during processing. The purification of betanin from these complex matrices was carried out by seven different methods. Ion exchange chromatography was the most efficient method for the purification of betanin from all samples; however, fractions contain high amounts of salt. Reversed-phase HPLC as well as reversed-phase column chromatography also produced good results at a much faster rate. The longer retention time of isobetanin when compared to betanin in reversed-phase conditions has been investigated by means of quantum-mechanical methods. (C) 2011 Elsevier Ltd. All rights reserved.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

Abstract Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Abstract Background Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.