Epoxy-activated Sepharose 6B Research Articles

A Serum D-Fucose Binding Lectin With B Cell Mitogenic Activity From the Grub of the Darkling Beetle Zophobas morio.

Lectins that can recognize and bind to carbohydrates and glycoconjugates are at the epicentre of research owing to their prospective applications. In the present study, a D-fucose binding lectin from the serum of darkling beetle, Zophobas morio was purified and their mitogenic potential over human B-cells was evaluated. Biochemical assays on the preliminary characterization revealed the occurrence of single D-fucose binding lectin. Through single step affinity chromatography using D-fucose coupled Epoxy-activated Sepharose 6B, lectin (ZmFBL) with a molecular mass of ~192 kDa from the serum of Z. morio was purified with homogeneity. The HA activity of the purified ZmFBL remained stable between the pH 7 and 12 and was thermo-tolerant up to a temperature of 40°C. MALDI-TOF-MS analysis of native lectin disclosed fucose-binding nature of the ZmFBL with mitogenic property. The results of functional analysis of purified ZmFBL on the effect on B-cell proliferation revealed that ZmFBL at the concentrations of 31.25 μg and 62.50 were the ideal concentrations that significantly enhanced (approximately 2.5-fold over control) the proliferation of the B-lymphocyte population up to 72 h of treatment without any cytotoxicity. The outcome of this study could possibly prove beneficial in the investigation on the potential use of ZmFBL as immunostimulant and in immunosuppressive treatments.

Purification and characterization of thermostable α-amylase from germinating Sword bean (Canavalia gladiata (Jacq.) DC.) seeds.

The thermostable α-amylase from germinating sword bean (Canavalia gladiata (Jacq.) DC.) seeds (CgAmy) was successfully purified by a combination of ammonium sulphate fractionation and Epoxy-activated Sepharose 6B affinity chromatography. The purified α-amylase showed 507.8 fold with a specific activity of 750.0 U/mg. SDS-PAGE of the purified enzyme revealed a single protein band of 50.0 kDa. Purified enzyme was confirmed as α-amylase type by LC-MS/MS analysis and activity on specific substrate of ethylidene-pNP-G7. The CgAmy revealed extreme activity at a high temperature of 50.0-70.0°C with optimum activity at 70.0°C. The optimal pH of enzyme activity was observed at 6.0. The CgAmy exhibited stability in pH range of 5.0-8.0 and highly thermostable with a temperature of 40.0-60.0°C. The kinetic parameters K m for hydrolysis of starch were found to be 3.12 mg/ml. The α-amylase activity was enhanced in the presence of Co2+ and β-mercaptoethanol. While, Na2+, K2+, Ca2+, Mg2+, Zn2+, Ba2+, Fe2+ and Cd2+ slightly inhibited α-amylase activity. Interestingly, the CgAmy displayed stability towards some organic solvents and detergents. Stability at high temperature and some metal ions, organic solvents and detergents indicated that this enzyme has potential for various applications.

Corrigendum

Corrigendum

A Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacter sp. 5K

Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe Thermoanaerobacter sp. 5K was purified and characterized. The enzyme was purified with ammonium sulfate precipitation followed by α-CD-bound, epoxy-activated Sepharose 6B affinity chromatography. Molecular weight of the purified enzyme was 70.6 kDa. The enzyme had optimal activity at 80-90°C and retained greater than 90% activity between 75°C and 95°C. Optimal pH activity was observed at 7.0, with at least 50% activity between pH 4.0 and 9.0. It was highly stable at elevated temperature, with no loss of activity after incubation at 80°C for 4 hours or at 90°C for 30 min. Km and Vmax values were 0.222 mg/mL and 0.206 mg β-CD/mL/min, respectively, with soluble starch. Amino acid composition of the enzyme was deduced from the sequence of the cloned CGTase gene. The mature enzyme has a deduced molecular weight of 75.63 kDa and contains residues conserved in the CGTase class of amylase enzymes. Keywords: Anaerobe; cyclodextrin glycosyltransferase, cyclodextrin, Thermoanaerobacter, thermophilic extremozyme.

Purification and identification of trichloroethylene induced proteins from Stenotrophomonas maltophilia PM102 by immuno-affinity-chromatography and MALDI-TOF Mass spectrometry

A novel bacterial isolate capable of growth on trichloroethylene as the sole carbon source was identified as Stenotrophomonas maltophilia PM102 by 16S rDNA sequencing (GenBank Acc.no. JQ797560). Serum was obtained from a rabbit immunized with the total protein extracted from the PM102 isolate grown in 0.2% TCE with 0.2% peptone. Antibodies to the common antigens were removed by preadsorbing the serum antibody on total protein extracted from the PM102 strain grown in 0.2% peptone. Western blot with the preadsorbed antibody reacted to a single band in TCE and TCE with peptone lane. No reaction was seen in peptone lane. This preadsorbed antibody specific for TCE inducible antigens was immobilised on epoxy activated sepharose 6B and total protein from PM102 cells grown in minimal medium with TCE as the sole carbon source was purified through the column. The bound protein fraction was eluted and resolved through 12% SDS PAGE. Four prominent bands observed in the protein profile were analysed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MS) and tandem mass spectrometry (MS/MS) after in gel digestion with 25 ng/μl trypsin. A number of mono/di-oxygenases that cometabolise TCE in presence of some other primary carbon source are present in literature but this is the first attempt in identification of TCE induced proteins linked to metabolic activity with oxidoreductase like function, from a bacterial isolate that utilises TCE as the sole carbon source.

English

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from a thermophilic anaerobic bacterium, Thermoanaerobacter sp. P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin epoxy activated-sepharose 6B column chromatography. Enzyme was purified 141 fold and had the specific activity of 143.8 U/mg proteins. Purification yields after ammonium sulfate precipitation and affinity chromatography were 25.8 and 17.8%, respectively. SDS-PAGE analysis showed that enzyme was purified successfully and had a single band. Molecular weight of the enzyme was determined as 68.7 kDa. The enzyme had optimum cyclization activity at 80 to 90°C and hydrolyzing activity at 90°C and maintained 87 and 95% of these activities at 95°C, respectively. Optimal pH was found as 7.0. It retained full activity at 80°C for 4 h. Enzyme was strongly inhibited by HgSO 4 and AgNO 3 . Addition of 1 mM CaCl 2 increased the enzymatic activity up to 7%. This novel enzyme could be a good candidate for industrial applications according to its characteristic found in the current study. Key words: Cyclodextrin glycosyltransferase, cyclodextrin production, Thermoanaerobacter sp. P4, thermophilic, enzyme purification, enzyme characterization.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Grass Degrading β-1,3-1,4-d-glucanases from Bacillus subtilis GN156: Purification and Characterization of Glucanase J1 and pJ2 Possessing Extremely Acidic pI

Purification of beta-1,3-1,4-glucanase from the cell-free culture fluid of Bacillus subtilis GN156 by affinity chromatography of epoxy-activated sepharose 6B and ultrafiltration technique resulted in homogeneous J1 and partially purified pJ2 enzymes. The molecular weight and pI of J1 were 25 kDa and 3.5, respectively, while those for J2 were 90 kDa and 3.6, respectively. Both beta-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6-6.5 and an optimum temperature of 60 degrees C. Both enzymes were not inhibited by Li(2+) but were inhibited significantly by Ca(2+), Cu(2+), Mn(2+) and Zn(2+). However, J1 was slightly inhibited by Fe(2+), while pJ2 was inhibited by Mg(2+) as well. They were highly specific to only barley beta-glucan. K (m) and V (max) values of J1 were 1.53 mg/ml and 8,511 microU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 microU/ml.min, respectively. Degradation of barley beta-1, 3-1,4-glucan resulted in four different oligosaccharides with 1,3 linkages triose, tetrose, pentose and a high molecular weight (HMW) with 1,3 linkage estimated from their mobilities. The quantitative degradation by the crude enzyme after of incubation yielded in descending order: triose, pentose and tetrose, while that of J1 in descending order yielded: pentose, triose and tetrose. The pJ2 showed low activity yielding a degradation pattern in descending order: pentose, triose, tetraose and a HMW polysaccharide.

Development of an Acetylcholinesterase-Based Detection Kit for the Determination of Organophosphorus and Carbamate Pesticide Residues in Agricultural Samples

The objective of this study was to develop a rapid, simple, and qualitative acetylcholinesterase (AChE)-detection kit, based on a modification of the Ellman and ELISA methods, for the detection of organophosphorus (OP) and carbamate (CB) pesticide. The developed kits were used to screen a large number of agricultural samples (spiked and real) for OP and CB pesticide residues. AChE was extracted from the heads of honeybees (Apis mellifera L.) using Triton X-100, and was purified through 3 steps: diethylaminoethylcellulose chromatography (DEAE), affinity chromatography and membrane filtering, and Mono-Q column chromatography. Epoxy-activated Sepharose 6B affinity chromatography was used for large-scale purification. The presence of OP and CB pesticide residues in agricultural samples was assayed on the basis of AchE inhibition value. The presence (6 bands) or absence of some colored bands on the test line indicated a negative or positive result, respectively. The limits of detection for measured organophosphorus (OP) and carbamates (CB) pesticide residues in standard pesticide solutions and fortified samples were ranged from 0.50 to 2.50 ppm and 0.50 to 4.75 ppm, respectively.

Isolation, Cloning, and Characterization of a Novel Phosphomannan-binding Lectin from Porcine Serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

Affinity-Purified Human Immunoglobulin G That Binds a Lacto-N-Neotetraose-Dependent Lipooligosaccharide Structure Is Bactericidal for Serogroup BNeisseria meningitidis

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.

Immobilized Zinc Affinity Chromatography of Pectin Hydroxamic Acids for Purification of Trypsin Inhibitors from Soybean and Sweet Potato

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.

Monoacetylcurcumin: A new inhibitor of eukaryotic DNA polymerase λ and a new ligand for inhibitor-affinity chromatography

We previously reported that a phenolic compound, curcumin (diferuloylmethane), was a selective inhibitor of DNA polymerase λ (pol λ) in vitro [Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki, M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai, K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA polymerase λ-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935–1944.]. We also found that monoacetylcurcumin ([1 E,4 Z,6 E]-7-(4″-acetoxy-3″-methoxyphenyl)-5-hydroxy-1-(4′-hydroxy-3′-methoxyphenyl)hepta-1,4,6-trien-3-on), a chemically synthesized derivative of curcumin, was a stronger pol λ inhibitor than curcumin, achieving 50% inhibition at a concentration of 3.9 μM. Monoacetylcurcumin did not influence the activities of replicative pols such as α, δ, and ε, and showed no effect even on the activity of pol β, the three-dimensional structure of which is thought to be highly similar to that of pol λ. The compound-induced inhibition of pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Monoacetylcurcumin did not inhibit the activity of the C-terminal catalytic domain of pol λ including the pol β-like core, in which the BRCT motif was deleted. The compound did not influence the activities of prokaryotic pols or other mammalian DNA metabolizing enzymes such as calf primase of pol α, calf terminal deoxynucleotidyl transferase, human telomerase, human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. Therefore, we concluded that monoacetylcurcumin is a selective inhibitor of pol λ and could be used as a chromatographic ligand to purify pol λ. We then made a monoacetylcurcumin-conjugated column with epoxy-activated Sepharose 6B. In the column, pol λ of full length was selectively adsorbed and eluted.

Novel lectins from rhizomes of two Acorus species with mitogenic activity and inhibitory potential towards murine cancer cell lines

Two novel lectins were purified from rhizomes of two sweet flag species, namely Acorus calamus (Linn.) and Acorus gramineus (Solandin Ait.) by affinity chromatography on mannose linked epoxy-activated Sepharose 6B. The apparent molecular mass of the lectins, as determined by gel filtration chromatography, was 56 kDa for ACL and 55 kDa for AGL. In SDS–PAGE, pH 8.3, both lectins migrated with a subunit molecular mass of 13.6 kDa and 13.5 kDa, respectively, under reducing and non-reducing conditions thus indicating the absence of disulphide linkages. Acorus lectins readily agglutinated rabbit, rat and guinea pig erythrocytes. Both ACL and AGL also reacted with RBCs from sheep, goat and human ABO blood groups after neuraminidase treatment. ACL and AGL were inhibited by mannose/glucose and their derivatives. The most effective inhibitor was methyl-α- d-mannopyranoside. Acorus lectins were stable up to 55 °C, did not require metal ions for their activity and were also affected by high concentrations of denaturants like urea, thiourea and guanidine–HCl. These lectins showed potent mitogenic activity towards mouse splenocytes and human lymphocytes. Both ACL and AGL also significantly inhibited the growth of J774, a murine macrophage cancer cell-line and to lesser extent WEHI-279, a B-cell lymphoma.

Detection of specific IgE to quinolones

Rationale Immediate adverse reactions to quinolones (IARQ) like urticaria, angioedema and shock are observed with increasing frequency. Our aim was to assess if these reactions are IgE-mediated. Methods We studied 55 patients with IARQ. Sixty-two percent of the patients recalled a previous exposure to quinolones. Two control groups were tested: 22 atopic individuals without history of ADR and 10 individuals who tolerated quinolones. We did not perform skin tests because they induce false positive results. Because of the severity of the reactions no provocation test was performed. IgE to quinolones was tested by a RIA using epoxy-activated sepharose 6B as solid phase, to which quinolones (ciprofloxacin, cinoxacin, lomefloxacin, ofloxacin, pipemidic acid, pefloxacin, norfloxacin, nalidixic acid, rufloxacin) were covalently coupled. Results The test yielded positive results in 30 patients (54.5%), tested 1-48 months after the reaction, and negative in the controls. In atopic patients, the quinolone-specific IgE seems to persist longer. The test negative patients may have lost drug specific IgE with time, reacted with an epitope of the quinolone not detectable in the test system or had actually pseudoallergic reactions. Inhibition of the uptake by various quinolones, but not by non related molecules (amoxicillin, penicillin G, cefuroxim, rifampicin and 4-aminoantipirin) demonstrated the specificity of binding of IgE to quinolones. These inhibition studies also allowed to identify a common epitope, which might explain the broad serological and clinical cross-reactivity. Conclusions A substantial part of IARQ appear to be IgE-mediated. Cross-reactivity is frequent and suggests that all quinolones should be avoided in these patients.

Detection of specific IgE to quinolones

Background In the last years, immediate reactions to quinolone antibiotics have been observed with increasing frequency, mainly urticaria, angioedema, and shock. No test was available because of the high incidence of false-positive results on skin tests. Thus the pathogenesis, value of diagnostic procedures, and cross-reactivity have not been evaluated in a systematic way. Objective We sought to assess whether these reactions are IgE mediated and whether an in vitro test for quinolone-specific IgE is useful in the diagnosis and understanding of cross-reactivity. Methods We assayed specific serum IgE to quinolones using epoxy-activated sepharose 6B as the solid phase in 55 patients with immediate adverse reactions; specificity of IgE binding was demonstrated by inhibition tests. Results The test yielded positive results in 30 (54.5%) patients who were tested 1 to 48 months after the reaction had occurred. The quinolone-specific IgE seems to disappear more slowly in atopic patients. The cross-reactivity between various quinolones allowed us to identify a common structural motif within quinolones that might be responsible for clinical and serologic cross-reactivity. Conclusion A substantial portion of immediate reactions to quinolones appear to be IgE mediated. Cross-reactivity of IgE among different quinolones is frequent and suggests that a common avoidance of quinolones should be attempted in all patients with respective symptoms.

Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor

Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3- c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.

Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase α and a new carrier for inhibitor-affinity chromatography

Two flavonoid glycosides, kaempferol 3- O-(6″-acetyl)-β-glucopyranoside (KAG) and quercetin 3- O-(6″-acetyl)-β-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol α, δ, and ε), but not other pol β, η, κ, and λ activities. KAG was a stronger inhibitor and more selective to pol α than QAG. The IC 50 values of KAG for pol α, δ, and ε were 41, 164, and 127 μM, respectively. The pol α inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol α, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol α was selectively adsorbed and eluted.

Glutathione S‐transferase isoenzymes in the two‐spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae)

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch.

Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.

We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (2) combined use of two beta-lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.