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1.
Bioorg Med Chem ; 9(11): 2943-53, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11597476

ABSTRACT

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.


Subject(s)
Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Disaccharides/pharmacology , Monosaccharides/pharmacology , Polystyrenes/chemistry , Animals , Binding Sites , Cell Adhesion/drug effects , Coated Materials, Biocompatible/metabolism , Diazomethane/chemistry , Disaccharides/chemistry , Disaccharides/metabolism , Electron Probe Microanalysis , Galactose/metabolism , Galactose/pharmacology , Hepatocytes/chemistry , Hepatocytes/drug effects , Hepatocytes/enzymology , Lactose/metabolism , Lactose/pharmacology , Lectins/metabolism , Monosaccharides/chemistry , Monosaccharides/metabolism , Rats , Sialyltransferases/metabolism , Spectrometry, Mass, Secondary Ion , Surface Properties
2.
Bioorg Med Chem Lett ; 11(18): 2503-6, 2001 Sep 17.
Article in English | MEDLINE | ID: mdl-11549456

ABSTRACT

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.


Subject(s)
Biochemistry/methods , Enzymes/chemistry , Epitopes/chemistry , Trisaccharides/chemical synthesis , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Galactosyltransferases/chemistry , Glucosyltransferases/chemistry , Molecular Sequence Data , N-Acetyllactosamine Synthase/chemistry , Polyethylene Glycols/chemistry , UDPglucose 4-Epimerase/chemistry
3.
Eur J Biochem ; 268(15): 4352-8, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11488931

ABSTRACT

beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1. The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.


Subject(s)
Amino Acids/chemistry , Galactosyltransferases/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Cattle , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glycosylation , Humans , Kinetics , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Plasmids/metabolism , Protein Isoforms , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Solubility
4.
J Biol Chem ; 276(24): 21608-17, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11279145

ABSTRACT

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.


Subject(s)
Sialyltransferases/chemistry , Sialyltransferases/metabolism , Animals , Binding Sites , CHO Cells , Catalytic Domain , Cloning, Molecular , Cricetinae , Cytidine Monophosphate N-Acetylneuraminic Acid/metabolism , Genetic Variation , Humans , Kinetics , Orosomucoid/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , Sialyltransferases/genetics , Substrate Specificity , Thyrotropin/metabolism , Transfection , Transferrin/metabolism , alpha-Fetoproteins/metabolism , beta-D-Galactoside alpha 2-6-Sialyltransferase
5.
J Org Chem ; 65(14): 4251-60, 2000 Jul 14.
Article in English | MEDLINE | ID: mdl-10891123

ABSTRACT

The radical C-glycosidation of (-)-(1S,4R,5R, 6R)-6-endo-chloro-3-methylidene-5-exo-(phenylseleno)-7-ox abi cyclo[2. 2.1]heptan-2-one ((-)-4) with 2,3,4, 6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide gave (+)-(1S,3R,4R, 5R,6R)-6-endo-chloro-5-exo-(phenylseleno)-3-endo-(1',3',4', 5'-tetra-O-acetyl-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-7-oxabi cyc lo[ 2.2.1]hept-2-one ((+)-5) that was converted into (+)-(1R,2S,5R, 6R)-5-acetamido-3-chloro-2-hydroxy-6-(1',3',4',5'-tetra-O-acetyl)-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)cyclohex -3-en- 1-yl acetate ((+)-10) and into (+)-(1R,2S,5R, 6S)-5-bromo-3-chloro-2-hydroxy-6-(1',3',4',5'-tetra-O-acetyl-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)cyclohex -3-en- 1-yl acetate ((+)-19). Ozonolysis of (+)-10 and further transformations provided 2-acetamido-2,3-dideoxy-3-C-(2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-D-galac tos e (alpha-C(1-->3)-D-mannopyranoside of N-acetylgalactosamine (alpha-D-Manp-(1-->3)CH(2)-D-GalNAc): 1). Displacement of the bromide (+)-19 with NaN(3) in DMF provided the corresponding azide ((-)-20) following a S(N)2 mechanism. Ozonolysis of (-)-20 and further transformations led to 2-acetamido-2,3-dideoxy-3-C-(2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-D-talose (alpha-C(1-->3)-D-mannopyranoside of N-acetyl D-talosamine (alpha-D-Manp-(1-->3)CH(2)-D-TalNAc): 2). The neutral C-disaccharide 1 inhibits several glycosidases (e.g., beta-galactosidase from jack bean with K(i) = 7.5 microM, alpha-L-fucosidase from human placenta with K(i) = 28 microM, beta-glucosidase from Caldocellum saccharolyticum with K(i) = 18 microM) and human alpha-1, 3-fucosyltransferase VI (Fuc-TVI) with K(i) = 120 microM whereas it 2-epimer 2 does not. Double reciprocal analysis showed that the inhibition of Fuc-TVI by 1 displays a mixed pattern with respect to both the donor sugar GDP-fucose and the acceptor LacNAc with K(i) of 123 and 128 microM, respectively.


Subject(s)
Disaccharides/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Fucosyltransferases/antagonists & inhibitors , Glycoside Hydrolases/antagonists & inhibitors , Mannose/chemical synthesis , Animals , Carbohydrate Conformation , Disaccharides/chemistry , Disaccharides/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Indicators and Reagents , Kinetics , Mannose/chemistry , Mannose/pharmacology , Models, Molecular , Stereoisomerism
6.
Biochem Biophys Res Commun ; 267(1): 169-73, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10623593

ABSTRACT

The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.


Subject(s)
Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Lactose Synthase/genetics , Lactose Synthase/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolism , Cloning, Molecular/methods , Humans , Kinetics , Mating Factor , Peptides/genetics , Peptides/physiology , Pichia , Recombinant Fusion Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Solubility , Substrate Specificity , beta-D-Galactoside alpha 2-6-Sialyltransferase
7.
Arch Biochem Biophys ; 374(1): 3-7, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10640389

ABSTRACT

The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.


Subject(s)
Baculoviridae/genetics , DNA Transposable Elements/genetics , Insecta/enzymology , Melitten/genetics , N-Acetyllactosamine Synthase/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Bees , Blotting, Western , Cell Line , Chromatography, Affinity , Cloning, Molecular , Culture Media, Conditioned/chemistry , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Enzymologic , Genetic Vectors/genetics , Insecta/cytology , Molecular Sequence Data , N-Acetyllactosamine Synthase/genetics , N-Acetyllactosamine Synthase/metabolism , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Staphylococcal Protein A/genetics
8.
Glycoconj J ; 16(2): 125-39, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10612412

ABSTRACT

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.


Subject(s)
Glycosyltransferases/biosynthesis , Recombinant Proteins/biosynthesis , Yeasts/enzymology , Biotechnology , Fermentation , Gene Expression , Genetic Vectors , Glycosyltransferases/genetics , Protein Engineering , Recombinant Proteins/genetics
9.
Glycoconj J ; 16(7): 327-36, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10619705

ABSTRACT

The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.


Subject(s)
N-Acetyllactosamine Synthase/chemistry , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate Sugars/chemical synthesis , Alkaline Phosphatase/chemistry , Animals , Carbohydrate Sequence , Cattle , Humans , Lactalbumin/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Milk/chemistry , Milk, Human/chemistry , Molecular Sequence Data , Substrate Specificity
10.
Glycoconj J ; 16(9): 487-97, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10815985

ABSTRACT

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.


Subject(s)
Epitopes/chemistry , Glycosyltransferases/chemistry , Mucins/chemistry , Oligosaccharides/chemical synthesis , Animals , Carbohydrate Sequence , Humans , Magnetic Resonance Spectroscopy , Membrane Glycoproteins/chemistry , Molecular Sequence Data , P-Selectin/chemistry , Rats , Sialyl Lewis X Antigen , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
11.
Carbohydr Res ; 313(1): 37-48, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9861700

ABSTRACT

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.


Subject(s)
Epitope Mapping , N-Acetyllactosamine Synthase/immunology , beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase/immunology , Antibodies, Monoclonal , Catalysis , Humans , Mutation , N-Acetyllactosamine Synthase/genetics , beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase/genetics
13.
Biochem Biophys Res Commun ; 240(3): 586-9, 1997 Nov 26.
Article in English | MEDLINE | ID: mdl-9398608

ABSTRACT

His6-tag technology has been introduced for easy purification of recombinant proteins expressed in Escherichia coli. Aiming at extending this technology to purification of glycoproteins expressed in Saccharomyces cerevisiae or in animal cells, respectively, we adapted this protocol to recombinant soluble beta-1,4-galactosyltransferase (rgal-T). A His6-tag was introduced to the N-terminus of the protein (hisGal-T). The Histagged enzyme expressed in yeast S. cerevisiae was enzymically active but could not be purified from the cell extract by virtue of the His6-tag. Binding efficiency of hisGal-T was found to be impaired by a bulky N-glycan close to the His-tag. Removal of the unique site of N-glycosylation using site-directed mutagenesis restored binding of hisGal-T to the Ni-NTA resin. In comparison N-glycosylated hisGal-T transiently expressed in COS cells was secreted as a soluble active enzyme and could be purified in one single step by virtue of the His6-tag.


Subject(s)
COS Cells/enzymology , N-Acetyllactosamine Synthase/genetics , N-Acetyllactosamine Synthase/isolation & purification , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Animals , Blotting, Western , Chromatography, Agarose , Enzymes, Immobilized , Gene Expression , Glycosylation , Histidine , Mutagenesis, Site-Directed , N-Acetyllactosamine Synthase/metabolism , Nickel/metabolism , Plasmids/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Solubility , Transfection/genetics
14.
Eur J Biochem ; 239(2): 340-8, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8706738

ABSTRACT

beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.


Subject(s)
Milk, Human/enzymology , N-Acetyllactosamine Synthase/isolation & purification , N-Acetyllactosamine Synthase/metabolism , Saccharomyces cerevisiae/enzymology , Binding Sites , Chromatography, Affinity , Circular Dichroism , Cloning, Molecular , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Female , Fermentation , Glycosylation , Humans , Isoelectric Focusing , Kinetics , Lactalbumin , Molecular Weight , N-Acetyllactosamine Synthase/chemistry , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
15.
FEMS Microbiol Lett ; 126(2): 105-11, 1995 Feb 15.
Article in English | MEDLINE | ID: mdl-7705601

ABSTRACT

In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 micrograms per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.


Subject(s)
Polysaccharide-Lyases/biosynthesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Molecular Sequence Data , Plasmids/genetics , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/genetics , Polysaccharide-Lyases/isolation & purification
16.
Protein Expr Purif ; 6(1): 72-8, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7756841

ABSTRACT

We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC 2.4.1.38) was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.


Subject(s)
N-Acetyllactosamine Synthase/genetics , Saccharomyces cerevisiae/genetics , Chromatography, Affinity , Cloning, Molecular , DNA, Complementary/genetics , Humans , N-Acetyllactosamine Synthase/biosynthesis , N-Acetyllactosamine Synthase/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility
18.
Acta Orthop Belg ; 60(2): 137-44, 1994.
Article in French | MEDLINE | ID: mdl-8053311

ABSTRACT

One hundred four severe knee sprains, treated by ligamentoplasty, according to Marshall-MacIntosh, with a Kennedy polypropylene ligament augmentation device, were reviewed with an average follow-up of 31 months. The functional result was evaluated as good or excellent in 3 out of the 4 cases; 87% of the sportsmen were satisfied. The result was unsatisfactory in 26% of the cases, with rupture or an elongation of the transplant in 9 cases (8.6%), due for some patients to premature resumption of sports; in 4 cases reoperation was necessary. A failure is always possible, even with an augmentation; however, with a ligament augmentation device, early rehabilitation and sports are possible (after an average of 6 months). There was no intolerance: 2 cases of synovitis, with a rupture, were observed. The authors underscore the ill effects of a delay between the accident and the stabilization, which causes numerous lesions of the menisci and the cartilage. This is generally admitted in the literature. The ligament augmentation device has been used for 3 years; the operation is performed after the acute phase of the first lesions and at the latest during the first year after the injury, before secondary lesions of the menisci occur.


Subject(s)
Athletic Injuries/surgery , Knee Injuries/surgery , Patellar Ligament/transplantation , Prostheses and Implants , Sprains and Strains/surgery , Adolescent , Adult , Bone Transplantation , Female , Humans , Joint Instability/physiopathology , Male , Polypropylenes/therapeutic use , Postoperative Complications/etiology , Reoperation , Sprains and Strains/physiopathology
19.
FEMS Microbiol Lett ; 110(1): 101-6, 1993 Jun 01.
Article in English | MEDLINE | ID: mdl-8319887

ABSTRACT

The recombinant plasmid pAL-A3 bears a (poly ManA) alginate lyase-encoding gene that originates from the marine bacterium ATCC 433367 (Brown et al., Appl. Environ. Microbiol. (1991) 57, 1870-1872). The alginate lyase produced by Escherichia coli TC4 harbouring pAL-A3 was purified to protein homogeneity and the corresponding gene sequenced, giving access to the first known primary structure of an alginate lyase. The 265-amino acid residue alginate lyase showed lytic activity on a Pseudomonas aeruginosa alginate isolated from a cystic fibrosis patient. Unexpectedly, the alginate lyase thus characterized differed from that isolated from the culture medium of the bacterium ATCC 433367 (Romeo and Preston, Biochemistry (1986) 25, 8385-8391).


Subject(s)
Alginates/metabolism , Bacterial Proteins/genetics , DNA, Bacterial/analysis , Polysaccharide-Lyases/genetics , Pseudomonas aeruginosa/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Glucuronic Acid , Hexuronic Acids , Molecular Sequence Data , Plasmids , Polysaccharide-Lyases/chemistry , Polysaccharides, Bacterial/metabolism , Recombinant Fusion Proteins , Sequence Analysis, DNA
20.
Med Biol Eng Comput ; 31(2): 180-3, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8331999

ABSTRACT

The analysis of the voltage curve V(t) picked up between the two electrodes of a vaginal plug used with a current source stimulation allowed us to suggest a simple electrical model for intravaginal tissue. It is made up of three cells in series: a resistor in parallel with a capacitor for the first two, and a simple resistor for the third. Measurements throughout the hormonal cycle exhibit large variations in resistance and capacitance, and justify the use of a current source device to keep reproducible conditions of stimulation. Subjective detection of sensitivity threshold currents shows results which are independent of the day in the cycle and thus confirms the use of current source stimulation. The curve representing dissipated energy as a function of the pulse duration tau exhibits a minimum value different from the chronaxie (here 800 microseconds). The minimum value is effectively obtained at lower values of tau between 450 and 500 microseconds, dependent on the day of the cycle and the electrical characteristics of vaginal tissue. Two values of tau (230 and 1000 microseconds) between which the energy dissipated is less than Emin + 10 per cent are determined. This gap of pulse duration seems to be a correct range according to the minimum energy criterion for electrostimulation applied to urogenital re-education.


Subject(s)
Electric Stimulation Therapy/methods , Models, Biological , Vagina/physiopathology , Electric Impedance , Electrophysiology , Female , Humans , Urination Disorders/therapy
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