Thermostable Pyrococcus woesei beta-D-galactosidase--high level expression, purification and biochemical properties

The gene encoding beta-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei beta-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei beta-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type beta-D-galactosidase (51100 U/mg). The activity of P. woesei beta-D-galactosidase was enhanced by thiol compounds, Mg(2+) ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca(2+) ions had no effect.     

Purified human liver acid beta-D-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide.

Our studies with purified human liver acid beta-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl beta-D-galactosidase and G(M1)-ganglioside beta-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincident elution from Sepharose 6B of all three enzyme activities.     

Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.     

Immobilized 4-aminophenyl 1-thio-beta-D-galactofuranoside as a matrix for affinity purification of an exo-beta-D-galactofuranosidase

An alternative and fast method for the purification of an exo-beta-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-beta-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM D-galactono-1,4-lactone in a 100-500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-beta-D-galactofuranosidase was ascertained through binding to Concanavalin A-Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 mumol h-1 microgram-1 respectively, when 4-nitrophenyl beta-D-galactofuranoside was employed as the substrate.     

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells

An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D- galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.     

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.     

A bireactant, irreversible, active-site-directed inhibitor of beta-D-galactosidase (Escherichia coli). Synthesis and properties of (1/2,5,6)-2-(3-azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol

(1/2,5,6)-2-(3-Azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol (1) was synthesized and was found to irreversibly inactivate beta-D-galactosidase (Escherichia coli). The inactivation was prevented by the presence of isopropyl 1-thio-beta-D-galactopyranoside (IPTG). The vinyloxirane group of 1 reacted with water and other nucleophiles, especially at higher pH values. Reaction of 1 with beta-D-galactosidase was slow enough so that a competitive-inhibition constant (Ki) of 29mM could be determined. The inhibition constant for (1,2/3,6)-6-(3-azibutylthio)-2-bromo-4-cyclohexene-1,3-diol (2), the precursor of the bireactant inhibitor 1, was 13 mM, while that of (1,3/2,4)-3-(3-azibutylthio)-5-cyclohexene-1,2,4-triol (3), the product formed when the reactant is allowed to react with water, was 23mM. After irradiation by light, beta-D-galactosidase that had initially been treated with the bireactant compound and then digested with trypsin, showed a new pattern of elution from h.p.l.c., indicating that there was reaction at two regions of the beta-D-galactosidase molecule.     

Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma

The major proteins of bovine seminal plasma, BSP-A1, BSP-A2, BSP-A3, and BSP-30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p-aminophenyl phosphorylcholine-Agarose (PPC-Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP-like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n-butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN₃), dialyzed against the same buffer, and applied to a PPC-Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS-PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti-BSP antibodies. Anti-BSP cross-reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane. © 1993 Wiley-Liss, Inc.     

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.     

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists

The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core structure was a key feature for good receptor binding activity. SAR study of the 6-(4-aminophenyl) group suggests that hydrophobic substituents were preferred. The best compound from this series had binding affinity (K(i)) of 0.4 nM to the human GnRH receptor.     

Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma

To replace conventional affinity ligand like protein A or protein G, a pseudobioaffinity ligand seems to be an alternative for the purification of immunoglobulin G (IgG). In this study, sulfamethazine (SMZ) was chosen as novel affinity ligand for investigating its affinity to human IgG. Monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) (PGMA) beads were employed as the support for high-performance affinity chromatography. SMZ was immobilized on PGMA beads using bisoxirane (ethanediol diglycigyl ether) as spacer. The resultant affinity media presented minimal non-specific interaction with other proteins. Results of high-performance frontal analysis indicated that the media showed specific affinity to human IgG with a dissociation constant on the order of 10(-6) M. The SMZ affinity column proved useful for a very convenient one-step purification of IgG from human plasma. Antibody purity after a one-step purification was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction under reducing condition. The results obtained indicate that SMZ is a valuable affinity ligand for purification of human IgG.     

A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaacetic acid and their coordination chemistry with yttrium(III)

The bifunctional chelating agents N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)methyl]-diethylenetriamine and N,N,N',N',N'-pentakis(carboxymethyl)-1-[(4-aminophenyl)methyl]-4- methyldiethylenetriamine were prepared in six-step syntheses in overall yields of 38% and 31%, respectively. The use of bromoacetate esters in the synthesis allowed large-scale flash chromatographic purification of reaction products. The synthesis of N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)-methyl]-4-methyldiethylenetriamine resulted in a mixture of two diastereomers. Chelation of yttrium-(III) with these bifunctional chelating agents resulted in 1:1 chelates. In the case of N,N,N',N',N'-pentakis(carboxymethyl)-1-[4- aminophenyl)methyl]diethylenetriamine, two diastereomers were observed upon chelation, as expected. In the case of N,N,N',N',N'- pentakis(carboxymethyl)-1-[(4-aminophenyl)-methyl]-4- methyldiethylenetriamine, only three of the four anticipated diastereomers were observed.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

An enzyme-linked immunosorbent assay (ELISA) to determine the specificity of the sugar-binding protein NgcE, a component of the ABC transporter for N-acetylglucosamine in Streptomyces olivaceoviridis

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.     

Properties and kinetics of a neutral beta-galactosidase from rabbit kidney

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.     

Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.     

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.