CH685057A5 - A process for producing glycosyltransferases. - Google Patents

Description

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description

The invention relates to the field of recombinant DNA technology and provides an improved method for producing glycosyltransferases by means of transformed yeast strains.

Glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide sugar, to a specific acceptor sugar to form a glycosidic bond. Depending on the type of sugar transferred, these enzymes are divided into families, e.g. Gaiactosyltransferases, sialyltransferases and fucosyltransferases. As membrane-bound proteins, which are mainly found in the Golgi apparatus, the glycosyltransferases have a common domain structure, which consists of a short amino-terminal cytoplasmic tail, a signal anchor domain and an extended stem region, followed by a large carboxyl-terminal catalytic domain. The signal anchor domain functions both as a non-cleavable signal peptide and as an anchor extending across the membrane and aligns the catalytic domain of the glycosyltransferase within the lumen of the Golgi apparatus. The luminal stem region, also called the spacer region, is believed to have the function of a flexible chain which enables the catalytic domain to glycosylate carbohydrate groups of membrane-bound and soluble proteins of the "secretory pathway" that pass through the Golgi apparatus . In addition, it was discovered that the stem section functions as a retention signal to keep the enzymes bound to the Golgi membrane (PCT Application No. 91/06 635). Soluble forms of glycosyltransferases can be found in milk, serum and other body fluids. It is believed that these soluble glycosyltransferases result from the proteolytic release of the corresponding membrane-bound forms of the enzymes by endogenous proteases, presumably through cleavage between the catalytic domain and the domain protruding through the membrane.

The enzymatic synthesis of carbohydrate structures has the advantage that stereoselectivity and regioselectivity are high, which makes the glycosyltransferases a valuable tool for the modification or synthesis of glycoproteins, glycolipids and oligosaccharides. In contrast to chemical methods, the time-consuming introduction of protective groups is unnecessary.

Since glycosyltransferases occur in very small quantities in nature, their isolation from natural substances and their subsequent purification are difficult. It was therefore worked on their production using the recombinant DNA method. For example, gaiactosyltransferases have been expressed in cells of E. coli (PCT 90/07 000) and Chinese hamster ovaries (CHO) (Smith, DF et al. (1990) J. Biol. Chem. 265, 6225-34), sialyltransferases were in CHO cells (Lee, EU (1990) Diss. Abstr. Int. B. 50, 3453-4) and COS-1 cells (Paulsen, JC et al. (1988) J. Cell. Biol. 107, 10A) and fucosyl transferases were expressed in COS-1 cells (Goelz, SE et al. (1990) Cell 63, 1349-1356; Larsen RD et al. (1990) Proc. Nati. Acad. Sci. USA 87, 6674 -6678) and CHO cells (Potvin, B. (1990) J. Biol. Chem. 265, 1615-1622). Considering that heterologous expression in prokaryotes has the disadvantage that non-glycosylated products are obtained, but glycosyltransferases are glycoproteins, and that the production of glycosyltransferases by mammalian hosts is very expensive and due to the presence of many endogenous glycosyltransferases that would contaminate the desired product , is complicated, there is a need for improved processes which enable the economical production of glycosyltransferases on a large scale.

It is an object of the present invention to provide such methods.

The present invention provides a method for the production of biologically active glycosyltransferases using recombinant DNA technology, in which a yeast vector expression system is used.

More specifically, the present invention provides a method for the production of a membrane-bound glycosyltransferase from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or one of their variants, which method is characterized in that a yeast strain is cultivated which has been transformed with a hybrid vector which contains an expression cassette consisting of a promoter and a DNA sequence coding for said glycosyltransferase or its variant, said DNA being controlled by said promoter, and the enzymatic activity is obtained.

In a first embodiment, the invention provides a process for the production of a membrane-bound glycosyltransferase from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or one of their variants, the process being characterized in that a yeast strain is cultivated which has been transformed with a hybrid vector which contains an expression cassette comprising a promoter, a DNA sequence coding for said glycosyltransferase or its variant, this DNA being controlled by said promoter, and a DNA sequence with yeast transcription termination signals , and the enzymatic activity is obtained.

In a second embodiment, the invention relates to a method for the production of a membrane-bound glycosyltransferase from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase or one of their variants, the method mentioned being characterized in that a yeast strain is cultured with a Hybrid2

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vector which comprises an expression cassette comprising a yeast promoter which is functionally linked to a first DNA sequence coding for a signal peptide and which is linked in the correct reading frame to a second DNA sequence coding for the glycosyltransferase mentioned or its variant, and a DNA sequence Sequence with yeast transcription termination signals, has been transformed, and the enzymatic activity is obtained.

The term "glycosyltransferase" whenever used in the preceding or following text is to be understood to include the family of gaiactosyltransferases, the family of sialyltransferases and the family of fucosyltransferases. The glycosyltransferases mentioned are naturally occurring enzymes which are found in mammals, e.g. Cattle, mice, rats and humans. Preferred glycosyltransferases are naturally occurring human glycosyltransferases in their full length, including those enzymes which are referred to in the text below with their EC numbers.

The membrane-bound gaiactosyl transferases and their variants, which are obtainable by the inventive method, catalyze the transfer of a galactose residue from an activated donor, usually a nucleotide activated donor, e.g. Uridine diphosphate galactose (UDP-Gal), on a carbohydrate group.

Examples of membrane-bound gaiactosyltransferases are UDP-galactose: β-galactoside-a (1-3) -ga-lactosyltransferase (EC 2.4.1.151), for which galactose serves as an acceptor substrate with formation of an a (1-3) bond, and UDP- Galactose: ß-N-acetylglucosamine-ß (1-4) -galactosyltransferase (EC 2.4.1.22), which transfers galactose to N-acetylglucosamine (GIcNAc) to form a ß (1-4) bond, including their respective variants. In the presence of a-lactalbumin, the β (1 ^ -galactosyltransferase also serves as glucose as an acceptor substrate, thereby catalyzing the laclos synthesis.

The most preferred membrane bound galactosyltransferase is the enzyme with the amino acid sequence listed under SEQ ID NO. 1 is specified in the sequence listing.

The membrane-bound sialyltransferases and their variants, which are obtainable by the process according to the invention, catalyze the transfer of sialic acids (for example N-acetylneuraminic acid (NeuAc)) from an activated donor, usually a cytidine monophosphatsialic acid (CMP-SA), to a carbohydrate acceptor residue. An example of a membrane-bound siaiyitransferase which can be obtained by the process according to the invention is CMP-NeuAc: β-galactoside-a (2-6) sialyltransferase (EC 2.4.99.1), which the NeuAc-a (2-6) Gal-β (1-4) GlcNAc ~ sequence that is found in many N-linked carbohydrate groups.

The most preferred membrane-bound siaiyitransferase is the enzyme with the amino acid sequence, which is listed in the sequence listing under SEQ ID no. 3 is listed.

The membrane-bound fucosyltransferases and their variants, which are obtainable by the inventive method, catalyze the transfer of a fucose residue from an activated donor, usually a nucleotide activated donor such as e.g. Guanosine diphosphate fucose (GDP-Fuc), on a carbohydrate group. Examples of such fucosyltransferases are GDP-fucose: β-galactoside-a (1 ^ -fucosyltransferase (EC 2.4.1.69) and GDP-fucose: N-acetylamine-a (1-3 / 4) -fucosyltransferase (EC 2.4.1.65).

The most preferred membrane-bound fucosyltransferase is the enzyme with the amino acid sequence, which is shown in the sequence listing under SEQ ID no. 5 is listed.

The term variants means both membrane-bound and soluble variants of the naturally occurring membrane-bound glycosyltransferases, with the proviso that these variants are enzymatically active. Variants occurring in humans are preferred.

For example, the term "variants" is to be understood to include naturally occurring membrane-bound variants of membrane-bound glycosyltransferases that are found within a certain type, e.g. a variant of a galactosyltransferase, which is derived from the enzyme with the SEQ ID no. 1 given amino acid sequence in that it lacks the serine in position 11 and in positions 31 and 32 valine and tyrosine occur in place of alanine and leucine, respectively. Such a variant can be encoded by a related gene of the same gene family or by an allelic variant of a specific gene. The term “variants” also includes glycosyltransferases that are produced by an in vitro mutated DNA, as long as the protein encoded by said DNA has the enzymatic activity of the native glycosyltransferase. Such modifications can consist of an addition, an exchange and / or a deletion of amino acids, in which case shortened variants arise.

Preferred variants which are produced by the method according to the invention are shortened variants, in particular soluble variants, i.e. Variants that are not membrane-bound. Shortened variants are, for example, soluble forms of membrane-bound glycosyltransferases and their membrane-bound variants, e.g. those variants mentioned above which can be secreted by a transformed yeast strain used in the method according to the invention. According to the present invention, these soluble enzymes are the preferred shortened variants.

The invention also relates to a method for producing a soluble variant of a membrane-bound glycosyltransferase from the group consisting of a galactosyltransferase, a siaiyitransferase and a fucosyltransferase, this method being characterized in that a yeast strain is cultured which is associated with a hybrid vector comprising an expression cassette a promoter, a DNA sequence which is functionally linked to this promoter and which is responsible for a signal peptide

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encoded, and which is connected in the correct reading frame to a second DNA sequence coding for the said variant, and a DNA sequence which contains yeast transcription termination signals has been transformed, and that said variant is isolated.

The soluble form of a glycosyltransferase is by definition a shortened variant, which differs from the corresponding form with the full length, i.e. the membrane-bound form, which is naturally found in the endoplasmic reticulum or in the Golgi complex, due to the absence of the cytoplasmic tail, the signal anchor and, if necessary, part of the stem region. The term “part of the stem region” is defined in the context of the present text in such a way that it denotes a smaller part of the N-terminal side of the stem region and consists of up to 12 amino acids. In other words, the soluble variants which are produced according to the present invention consist of the essentially entire stem region and the catalytic domain.

The soluble variants are enzymatically active enzymes which differ from the respective full-length forms in that an NH2-terminal peptide consisting of 26 to 61 amino acids is missing, with the proviso that those forms in which part of the stem region missing, only the smaller part of this region defined above is missing. Preferred soluble variants are those which are obtainable from the membrane-bound glycosyltransferases for which EC numbers are indicated, and additionally soluble variants which are from the membrane-bound fucosyltransferase with the SEQ. ID no. 5 are available.

Preferred soluble variants of the gaiactosyl transferases differ from the respective full-length forms in that they lack an NH2-terminal peptide which consists of 37 to 55, in particular 41 to 44, amino acids. The most preferred soluble variant, which is produced according to the inventive method, is the enzyme with the amino acid sequence, which is under the SEQ ID no. 2 is listed in the sequence listing.

Preferred soluble variants of sialyltransferases lack an NH2-terminal peptide, which consists of 26 to 38 amino acids, compared to the full-length forms. The most preferred soluble variant, which is prepared according to the inventive method, is the enzyme with the amino acid sequence, which is in the sequence listing under SEQ ID no. 4 is listed. Also preferred is the soluble variant designated ST (Lys27-Cys406), which consists of amino acids 27 to 406 of the sequence shown in SEQ ID no. 3 given amino acid sequence exists.

Preferred soluble variants of the fucosyltransferases differ from the respective full-length enzymes in that they lack an NH2-terminal peptide which consists of 56 to 67, in particular 56 to 61, amino acids. Particularly preferred is the soluble variant designated FT (Arg62-Arg4os), which consists of amino acids 62 to 405 of the sequence shown in SEQ ID no. 5 amino acid sequence listed exists.

The yeast host strains and the components of the hybrid vectors are those given below.

The transformed yeast strains are grown using methods known to those skilled in the art.

Thus the transformed yeast strains according to the invention are grown in a liquid medium which contains assimilable sources of carbon, nitrogen and mineral salts.

A number of carbon sources can be used. Examples of preferred carbon sources are assimilable carbohydrates such as e.g. Glucose, maltose, mannitol, fructose or lactose, or an acetate such as sodium acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids such as casamino acids, peptides and proteins and their breakdown products such as trypton, peptone or meat extracts, furthermore yeast extract, malt extract, corn steep liquor, as well as ammonium salts such as ammonium chloride, ammonium sulfate or ammonium nitrate, which are used either alone or in suitable mixtures can be. The inorganic salts that can be used include, for example, sodium, potassium, magnesium and calcium sulfates, chlorides, phosphates and carbonates. The nutrient medium can also contain growth-promoting substances. The growth-promoting substances include, for example, trace elements such as iron, zinc, manganese and the like, or individual amino acids.

Because of the incompatibility between the endogenous two micron DNA and hybrid vectors with their replicon, yeast cells transformed with such hybrid vectors show a tendency to lose them. Such yeast cells must be grown under selective conditions, i.e. Conditions under which expression of a plasmid-encoded gene is required for growth. Most of the currently used selective markers which are present in the hybrid vectors according to the invention (see below) are genes which code for enzymes for amino acid or purine biosynthesis. Therefore minimal synthetic media, in which the respective amino acid or purine base is missing, must be used. However, it is also possible to use genes that confer resistance to a suitable biocid [e.g. a gene that confers resistance to the aminoglycoside G418]. Yeast cells transformed with vectors containing genes for antibiotic resistance are grown in complex media that contain the respective antibiotic, resulting in higher growth rates and higher cell density.

Hybrid vectors containing the complete two micron DNA (including a functional replication start site) remain stable within Saccharomvces cerevisiae strains, which

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endogenous two-micron plasmids are missing (so-called cir ° strains), so that they can be grown under non-selective growth conditions, i.e. in a complex medium.

Yeast cells which contain hybrid plasmids with a constitutive promoter express the DNA which codes for a glycosyltransferase controlled by the abovementioned promoter, or a variant thereof, without induction. However, if the above-mentioned DNA is controlled by a regulated promoter, the composition of the culture medium must be changed in order to obtain a maximum of mRNA transcripts, for example, when the PH05 promoter is used, the concentration of inorganic phosphate in the culture medium must be low to dereprize this promoter.

Breeding is carried out using conventional techniques. The breeding conditions such as temperature, pH of the medium and fermentation time are chosen so that a maximum of heterologous protein is produced. A selected yeast strain is preferably grown under aerobic conditions in a submerged culture with shaking or stirring at a temperature of about 25 ° to 35 ° C, preferably at about 28 ° C, and at a pH of 4 to 7, e.g. approximately pH 5, cultured for at least 1 to 3 days, preferably as long as satisfactory protein yields are obtained.

After its expression in yeast, the membrane-bound glycosyltransferase or its variant is either enriched within the cells or secreted into the culture medium and isolated in a conventional manner.

For example, the first step usually consists in separating the cells from the culture liquid by centrifugation. If the membrane-bound glycosyltransferase or its variant has accumulated within the cells, the protein must be released from the cell interior by cell disruption. Yeast cells can be disrupted in various ways that are known to those skilled in the art: e.g. by exerting mechanical forces such as shaking with glass beads, by ultrasonic vibration, osmotic shock and / or by enzymatic digestion of the cell wall. If the desired membrane-bound glycosyltransferase or its variant is associated or bound to a membrane fraction, further enrichment e.g. can be achieved by subjecting the cell extract to a differential centrifugation and, if appropriate, then the respective fraction subsequently with a detergent such as e.g. Triton is being treated. The methods suitable for the purification of the "raw" glycosyltransferase, or its variant, include the usual chromatographic methods such as affinity chromatography, for example with a suitable substrate, with antibodies or with Concanavalin A, ion exchange chromatography, gel filtration, partition chromatography, HPLC, electrophoresis, precipitation steps such as ammonium sulfate precipitation, and other processes, in particular the processes known from the literature.

If the glycosyltransferase or its variant is secreted from the yeast cell into the periplasmic space, a simplified isolation protocol can be used: the protein is removed without cell lysis by enzymatic removal of the cell wall or by chemicals, e.g. Thiol reagents or EDTA, which induce cell wall damage and thus enable the release of the glycosyltransferase produced, are obtained. If the glycosyltransferase, or the variant, is secreted into the culture liquid, it can be obtained directly therefrom and purified using the methods mentioned above.

Tests known from the literature can be used to detect glycosyltransferase activity. Galactosyltransferase activity can e.g. be determined by measuring the amount of radiolabelled galactose that is incorporated into a suitable acceptor molecule such as a glycoprotein or a free sugar residue. Analogously, sialyltransferase activity can be tested by e.g. Sialic acid is incorporated into suitable substrates, and fucosyltransferase activity can be tested by transferring fucose to a suitable acceptor.

The transformed yeast host cells according to the invention can be produced using recombinant DNA techniques according to the following steps:

Production of a hybrid vector comprising a yeast promoter and a DNA sequence coding for a membrane-bound glycosyltransferase or its variant, this DNA sequence being controlled by said promoter,

- transformation of a yeast host strain with said hybrid vector,

- and selection of the transformed yeast cells from the untransformed yeast cells.

Expression vectors

The yeast hybrid vector according to the invention contains an expression cassette which comprises a yeast promoter and a DNA sequence coding for the membrane-bound glycosyltransferase or a variant thereof, this DNA sequence being controlled by said promoter.

In a first embodiment, the yeast hybrid vector according to the invention contains an expression cassette which contains a yeast promoter, a DNA sequence coding for a membrane-bound glycosyltransferase or one of its variants, this DNA sequence being controlled by the promoter mentioned, and a DNA sequence, which contains yeast transcription termination signals.

In a second embodiment, the yeast hybrid vector according to the invention contains an expression cassette which contains a yeast promoter which is functionally linked to a first DNA sequence.

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which encodes a signal peptide and which is linked in the correct reading frame to a second DNA sequence which codes for a membrane-bound glycosyltransferase or a variant thereof and which comprises a DNA sequence which contains yeast transcription termination signals.

The yeast promoter is a regulated (inducible) or constitutive promoter, which is preferably derived from a highly expressed yeast gene, in particular a Saccharomvces cerevisiae gene. So the promoters of the TRP1 gene. of the ADHI or ADHII gene. of the acid phosphatase (PH051 gene, a promoter of the yeast mating pheromone genes which code for the a or a factor, or a promoter of a gene coding for a glycolytic enzyme, such as the promoter of the enolase, glyceraldehyde-3 phosphate dehydrogenase (GAP) -. 3-phosphoglycerate kinase (PGKK hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triophosphate isomerase, or gene glucose isase is used further it is possible to use hybrid promoters containing “upstream activation sequences” (UAS) from a yeast gene and “downstream promoter elements” with a functional “TATA box” of another yeast gene, for example a hybrid promoter with the UAS of the yeast PH05 gene and “downstream promoter elements” with a functional “TATA box” of the yeast GAP gene (PH05-GAP hybrid promoter) A preferred promoter is the promoter of the GAP gene, in particular whose functional fragments begin with nucleotides between positions -550 and -180, in particular with nucleotide -540, -263 or -198, and end with nucleotide -5 of the GAP gene. Another preferred promoter of the regulated type is the PH05 promoter. A preferred constitutive promoter is a truncated acid phosphatase PH05 promoter. which lack the upstream regulatory elements (UAS), as well as the PH05M73) promoter element, which begins at nucleotide -173 of the PH05 gene and ends at nucleotide -9 of this gene.

The DNA sequence encoding a signal peptide ("signal sequence") preferably originates from a yeast gene which codes for a polypeptide which is normally secreted. Other signal sequences of heterologous proteins that are normally secreted can also be chosen. Yeast signal sequences are, for example, the signal and prepro sequences of the yeast invertase gene, a-factor gene, pheromone peptidase (KEXI) gene, “killer toxin” gene and the repressible acid phosphatase (PH05) gene . and the Aspergillus awamori glucoamylase signal sequence. Alternatively, combined signal sequences can be constructed by ligating part of the signal sequence (if present) of the gene naturally associated with the promoter used (for example PH05) with part of the signal sequence of another heterologous protein. Preferred combinations are those that allow precise cutting between the signal sequence and the glycosyltransferase amino acid sequence. Additional sequences such as pro- or spacer sequences with or without special processing signals can also be included in the constructions in order to enable precise processing of precursor molecules. However, it is also possible to produce fusion proteins with internal processing signals that enable maturation in vivo or in vitro. For example, the processing signals contain Lys-Arg, which is recognized by a yeast endopeptidase in the Golgi membranes. The preferred signal sequence according to the present invention is that of the yeast invertase gene.

When a full length glycosyltransferase or one of its membrane bound variants is expressed in yeast, the preferred yeast hybrid vector contains an expression cassette which contains a yeast promoter, a DNA sequence coding for said glycosyltransferase or variant and controlled by said promoter, and a DNA Sequence comprising yeast transcription termination signals.

If a soluble variant of a glycosyltransferase is expressed in yeast, the preferred yeast hybrid vector contains an expression cassette which contains a yeast promoter which is functionally linked to a first DNA sequence which codes for a signal peptide and which is in the correct reading frame with a second DNA Sequence is connected, which codes for the variant mentioned, and comprises a DNA sequence which contains yeast transcription termination signals.

DNA encoding a membrane-bound glycosyltransferase or one of its variants can be produced by methods known to those skilled in the art and contains genomic DNA, e.g. DNA derived from a mammalian genomic DNA library, e.g. Rat, mouse and bovine or human cells. If necessary, the introns that occur in the genomic DNA encoding the enzyme are removed. A DNA coding for a membrane-bound glycosyltransferase or one of its variants also comprises cDNA which can be isolated from a mammalian cDNA library or prepared from the corresponding mRNA. The cDNA library can be made from cells of different tissues, e.g. Placental cells or liver cells. The cDNA is produced via the mRNA using customary methods such as the polymerase chain reaction (PCR).

For example, poly (A) + RNA is isolated from mammalian cells, e.g. HeLa cells, and the subsequent synthesis of the first strand of the cDNA according to standard methods known in the art. Starting from this synthesized DNA template, the target sequence, i.e. the glycosyltransferase DNA, or one of its fragments, is amplified while minimizing the amplification of the excess undesirable sequences. For this purpose the sequence of a small nucleotide section on both sides of the target sequence must be known. These flanking sequences are used to build up two synthetic single-stranded primer oligonucleotides, the sequence of which is chosen such that the bases are complementary to the flanking sequence in question.

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PCR begins with the denaturation of the mRNA-DNA hybrid strand, followed by the attachment of the primer to the sequences flanking the target sequence. By adding a DNA polymerase and deoxynucieoside triphosphates, two pieces of double-stranded DNA are formed, each beginning with the primer and extending over the target sequence, the latter being copied. Each of the newly synthesized products can be used as a template for primer attachment and extensions (in the next cycle), resulting in an exponential increase in double-stranded fragments of discrete length.

In addition, a DNA encoding a membrane-bound glycosyltransferase or one of its variants can be synthesized enzymatically or chemically. A variant of a membrane-bound glycosyltransferase with enzymatic activity and an amino acid sequence in which one or more amino acids are deleted (DNA fragments) and / or exchanged for one or more other amino acids is encoded by a DNA mutant. A mutated DNA is also to be understood as a silent mutant in which one or more nucleotides are replaced by other nucleotides, the new codons coding for the same amino acid (s). Such a mutated sequence can also be a degenerate DNA sequence. The latter are degenerate in genetic code in that an unlimited number of nucleotides can be replaced by other nucleotides without changing the initially encoded amino acid sequence. Such degenerate DNA sequences can be useful because they have different restriction sites and / or frequencies of certain codons that are preferred by a specific value for optimal expression of a glycosyltransferase. Such DNA sequences preferably have codons, as are preferably used by yeast.

A DNA mutant can also be obtained by mutating a naturally occurring genomic DNA or a cDNA in vitro according to methods known in the art. For example, from a full-length DNA encoding the particular membrane-bound glycosyltransferase, a partial DNA encoding a soluble form of a glycosyltransferase can be excised using restriction enzymes. For this purpose it is advantageous if a suitable restriction site is available.

A preferred DNA sequence that contains yeast transcription termination signals is the 3 'flanking sequence of a yeast gene that contains correct signals for transcription termination and polyadenyla- tion. Suitable 3-flanking sequences are e.g. the sequences of the yeast gene which are naturally linked to the promoter used. The preferred flanking sequence is that of the yeast PH05 gene.

The yeast promoter, the optional DNA sequence encoding the signal peptide, the DNA sequence encoding a membrane bound glycosyltransferase or one of its variants, and the DNA sequence containing the yeast transcription termination signals are functional in a tandem arrangement connected, ie they are placed side by side in such a way that their normal functions are retained. They are arranged in such a way that the promoter effects correct expression of the DNA sequence encoding a membrane-bound glycosyltransferase or one of its variants (if appropriate with an upstream signal sequence), that the transcription termination signals bring about a correct termination of the transcription and the polyadenylation, and that the optionally present signal sequence is connected in the correct reading frame to the above-mentioned DNA sequence in such a way that the last codon of the signal sequence is connected directly to the first codon of the said DNA sequence and the protein is secreted. If the promoter and the signal sequence are from different genes, the promoter is preferably added to the signal sequence at a location between the main mRNA start and the ATG of the gene naturally associated with the promoter. The signal sequence should have its own ATG for translation initiation. These sequences can be combined with the recognition sequence of an endonuclease by means of synthetic oligodeoxynucleotide linkers.

Vectors suitable for replication and expression in yeast contain a yeast repositioning start site. Hybrid vectors containing a yeast replication start site, for example the chromosomal autonomously replicating segment (ARS), remain extrachromosomally in the yeast cell after transformation and are replicated autonomously during mitosis. Hybrid vectors containing sequences homologous to yeast 2 | i plasmid DNA can also be used. Such hybrid vectors are integrated by recombination into 2p. Plasmids that already exist in the cell, or they replicate autonomously.

The hybrid vectors according to the invention preferably contain one or more, in particular one or two, selective genetic markers for yeast and such a marker and a start of replication for a bacterial host, in particular Escherichia coli.

As far as the selective gene markers for yeast are concerned, all marker genes can be used which enable selection for transformants on the basis of the phenotypic expression of the marker gene. Examples of suitable markers for yeast are those which express resistance to antibiotics or, in the case of auxotrophic yeast mutants, genes which compensate for deficiencies in the host. The genes in question confer resistance, for example, to the antibiotics G418, hygromycin or bleomycin, or mean prototrophy in an auxotrophic yeast mutant, for example the URA3-. I FU? -. I.YS2 or TRP1 gen.

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Since the amplification of the hybrid vectors can be carried out conveniently in E. coli, it is advantageous to incorporate a genetic marker for E. coli and an E. coli repeat site. These can be derived from E. coli plasmids such as e.g. pBR322, or a pUC plasmid, for example pUC18 or pUC19, can be obtained which contain both an E. coli replication start site and a genetic marker for E. coli which confers resistance to antibiotics such as ampicillin.

The hybrid vectors according to the invention are produced by methods known to those skilled in the art, for example by the expression cassette comprising a yeast promoter and a DNA sequence coding for a glycosyltransferase, or a variant, which is controlled by the promoter mentioned, or several components of the expression cassette , with the DNA fragments containing selective genetic markers for yeast and for a bacterial host and replication starting parts for yeast and for a bacterial host, in the predetermined order.

Yeast strains and their transformation

Suitable yeast host organisms are strains of the genus Saccharomvces. especially strains of Saccharomvces cerevisiae. The yeast strains mentioned include yeast strains which have optionally been freed from endogenous two-micron plasmids and / or which may have no yeast peptide activity (s), e.g. Peptidase have yscoa, yscA, yscB, yscY, and / or yscS activity.

The invention further relates to a yeast strain which has been transformed with a hybrid vector which contains an expression cassette comprising a yeast promoter and a DNA sequence coding for a membrane-bound glycosyltransferase or one of its variants, this DNA being controlled by the promoter mentioned.

In a first embodiment, the yeast strain according to the invention with a hybrid vector comprising an expression cassette comprising a yeast promoter, a DNA sequence coding for a membrane-bound glycosyl transferase or one of its variants, this DNA sequence being controlled by the promoter mentioned, and one DNA sequence containing yeast transformation termination signals transformed.

In a second embodiment, the yeast strain according to the invention was transformed with a hybrid vector which has an expression cassette comprising a yeast promoter which is functionally linked to a first DNA sequence which codes for a signal peptide and which is linked in the correct reading frame to a second DNA sequence, which codes for a membrane-bound glycosyltransferase or one of its variants, and transforms a DNA sequence which contains yeast transcription termination signals.

The transformation of yeast with the hybrid vectors according to the invention is carried out according to methods known in the art, for example according to the method described by Hinnen et al. (Proc. Nati. Acad. Sci. USA (1978) 75, 1929) and Ito et al. (J. Bact. (1983) 153, 163-168).

The membrane-bound glycosyltransferases produced by the process according to the invention and their variants can be used in a manner known per se, e.g. for the synthesis and / or modification of glycoproteins, oligosaccharides and glycolipids (US patent 4 925 796; EP application 414 171).

The invention relates in particular to the process for the preparation of membrane-bound glycosyltransferases and their variants, the hybrid vectors and the transformed yeast strains, as described in the examples.

The following abbreviations are used in the examples: GT = galactosyltransferase (EC 2.4.1.22); PCR ê polymerase chain reaction; ST = Siaiyitransferase (EC 2.4.99.1) variants; FT = fucosyltransferase

Example 1:

Cloning of galactosyltransferase (GTi cDNA from HeLa cells

GT cDNA is isolated from HeLa cells (Watzele, G. and Berger, E.G. (1990) Nucleic Acids Res. 18, 7174) using the polymerase chain reaction (PCR) method:

1.1 Preparation of PoIy (A) + RNA from HeLa cells.

For the RNA preparation, HeLa cells are grown on 5 plates (23 × 23 cm) in a single cell layer culture. RNA is quickly and effectively isolated from the cultured cells by means of extraction with guani-din-HCl as described by MacDonald, R.J. et al (Meth. Enzymol. (1987) 152, 226-227). In general, the yields are approximately 0.6 to 1 mg of total RNA per plate of confluently grown cells. The PoIy (A) + RNA is enriched by affinity chromatography on oligo (dT) cellulose according to the method described in the Maniatis manual (Sambrook, J. Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd édition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA), with 4 mg of total RNA being applied to a 400 nl column. 3% of the 8

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Carried RNAs are recovered as enriched Poiy (A) + RNA, precipitated with three times the volume of ethanol and stored aliquoted at -70 ° C until use.

1.2 Synthesis of first strand cDNA for PCR

Poly (A) + RNA (mRNA) is reverse transcribed in DNA using Moloney Murine Leukemia Virus RNase H-Reverse Transcriptase (M-MLV H-RT) (BRL). The 20 nl reaction mixture is prepared with slight deviations according to the protocol provided by BRL: 1 ng HeLa cell poly (A) + RNA and 500 ng oligo (dT) i2-i8 (Pharmacia) in 11.5 pl sterile H2O are heated to 70 ° C for 10 minutes and then quickly cooled on ice. Then 4 nl reaction buffer (BRL; 250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCb), 2 ml 0.1 M dithiothreitol, 1 | il mixed dNTP (each 10 mM dATP, dCTP, dGTP, TTP, Pharmacia), 0.5 jaI (17.5 U) RNAguard (RNase inhibitor from Pharmacia) and 1 nl (200 U) M-MLVH-RT added. The reaction is carried out at 42 ° C and stopped after one hour by heating the tube to 95 ° C for 10 minutes.

To check the effectiveness of the reaction, an aliquot of the mixture (5 | il) is incubated in the presence of 2 p.Ci a-32P dCTP. The amount of cDNA synthesized is calculated by measuring the built-in dCTP. The first strand synthesis yield is routinely between 5 and 15%.

1.3 Polymerase chain reaction

The oligodeoxynucleotide primers used for the PCR are applied in vitro using the phosphoramidite method (MH Caruthers, in Chemical and Enzymatic Synthesis of Gene Fragments, HG Gassen and A. Lang, ed. Verlag Chemie, Weinheim, FRG) in an Applied Biosystems Synthesizer , Model 380B, synthesized. They are listed in Table 1.

Table 1: PCR primers accordingly

Primer sequence (5 'to 3') 1} bp in GT cDNA2)

Plup (Kpnl)

cacaatACCCTTCTTAAAGCGGCGGCGGGAAGATG

(-26) -

3rd

PI (EcoRI)

gccsaattcATGAGGCTTCGGGAGCCGCTCCTGAGCG

1 -

28

P3 (Sacl)

CTGGAGCTCGTGGCAAAGCAGAACCC

448-

473

P2d (EcoRI)

scceaaTTCAGTCTCTTATCCGTGTACCAAAACGCCTA

1222-

1192

P4 (HindIII)

cccaaactTGGAATGATGATGGCCACCTTGTGAGG

546-

520

'Upper case letters stand for GT sequences, lower case letters are additional sequences, digits for

Restriction enzymes are underlined, "start" and "stop" codons for RN A translation are in bold. ^ DT-cDN A sequence from the human placenta as published in GenBank (entry no. M22921).

The following are standard PCR conditions for a 30 μl incubation batch: 1 μl of the reverse transcriptase reaction (see Example 1.2), which contains approx. 5 ng first-strand cDNA, in each case 15 pmol of the relevant primers, in each case 200 pmol the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and TTP) in PCR buffer (10 mM Tris-HCl pH 8.3 (at 23 ° C), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin) and 0.5 U AmpliTaq polymerase (Perkin Elmer). The amplification is carried out in the Thermocycler 60 (Biomed) under the following conditions: 0.5 minutes denaturation at 95 ° C, 1 minute addition at 56 ° C and 1 minute 15 seconds extension at 72 ° C, over a total of 20-25 cycles. In the last cycle, primer extension takes 5 minutes at 72 ° C.

For sequencing and subcloning, the HeLa-GT cDNA is amplified in two overlapping sections, using different primer combinations:

(1) Fragment P1-P4: The primers P1 and P4 are used for the amplification of a 0.55 kb DNA fragment which extends over the nucleotide positions 7-556 in HeLa-GT cDNA (SEQ ID NO. 1)

(2) Fragment P3-P2d: The primers P3 and P2d are used for the amplification of a 0.77 kb fragment which extends over the nucleotide positions 457-1232 (SEQ ID No. 1).

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In order to avoid errors during the amplification, 4 independent PCRs are carried out per fragment. Primer P1up (Kpnl) is used together with primer P4 to determine the DNA sequence, which is followed by the «start» codon.

After the PCR amplification, fragment P1-P4 is digested with the restriction enzymes EcoRI and Hindill, the digestion is analyzed on a 1.2% agarose gel, eluted from the gel using GENECLEAN (BIO 101) and into the vector pUC18 (Pharmacia), which contains was digested with the same enzymes, sub-cloned. Fragment P3-P2d is digested with Sacl and EcoRI, the digest is analyzed on a 1.2% gel, eluted and subcloned into pUC18, which was digested with Sacl and EcoRI. The subclones obtained are pUC18 / P1-P4 and pUC18 / P3-P2d. Standard protocols as described in Example 2 are followed for subcloning, ligation and transformation of E. coli strain DH5a. Mini preparations of the plasmids pUC18 / P1-P4 or pUC18 / P3-P2d are used for the dideoxy sequencing of denatured double-stranded DNA with the T7 polymerase sequencing kit (Pharmacia). M13 / pUC sequencing primers and reverse sequencing primers (Pharmacia) are used for sequencing overlapping fragments which have been produced from both DNA strands by digestion with different restriction enzymes. Further subcloning of restriction fragments of the GT gene is required for extensive sequencing of overlapping fragments of both strands. The sequence of fragments amplified by independent PCR shows that the amplification error is less than 1 in 3000 nucleotides. The complete nucleotide sequence of the HeLa cell GT cDNA, which is shown in SEQ ID no. 1 is 99.2% homologous to that of the human placenta (Genbank entry no. M22921). There are three differences:

(a) Three additional base pairs in nucleotide positions 37-39 (SEQ ID NO: 1) which give an additional amino acid (Ser) in the N-terminal region of the protein; (b) bp 98-101 are "CTCT" instead of "TCTG" in the sequence of the human placenta, which results in two conservative amino acid substitutions (Ala Leu instead of ValTyr) in amino acid positions 31 and 32 in the GT membrane-wide domain; (c) the nucleotide at position 1047 changes from "A" to "G" without causing changes in the amino acid sequence.

The two overlapping DNA fragments P1-P4 and P3-P2d, which encode the HeLa-GT cDNA, are joined via the NotI restriction site in nucleotide position 498, which exists in both fragments.

The complete HeLa cell GT cDNA (SEQ ID No. 1) is cloned as a 1.2 kb EcoRI-EcoRI restriction fragment in plasmid plC-7, where plC-7 is a derivative of pUC8 with additional restriction sites in the multiple cloning site (Marsh, J.L. "Erfle, M. and Wykes, EJ (1984) Gene 32, 481-485), which gives the vector p4AD113. To construct the GT expression cassette, the EcoRI restriction site (bp 1227) at the 3 'end of the cDNA sequence is eliminated as follows: Vector p4AD113 is first linearized by digestion with EcoRV and then treated with alkaline phosphatase. In addition, 1 ng of the linearized plasmid DNA is partially digested with 0.25 U EcoRI for 1 hour at 37 ° C. After electrophoresis on agarose gel, a fragment is isolated from the gel using GENECLEAN (Bio 101), which corresponds to the size of the linearized plasmid (3.95 kb). The protruding EcoRI end is filled in with Klenow polymerase as described in the Maniatis manual (see above). After phenolization and precipitation with ethanol, the plasmid is ligated again and used for the transformation of E. coli DH5a (Gibco / BRL). Plasmid minipreps are made from six transformants. The plasmids obtained are checked by restriction analysis for the absence of the EcoRI and EcoRV restriction sites at the 3 end of the HeLa-GT cDNA. The plasmid designated p4AE113 is selected for the following experiments since its DNA sequence is identical to that of plasmid p4AD113, with the exception that bp 1232-1238 with the EcoRI-EcoRV restriction sites are eliminated.

Example 2:

Construction of extra cassettes for full GT

For heterologous expression in Saccharomvces cerevisiae, the complete HeLa-GT cDNA sequence (SEQ ID No. 1) is combined with transcriptional control signals from the yeast in order to achieve effective initiation and termination of the transcription. The promoter and terminator sequences come from the yeast acid phosphatase gene (PH05Ì (EP 100 561). The complete PH05 promoter is regulated by the supply of inorganic phosphate in the culture medium. High P, concentrations lead to repression of the promoter during an low Pj content has an inductive effect, but a short 173 bp PH05 promoter fragment can be used, which lacks all the regulating elements and which therefore behaves like a constitutive promoter.

2.1 Construction of a phosphate-inducible expression cassette

The GT cDNA sequence is combined as follows with the yeast PH05 promoter and transcription termination sequences:

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(a) Complete HeLa-GT cDNA sequence:

Vector p4AE113 with the complete GT cDNA sequence is digested with the restriction enzymes EcoRI and Bgiii. The DNA fragments are separated electrophoretically on a 1% agarose gel. A 1.2 kb DNA fragment, which contains the complete cDNA sequence for HeLa-GT, is isolated from the gel by adsorption on "glass milk" using the GENECLEAN kit (B10 101). On this fragment, the "ATG" start codon for the protein synthesis of GT is located directly behind the interface for EcoRI, while the "TAG" stop codon is followed by 32 bp, the composition of which is the 3'-untranslated region of the HeLa-GT and the multiple cloning site of the vector with the Bglli restriction site are involved.

(b) Vector for amplification in E. coli:

The amplification vector, plasmid p31R (cf. EP 100 561), a pBR322 derivative, is digested with the restriction enzymes BamHI and Sali. The restriction fragments are separated on a 1% agarose gel and a 3.5 kb vector fragment is isolated from the gel as described above. This DNA fragment contains the large Sall-HindIII vector fragment of the pBR322 derivative and a 337 bp PH05 transcription termination sequence instead of the HindIII-BamHI sequence from pBR322.

(c) Sequence for the inducible PH05 promoter:

The PH05 promoter fragment containing the regulatory elements (UASp) for phosphate induction is isolated from plasmid p31R (cf. EP 100 561) by digestion with the restriction enzymes Sali and EcoRI. The 0.8 kb Sall-EcoRI-DNA fragment contains the 276 bp Sail-BamHI pBR322 sequence and the 534 bp BamHI-EcoR! PH05 promoter fragment with the EcoRI linker (5'-GAATTC-3 ') introduced in position -8 of the PH05 promoter sequence.

(d) Construction of plasmid pGTA 1132

The three DNA fragments (a) to (c) are ligated in a 12 nl ligation mixture: 100 ng DNA fragment (a) and 30 ng each of fragments (b) and (c) are kept at 15 ° C. for 18 hours 0.3 U T4 DNA ligase (Boehringer) in the ligase buffer supplied (66 mM Tris-HCl pH 7.5, 1 mM dithioerythritol, 5 mM MgCb, 1 mM ATP) ligated. Competent cells from the E. coli strain DH5a (Gibco / BRL) are transformed with half of the ligation mixture. For the production of competent cells and for the transformation, the standard protocol as described in the Maniatis manual (see above) is used. The cells are spread on selective LB medium supplemented with 75 μg / ml ampicillin and incubated at 37 ° C. About 120 transformants are obtained. Plasmid minipreparations are carried out from six independent transformants according to the modified alkaline lysis protocol of Birnboim, H.C. and Doly, J., as specified in the Maniatis manual (see above). The isolated plasmids are characterized by restriction analysis with 4 different enzymes (EcoRI, Pstl, HindIII, Sali, and combinations). All six plasmids show the expected restriction fragments. One of the clones is selected and designated pGTA 1132. Plasmid pGTA 1132 contains the expression cassette with the complete HeLa-GT cDNA, which is under the control of the phosphate-regulated PH05 promoter, and the PH05 transcription termination sequence. This expression cassette can be cut out of pGTA 1132 as a 2.35 kb SalI HindIII fragment and is referred to as DNA fragment (1A).

2.2 Construction of a constitutive expression cassette:

For the construction of an expression cassette with a constitutive, unregulated promoter, a 5'-shortened PH05 promoter fragment without phosphate regulatory elements is used, which is isolated from plasmid p31 / PH05M73) RIT.

(a) Construction of plasmid p31 / PH05f-173) RIT

Plasmid p31 RIT12 (EP 288 435) contains the fully regulated PH05 promoter (with an EcoRI site introduced in the nucleotide position -8 on a 534 bp BamHl-EcoRI fragment), which is followed by the coding sequence for the yeast invertase signal sequence (72 bp EcoRI-Xhol) and the PH05 transcription termination signal (135 bp Xhol-HindIII), cloned in tandem between BamHI and HindIII of the vector derived from pBR322.

The constitutive PH05M 73) promoter element from plasmid pJDB207 / PH05M 73VYHIR (EP 340 170) comprises the nucleotide sequence of the yeast PH05 promoter from nucleotide position -9 to -173 (BstEil restriction site), but has no regulatory sequences "upstream" ( UASp). The PH05M731 promoter therefore behaves like a constitutive promoter. This example describes the replacement of the

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regulated PH05 promoter in plasmid p31RIT12 by the short, constitutive PH05M 73) promoter element to obtain plasmid p31 / PH05f-173) RIT.

The plasmids p31RIT12 (EP 288, 435) and pJDB207 / PH05M73) -YHIR (EP 340, 170) are digested with the restriction endonucleases Sali and EcoRI. The respective 3.6 kb and 0.4 kb Sall-EcoRI fragments are isolated on a 0.8% agarose gel, eluted from the gel, precipitated with ethanol and resuspended in H2O at a concentration of 0.1 pmol / nl. The two DNA fragments are ligated and aliquots of the 1 nl ligation mixture are used for the transformation of competent E. coli HB101 (ATCC) cells. Colonies resistant to ampicillin are grown individually in LB medium supplemented with ampicillin (100 p.g / ml). Plasmid DNA is prepared using the method of Holmes, D.S. et al. (Anal. Biochem. (1981) 144, 193) isolated and analyzed by restriction digestion with Sali and EcoRI. The plasmid of a clone with the correct restriction fragments is called p31 / PH05 (-173) RIT.

(b) Construction of plasmid pGTB 1135

Plasmid p31 / PH05M73iRIT is digested with the restriction enzymes EcoRI and Sali. After separation on a 1% agarose gel, a 0.45 kb SalI EcoRI fragment is isolated from the gel using GENECLEAN (BIO 101). This fragment contains the 276 bp Sal-BamHI sequence from pBR322 and the 173 bp BamHI (BstEII) EcoRI fragment of the constitutive PH05 promoter. The 0.45 kb Sall-EcoRI fragment is ligated with the 1.2 kb EcoRI-BglII GT cDNA (fragment (a)) and the 3.5 kb BamHI-Sall vector part is used for amplification in E. coli with the in Example 2.1. described PH05 terminator (fragment (b)) ligated. Ligation and transformation of E. coli strain DH5a are carried out as above, whereby 58 transformants are obtained. Plasmids are isolated from six independent colonies using mini preparations and characterized by restriction analysis. All six plasmids show the expected fragmentation pattern. A correct clone is designated pGTB 1135 and used for further cloning experiments to provide the HeLa-GT expression cassette under the control of the constitutive PH05M 73) promoter fragment. This expression cassette can be cut out from vector pGTB 1135 as a 2 kb SalI-HindIII fragment and is referred to as a DNA fragment (IB).

Example 3:

Construction of the expression vectors dDPGTA8 and pDPGTB5

The yeast vector used for heterologous expression is the episomal vector pDP34 (11.8 kb), which is a yeast E. coli shuttle vector with the ampicillin resistance marker for E. coli and the yeast-selective markers URA3 and dLEU2. Vector pDP34 (cf. EP 340 170) is digested with the restriction enzyme BamHI. The linearized vector is isolated using GENECLEAN and the protruding ends are filled in using Klenow polymerase treatment as described in the Maniatis manual (see above). The reaction is stopped after 30 minutes by heating at 65 ° C. for 20 minutes together with 10 mM EDTA. After ethanol precipitation, the plasmid is digested with saline and the digest is separated by gel electrophoresis on a 0.8% agarose gel. The (BamHI) blunt-ended vector pDP34 cut with saline is isolated from the gel as an 11.8 kb DNA fragment using the GENECLEAN kit.

Analogous to the vector preparation, the plasmids pGTA 1132 and pGTB 1135 are each digested with HindIII. The protruding ends of the linearized plasmids are filled in by means of Klenow polymerase treatment and then subjected to a Sall digestion, whereby

(2A) a 2.35 kb (HindIII) blunt-ended SalI fragment with the phosphate-regulated expression cassette or

(2B) a 2.0 kb (HindIII) blunt-ended Sall fragment with the constitutive expression cassette is formed.

The ligation of the blunt-ended Sali pDP34 vector part with fragment 2A or fragment 2B and the transformation of competent cells of the E. coli strain DH5a are carried out as described above in Example 2, using 80 ng of the vector part and 40 ng of fragment 2A or 2B . 58 and 24 transformants are obtained. Six plasmids from each transformation are prepared and characterized by means of restriction analysis. 2 plasmids have the restriction pattern (fragment 2A) expected for the construction with the regulated expression cassette. One of the clones is chosen and designated pDPGTA8. A plasmid has the restriction pattern expected for construction with the constitutive expression cassette (fragment 2B) and is referred to as pDPGTB5.

Example 4:

Transformation of the S. cerevisiae strain BT 150

DNA of the expression vectors pDPGTA8 and pDPGTB5 purified by means of CsCI is produced according to the protocol by R. Treisman in the Maniatis manual (see above). Each of the protease deficient

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cerevisiae strains BT 150 (MATa, his4, Ieu2, ura3, pral, prbl, prcl, cpsl) and H 449 (MATa, prbl, cpsl, ura3A5, leu 2-3, 2-112, cir °) each have 5 the plasmids pDPGTA8, pDPTGB5 and pDP34 (control without expression cassette) were transformed according to the lithium acetate transformation method (Ito, H. et al., see above). Ura + transformants are isolated and used for screening for GT activity (see above). Individual transformed yeast cells are selected and labeled as follows:

Saccharomvces cerevisiae BT 150 / pDPGTA8 Saccharomvces cerevisiae BT 150 / pDPGTB5 Saccharomvces cerevisiae BT 150 / pDP34

Saccharomvces cerevisiae H 449 / pDPGTA8 Saccharomvces cerevisiae H 449 / pDPGTB5 Saccharomvces cerevisiae H 449 / pDP34

Example 5:

Enzyme activity of complete GT expressed in yeast

5.1 Production of cell extracts

Cells of the transformed Saccharomvces cerevisiae strains BT 150 and H 449 are each grown with uracil selection in minimal yeast media (Difco) enriched with histidine and leucine. The growth rate of the lines is in no way influenced by the introduction of an expression vector. Exponentially growing cells (with an OD578 of 0.5) or stationary cells are harvested by centrifugation, once with 5 mM Tris-HCl buffer, pH 7.4 (buffer 1 ), washed and suspended in a concentration corresponding to 0.1-0.2 OD578 in buffer 1. The cells are broken up mechanically by shaking vigorously for 4 minutes on a vortex mixer with glass balls (0.45-0.5 mm in diameter), with cooling in between. The crude extracts are used directly for the determination of the enzyme activity.

Fractionation of the cell components is achieved by differential centrifugation of the extract. The extract is first centrifuged at 450 g for 5 minutes; the supernatant obtained is then centrifuged at 20,000 g for 45 minutes.

The supernatant is drawn off and the pellets are suspended in buffer 1.

For the phosphate induction of GT expression under the control of the inducible PH05 promoter, cells of the transformants BT 150 / pDPGTA8 and H 449 / pDPGTA8 are converted in bulk to a minimal medium with a low phosphate content (Meyhack, B. et a. Embo J. (1982) 1, 675-680). The cell extracts are prepared as described above.

5.2 Protein test

Protein concentration is determined using a BCA protein assay kit (Pierce).

5.3 Test for GT activity

GT activity can be measured by radiochemical methods either using ovalbumin, a glycoprotein that has only GIcNAc as the acceptor, or free GIcNAc as the acceptor substrate. Cell extracts (with 1-2 cell-related ODS578) are left for 45 or 60 minutes at 37 ° C in 100 (il incubation mixture with 100 mM Tris-HCl pH 7.4, 50 nCi UDP-i4C-Gal (325 mCi / mmol), 80 nmol UDP-Gal, 1 µmol MnCk, 1% Triton X-100 and 1 mg ovalbumin or 2 nmol GIcNAc tested as acceptor. If a glycoprotein acceptor substrate is used, the reaction is stopped by acid precipitation of the protein and the amount of 14C- Galactose, which was incorporated into ovalbumin, is determined by liquid scintillation counting (Berger, EG et al. (1978) Eur. J. Biochem. 90, 213-222) ml ice-cold H2O is stopped, and the unabsorbed UDP-14C-galactose is separated from the ^ products on an anion exchange column (AG X1-8, BioRad) as described (Masibay, AS and Qasba, PK (1989) Proc. Nati Acad Sci. USA 86, 5733-5737) Tests with and without acceptor molecules are carried out to determine the extent of the Hy Drolysis of UDP-Gal to be assessed by nucleotide pyrophosphatases. The results are shown in Table 2.

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Table 2:

GT activity in the S. cerevisiae strain BT 150 transformed with various plasmids.

Plasmid

PH05 promoter

GT specific activity high Pi

(mU / mg protein) low Pi pDP34

-

<0.01

n.b.1)

pDPGTA8

phosphate regulated

0.1

0.6-1

pDPGTB

constitutive

0.6

n.b.1)

1) not determined

The GT activity of cultures switched to inducible conditions (minimal medium with low Pj content) is approximately equal to the activity of cultures in minimal media which constitutively express GT (Table 2). As expected, there is no enzyme activity in cells that are only transformed with the vector.

During the fractionation, the most GT activity (70-90%, see Table 3) and the highest specific activity are always found in the pellet obtained at the high acceleration (20,000 g). Enzyme activity can be increased by adding 1-2% Triton X-100 to the enzyme test. Both results suggest that recombinant GT is membrane-bound in yeast cells as well as in HeLa cells.

Table S. Distribution of GT activity during fractionation of BT 150 / pDPGT5 cells

fraction

GT activity in GIcNAc-built UDP-i4C-Gal, in cpm

%

Raw act

19400

400 g pellet

1000

4.6%

20,000 g pellet

15800

72.5%

20,000 g of supernatant

5000

22.9%

total

21800

100%

Example 6:

Purification of the recombinant GT

To release the membrane bound enzyme, the 20,000 g pellet fraction of the BT 150 / pDPG-TB5 cells is treated with 1% (w / v) Triton X-100 at room temperature for 10 minutes and with 50 mM Tris HCl pH 7.4 , 25 mM MgCl2, 0.5 mM UMP and 1% (w / v) Triton X-100 equilibrated. The supernatant obtained after centrifugation is filtered 0.2 .mu.m and a filtrate is charged to a GIcNAc-p-aminophenyl-Sepharose column (Berger, EG et al. (1976) Experientia 32, 690-691), the bed volume being 5 ml and the flow rate is 0.2 ml / min at 4 ° C. The enzyme is eluted with 50 mM Tris-HCl pH 7.4, 5 mM GIcNAc, 25 mM EDTA and 1% Triton X-100. A single peak with enzyme activity is eluted within five fractions (size: 1 ml). These fractions are pooled and dialyzed against 2 x 1150 mM Tris-HCl pH 7.4, 0.1% Triton X-100. The cleaned GT is enzymatically active.

Example 7:

Construction of an expression cassette for soluble GT

Galactosyltransferase expressed in yeast can be secreted into the culture medium if a yeast promoter is functionally linked to a first DNA sequence which codes for the signal sequence of the yeast invertase gene SUC2 and which is linked in the correct reading frame to a cDNA which is used for a soluble GT encoded.

(a) HeLa GT cDNA sequence

GT cDNA is released from plasmid p4AD113 (Example 1) by means of EcoRI digestion. The 1.2 kb fragment with the complete GT cDNA is isolated and partially digested with Mvnl (Boehringer), the GT

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Sequence in positions 43, 55, 140 and 288 of the in SEQ ID no. 1 shown nucleotide sequence is cut. When 0.2 µg of the EcoRI-EcoRI fragment is digested with 0.75β Mvnl for one hour, the GT cDNA is cut only once at nucleotide position 134, with a 1.1 kb Mvnl EcoRi fragment arises.

(b) Vector for amplification in E. coli

Plasmid pUC18 (Pharmacia) is cut with BamHI and EcoRI at the multiple cloning site. The plasmid is then treated with alkaline phosphatase as described in the Maniatis manual (see above), subjected to agarose gel electrophoresis, and isolated from the gel as a 2.7 kb DNA fragment.

(c) The constitutive PH05M 73) promoter and the SUC2 sianal sequence

The vector p31 / PH05f-173l RIT (Example 2) is digested with the restriction enzymes BamHI and Xhol. A 0.25 kb BamHI-Xhol fragment with the constitutive PH05H 73) promoter and the adjacent sequence, which codes for the invertase signal sequence (inv ss), is isolated. The fragment is then cut again with Hgal (BioLabs). The Hgal recognition sequence is on the DNA antisense strand. The restriction enzyme cuts "upstream" of the recognition sequence in such a way that the 5-protruding end of the antisense strand coincides with the end of the coding sequence of the invertase signal sequence. The 0.24 kb DNA fragment cut with BamHI and Hgal contains the constitutive PH05 (-173) promoter sequence which is linked to the yeast invertase signal sequence.

(d) adapter

Fragment (a) is linked to fragment (c) by means of an adapter sequence which is produced from equimolar amounts of the synthetic oligonucleotides (microsynth) 5'CT GCA CTG GCT GGC CG 3 'and 5'CG GCC AGC CAG 3' for the complementary strand. The oligonucleotides are attached to one another by first heating them to 95 ° C. and then slowly cooling them to 20 ° C. The attached adapter is kept frozen.

(e) Construction of the plasmid psGT

For the ligation, the linearized vector (b), the GT cDNA fragment (a), fragment (c) with the promoter and the sequence coding for the signal peptide, and adapter (d) in a molar ratio of 1: 2 : 2: 30-100. The ligation is carried out in 12 ni ligase buffer (66 mM Tris-HCl pH 7.5, 1 mM dithioerythritol, 5 mM MgCfe, 1 mM ATP) at 16 ° C. for 18 hours. The ligation mixture is used for the transformation of competent E. coli strain DH5a cells (see above). Plasmid mini-preparations are carried out from 24 independent transformants. The isolated plasmids are characterized by restriction analysis with four different enzymes (BamHI, Pstl, EcoRI, Xhol, also in combination). A single clone with the expected restriction pattern is called psGT.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide with the cDNA encoding soluble GT is confirmed for plasmid psGT by the fact that the T7 sequencing kit (Pharmacia) and primer 5 'AGTCGAGGTTAGTATGGC 3', beginning in position -77 in the constitutive PH05M 73i promoter can be used.

DNA Mvnl

Sequence ^^: 5 'AAA ATA Tct gca ctg gct ggc cgC GAC CTG AGC 3'

3 'TTT TAT AGA CGT Protein: Lys Ile Ser Ala

16 19

inv ss gac cga ccg gcG CAG GAC TCG 5 'Leu Ala Gly Arg Asp Leu Ser 42

GT sequence interface for signal endopeptidase

Lower case letters denote the adapter sequence

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The expression cassette for soluble GT, which contains the constitutive PH05M 73) promoter, the DNA sequence coding for inverted signal peptide and the partial GT cDNA from the plasmid psGT, can be in the form of a 1.35 kb Sali (BamHI) -EcoRI fragments are cut out. The expression cassette is still missing the PH05 terminator sequences. which are added in the subsequent cloning step.

Example 8:

Construction of the expression vector dDPGTS

The following fragments are assembled to construct the soluble GT expression vector:

(a) Vector part

Plasmid pDP34 is digested and pretreated as described in Example 3, producing a blunt-digested Sall vector fragment of 11.8 kb.

(b) Expression cassette

Plasmid psGT is first linearized by digestion with Sali (at the multiple cloning site) and then partially digested with EcoRI. A 1.35 kb DNA fragment containing the constitutive PH05f-173i promoter, a DNA sequence encoding the yeast invertase signal peptide and the partial GT cDNA is isolated.

(c) PH05 terminator sequence

The PH05 terminator sequence is isolated from plasmid p31, which is constructed from plasmid p30 as described in EP 100 561. After digestion with the restriction enzyme HindIII, the protruding ends are filled in using Klenow polymerase treatment as described above. The plasmid is then digested with EcoRI and a blunt-ended EcoRI fragment of 0.39 kb with the PH05 terminator sequences is isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells of the E. coli strain DH5a are carried out as described in Example 2. Plasmids are isolated from the transformants obtained and characterized by restriction analysis. The plasmid of a clone with the correct restriction fragments is called pDPGTS.

Example 9:

Expression of soluble GT in yeast

Analogously to Example 4, DNA of the expression vector pDPGTS purified by means of CsCI is used for the transformation of the S. cerevisiae strains BT 150 and H 449. Ura + transformants are isolated and screened for GT activity. One transformant each is selected and referred to as Saccharomvces cerevisiae BT 150 / pDPGTS and Saccharomvces cerevisiae H 449 / pDPGTS, respectively. When the test described in Example 5 is carried out, GT activity is found in the culture broths of both transformants.

Example 10:

Cloning of siaiyitransferase (STi cDNA from human HepG2 cells

ST-cDNA is isolated from HepG2 cells by means of PCR analogous to GT-cDNA. The preparation of poly (A) + RNA and the synthesis of first-strand cDNA are carried out as described in Example 1. The primers (Microsynth) used for the PCR are listed in Table 5.

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Table 5: PCR primer corresponds to bp

Primer sequence (5 'to 3') 1} in ST cDNA2)

PslI / EcoRI

SIA1 cgctgcagaattcaaaATGATTCACACCAACCTGAAGAAAAAGT 1 - 28 BamHI '

SI A3 cgcggatCCTGTGCTTAGCAGTGAATGGTCCGGAAGCC 1228-1198

^ Upper case letters stand for ST sequences, lower case letters are additional sequnts with restriction points (underlined). "Start" and "stop" codons for protein synthesis are printed in bold.

^ ST cDNA sequence from human placenta (27) as published in EMBL Data Bank (Issue No.X 17247).

HepG2-ST cDNA can be amplified using the primers SIA1 and SIA3 as a 1.2 kb DNA fragment. The PCR is carried out as described for GT-cDNA under slightly changed cycle conditions: 0.5 minutes denaturing at 95 ° C, 1 minute 15 seconds attachment at 56 ° C, and 1 minute 30 seconds extension at 72 ° C, over a total of 25 to 35 cycles. In the last cycle, primer extension takes 5 minutes at 72 ° C.

After the PCR amplification, the 1.2 kb fragment is digested with the restriction enzymes BamHI and Pstl, the digestion is analyzed on a 1.2% agarose gel, eluted from the gel and subcloned into the vector pl) C18. The subclone obtained is called pSIA2.

Example 11:

Construction of the constitutive ST expression cassette

For constitutive heterologous expression, ST cDNA is ligated with the constitutive PH05 (-173) promoter fragment and the PH05 terminator sequences.

Plasmid pSIA2 is first linearized by digestion with the restriction enzyme BamHI and then partially digested with EcoRI. Since ST-cDNA also contains an internal restriction site for the enzyme EcoRI (at bp 144), a 1.2 kb fragment with the complete ST-cDNA (SEQ. ID No. 3) is produced by partial digestion with EcoRI, 1 p , g DNA and 0.25 U EcoRI can be used (1 h, 37 ° C). After gel electrophoresis, the 1.2 kb EcoRI-BamHI fragment with the complete ST cDNA (SEQ ID No. 3) is isolated. The «ATG» start codon for the translation of ST is located on this DNA fragment in the vicinity of the EcoRI restriction site. Three adenosine phosphates (see PCR primer SIA 1) provide an “A” in bp position 12, which is found in the consensus sequence for “ATG” from highly expressed genes in yeast (Hamilton, R. et al. (1987) Nucleic Acids Res. 14, 5125-5149). The BamHI site follows the stop codon “TAA” and 5 bp of the 3 ′ untranslated gene region.

The 1.2 kb EcoRI-BamHI-ST cDNA fragment is combined with the 0.45 kb Sall-EcoRI fragment with the constitutive PH05M73) promoter (Example 2.2 (b)) and with a 3.5 kb BamHI-Sall -Vector part for the amplification in E. coli, which contains the PH05 terminator sequence (cf. Example 2.1, fragment (b)) ligated. Ligation and transformation of E. coli strain DH5a are carried out as specified in Example 2.1. A clone that has the expected restriction pattern is called pST2.

Vector pST2 contains the expression cassette for HepG2-ST under the control of the constitutive PH05M 73) promoter in the form of a 2.0 kb SalI-HindIII fragment, which is referred to as a DNA fragment (IC).

Example 12:

Expression of ST in yeast

Vector pDP34 (cf. EP 340 170) is digested with the restriction enzyme BamHI. The linearized vector is isolated with GENECLEAN and the protruding ends are filled in using Klenow polymerase treatment as described in the Maniatis manual (see above). The reaction is stopped after 30 minutes by heating at 65 ° C for 20 minutes in the presence of 10 mM EDTA. After ethanol precipitation, the plasmid is digested with saline and subjected to gel electrophoresis on a 0.8% agarose gel. The (BamHI) blunt-ended, sali-cut vector pDP34 is isolated with the GENECLEAN kit in the form of an 11.8 kb DNA fragment.

Plasmid pST2 is digested with the restriction enzyme HindIII and analogous to the preparation of the vector

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tors filled by Klenow polymerase treatment at the Hindi I site, analogously to the preparation of the vector. The product is digested with sali, whereby a 2.0 kb (Hindlll) blunt-ended SalI fragment is formed with the constitutive ST expression cassette (2C).

The ligation of 80 ng of the pDP34 vector with 40 ng fragment 2C, and the transformation of competent cells from E. coli strain DH5a is carried out as described in Example 2. A clone that has the expected restriction pattern is selected and designated pDPSTo.

For the transformation of yeast, DNA of the expression vector pDPST5 purified with CsCI is produced according to the standard method given in the Maniatis manual (see above). The S. cerevisiae strains BT 150 and H 449 are each transformed with 5 ng plasmid DNA according to the lithium acetate transformation method (Ito, H. et al, see above). Ura + transformants are selected and screened for ST activity. In each case a positive transformant is selected and designated Saccharomvces cerevisiae BT 150 / pDPST5 and Saccharomvces cerevisiae H 449 / pDPST5.

Example 13:

Enzyme activity of complete ST expressed in yeast

13.1 Production of cell extracts

Saccharomvces cerevisiae BT 150 / pDPST5 cells are grown with uracil selection on minimal yeast media enriched with histidine and leucine. Exponentially growing cells (with an OD578 of 0.5) or stationary cells are harvested by centrifugation, washed once with 50 mM imidazole buffer pH 7.0 (buffer 1), and in again at a concentration corresponding to 0.1-0.2 OD578 Buffer 1 suspended. The cells are broken up mechanically by shaking vigorously for 4 minutes with glass beads (0.45-0.5 mm in diameter) on a vortex mixer, with cooling in between. The ST activity in the crude extracts can be measured using the test described below.

13.2 Test for ST activity

ST activity can be determined by measuring the amount of radiolabeled sialic acid that is transferred from CMP sialic acid to a glycoprotein acceptor. After completion of the reaction by acid precipitation, the precipitate is filtered through glass fiber filters (Whatman GFA), washed thoroughly with ice-cold ethanol and tested for radioactivity by liquid scintillation counting (Hesford et al. (1984), Glycoconjugate J. 1, 141-153). The cell extracts are tested for 45 minutes in an incubation mixture containing 37 nl cell extract, which corresponds to approximately 0.5 mg protein, and 3 n 'imidazole buffer 50 mmol / i, pH 7.0, 50 nmol CMP-N-acetylneuraminic acid ( Sigma) with the addition of CMP-3H-N-acetylneuraminic acid (Amersham) to finally obtain a specific activity of 7.3 Ci / mol and 75 ng Asialo-Fetuin (produced by acid hydrolysis with 0.1 M H2SO4 for 60 minutes 80 ° C and subsequent neutralization, dialysis and lyophilization) contains.

ST activity is found in the crude extracts made from S. cerevisiae BT 150 / pDPST5 and H449 / pDPST5 cells.

Example 14:

Construction of an expression cassette for soluble ST (Lvsaa-Cvsanfii

The soluble ST (Lys39-Cys406) is an N-terminal shortened variant and consists of 368 amino acids (SEQ. ID No. 4).

(a) HepG2-ST cDNA partial sequence

Plasmid pSIA2 is digested with EcoRI and a 1.1 kb EcoRI-EcoRi fragment is isolated.

(b) Vector for amplification in E. coli

Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRI at the multiple cloning site. The plasmid is then treated with alkaline phosphatase as described in the Maniatis manual (see above), subjected to agarose gel electrophoresis and isolated from the gel in the form of a 2.7 kb DNA fragment.

(c) The constitutive PH05f-173) promoter and the SUC2 sianal sequence

The vector p31 / PH05f-173ì RIT (Example 2) is digested with the restriction enzymes BamHI and Xhol. A 0.25 kb BamHI-Xhol fragment with the constitutive PH05M73) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is isolated. The fragment is then cut again with Hgal (BioLabs). The Hgal recognition sequence can be found on the DNA antisense strand. The restriction enzyme cuts to sol18 above the recognition sequence

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way that the 5-protruding end of the antisense strand coincides with the end of the coding sequence of the invertase signal sequence. The 0.24 kb DNA fragment cut with BamHI and Hgal contains the constitutive PH05H 73) promoter sequence bound to the yeast invertase signal sequence.

(d) adapter

Fragment (a) is linked to fragment (c) via an adapter sequence which consists of equimolar amounts of the synthetic oligonucleotides 5 'CTGCAAAATTGCAAACCAAGG 3' and

5 'AATTCCTTGGTTTGCAATTT 3' is produced in the case of the complementary strand. The oligonucleotides are attached to one another by first heating them to 95 ° C. and then slowly cooling them to 20 ° C. The attached adapter is stored in the frozen state.

(e) Construction of a plasmid psST

For the ligation, the linearized vector (b), the STcDNA fragment (a), fragment (c) with the promoter and the sequence coding for the signal peptide, and adapter (d) are used in a molar ratio of 1: 2: 2: 30-100 used. The ligation is carried out over 18 hours in 12 μl ligase buffer (66 mM Tris-HCl pH 7.5, 1 mM dithioerythritol, 5 mM MgCl 2, 1 mM ATP) at 16 ° C. Competent cells of the E. coli strain DH5a are transformed with the ligation mixture as described above. Plasmid mi-nip preparations are made from 24 independent transformants. A single clone that shows the expected restriction pattern after characterization by restriction analysis with four different enzymes (BamHI, Pstl, EcoRI, Xhol, also in combination) is called psST.

The correct sequence at the fusion site of the sequence coding for the invertase signal peptide with the cDNA coding for soluble ST (Lys39-Cys406) is determined for plasmid psST using the T7-Sequence-cing Kit (Pharmacia) and primer 5 'ACGAGGTTAATGGC 3', the starts at position -77 in the constitutive PH05 - (- 173) promoter, confirmed.

DNA

Sequence ^ - ^

Protein inv ss ST sequence

Interface for signal endopeptidase

^ Lower case letters denote the adapter sequence

The expression cassette for soluble ST with the constitutive PH05M 73V promoter. the DNA sequence coding for invertase signal peptide and the partial ST cDNA from plasmid psST can be excised in the form of a 1.35 kb Sali (BamHI) EcoRI fragment. The expression cassette still lacks the PH05 terminator sequences to be added in the subsequent cloning step.

Example 15:

Construction of the expression vector pDPSTS

The following fragments are assembled to construct the expression vector for soluble ST (Lys39-Cys406):

5 '

AAA

ATA

Tct gca aaa ttg caa acc aag gAA 3 '

3 '

TTT

DID

AGA

GTG

ttt aac gtt tgg ttc ctt 5 '

Lys

Ile

Ser

Ala

Lys

Leu

gin

Thr

Lys

Glu

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(a) Vector part

Plasmid pDP34 is digested and pretreated as described in Example 3, which results in a blunt-ended SalI vector fragment of 11.8 kb.

(b) Expression cassette

Plasmid psST is first linearized by digestion with Sali (at the multiple cloning site) and then partially digested with EcoRI. A 1.35 kb DNA fragment with the constitutive PH05M 73i promoter. a DNA sequence coding for the yeast invertase signal peptide and the partial ST cDNA is isolated.

(c) PH05 terminator sequence

The PH05 terminator sequence is isolated from plasmid p31, which is constructed from plasmid p30 as described in EP 100 561. After digestion with the restriction enzyme Hindill, the protruding ends are filled in using Klenow polymerase treatment as described above. The plasmid is then digested with EcoRI and a 0.39 kb blunt-ended EcoRI fragment with the PH05 terminator sequences is isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells of the E. coli strain DH5a are carried out as described in Example 2. Plasmids are isolated from the transformants obtained and characterized by means of restriction analysis. The plasmid of a clone with the correct restriction fragments is called pDPSTS.

Example 16:

Expression of soluble ST in yeast

Analogously to Example 4, S. cerevisiae strains BT 150 and H 449 are transformed by means of DNA of the expression vector pDPSTS purified with CsCI. Ura + transformants are isolated and screened for ST activity. One transformant each is selected and designated Saccharomvces cerevisiae BT 150 / pDPSTS and Saccharomvces cerevisiae H 449 / pDPSTS. With the test described in Example 13, ST activity is found in the culture broths of both transformants.

Example 17:

Construction of an expression cassette for soluble STfLvs ^ -Cvsdnfii

The soluble ST (Lys27-Cys406) is an N-terminal shortened variant that encompasses the entire stem region and the catalytic domain. It consists of 380 amino acids, that is amino acids 27 to 406 of the SEQ ID no. 3 given amino acid sequence.

(a) HepG2-ST cDNA partial sequence

Plasmid pSIA2 is digested with EcoRI and a 1.1 kb EcoRI-EcoRI fragment is isolated.

(b) Vector for amplification in U. coli

Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRI at the multiple cloning site. The plasmid is then treated with alkaline phosphatase as described in the Maniatis manual (see above), subjected to agarose gel electrophoresis and isolated from the gel as a 2.7 kb DNA fragment.

(c) The constitutive PH05M 73) promoter and the SUC2 signal sequence

The vector p31 / PH05 (-173l RIT (Example 2) is digested with the restriction enzymes BamHI and Xhol. A 0.25 kb BamHI-Xhol fragment with the constitutive PH05H 73) promoter and the adjacent coding sequence for the invertase signal sequence ( inv ss) is isolated. The fragment is then cut again with Hgal (BioLabs). The Hgal recognition sequence can be found on the DNA antisense strand. The restriction enzyme cuts "upstream" of the recognition sequence in such a way that the 5-protruding end of the antisense strand matches the end of the coding sequence of the invertase signal sequence. The 0.24 kb DNA fragment cut with BamHI and Hgal contains the PH05M 73) promoter sequence bound to the yeast invertase signal sequence.

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(d) adapter

Fragment (a) is linked to fragment (c) via an adapter sequence, which is produced from equimolar amounts of the synthetic oligonucleotides 5 'CTGCAAAGGAAAA GAAGAAAGGGAGTTACTATGATTCCTTT AAATTGCAAACCAAGG 3', and 5 'AATTCCTTGTTGCAATTTAAAGGAATTCTTaTtrT in the complTCCTT 3Rst case in the complTCCTT 3RT case. The oligonucleotides are attached to one another by first heating them to 95 ° C. and then slowly cooling them to 20 ° C. The attached adapter is stored in the frozen state.

(e) Construction of plasmid psST1

For the ligation, the linearized vector (b), the STcDNA fragment (a), fragment (c) with the promoter and the sequence coding for the signal peptide, and adapter (d) are used in a molar ratio of 1: 2: 2: 30-100 used. The ligation is carried out over 18 hours in 12 μl ligase buffer (66 mM Tris-HCl pH 7.5, 1 mM dithioerythritol, 5 mM MgCb, 1 mM ATP) at 16 ° C. With the ligation mixture, competent cells of the U. coli strain DH5ot are transformed as described above. Plasmid mi-nip preparations are made from 24 independent transformants. A single clone, which after characterization by means of restriction analysis with four different enzymes (BamHI, Pstl, EcoRI, Xhol, also in combination) shows the expected restriction pattern, is referred to as psSTI.

The correct sequence at the fusion site of the sequence coding for the invertase signal peptide with the cDNA coding for soluble ST (Lys27-Lys406) is for plasmid psSTI using the T7-Sequencing-Kit (Pharmacia) and primer 5 'AGTCGAGTAGTATGGC 3', the starts at position -77 in the constitutive PH05M 73 ^ promoter, confirmed.

DNA

Sequence-

protein

5 '

AAA

ATA

Tct gca aag gaa aag aag aaa ggg

3 '

3 '

TTT

DID

AGA

GTG

ttc ctt ttc ttc ttt ccc

5 '

Lys

Ile

Ser

Ala

Lys

Glu

Lys

Lys

Lys

Gly

16

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27

mv ss ST sequence

Interface for signal endopeptidase l) Lower case letters denote the adapter sequence

The expression cassette for soluble ST (Lys27-Cys406) with the constitutive PH05M 73) promoter, the DNA sequence coding for invertase signal peptide and the partial ST cDNA from plasmid psSTI can be in the form of a 1.35 kb Sali (BamHI) - EcoRI fragments are cut out. The expression cassette still lacks the PH05 terminator sequences to be added in the subsequent cloning step.

Example 18:

Construction of the expression vector pDPSTSI

The following fragments are combined to construct the expression vector for soluble ST (Lys27-Cys406):

(a) Vector part

Plasmid pDP34 is digested and pretreated as described in Example 3, which results in a blunt-ended SalI vector fragment of 11.8 kb.

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(b) Expression cassette

Plasmid psSTI is first linearized by digestion with Sali (at the multiple cloning site) and then partially digested with EcoRI. A 1.35 kb DNA fragment with the constitutive PH05M73) promoter, a DNA sequence coding for the yeast invertase signal peptide and the partial ST cDNA is isolated.

(c) PH05 terminal sequence

The PH05 terminator sequence is isolated from plasmid p31, which is constructed starting from plasmid p30 as described in EP 100 561. After digestion with the restriction enzyme HindIII, the protruding ends are filled in by means of Klenow polymerase treatment as described above. The plasmid is then digested with EcoRI and a 0.39 kb blunt-ended EcoRI fragment with the PH05 terminator sequences is isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells from U. coli strain DH5a are carried out as described in Example 2. Plasmids are isolated from the transformants obtained and characterized by means of restriction analysis. The plasmid of a clone with the correct restriction fragments is called pDPSTSI.

Example 19:

Expression of soluble ST (Lvs? 7-Cvs4nRÌ in yeast

Analogously to Example 4, S. cerevisiae strains BT 150 and H 449 are transformed by means of DNA of the expression vector pDPSTS 1 purified with CsCI. Ura + transformants are isolated and screened for ST activity. One transformant each is selected and referred to as Saccharomvces cerevisiae BT 150 / pDPSTS1 and Saccharomvces cerevisiae H 449 / pDPSTS1. With the test described in Example 13, ST activity is found in the culture broths of both transformants.

Example 20:

Cloning the FTcDNA from the human HL 60 cell line

Based on the published cDNA sequence (Goelz, SE et al. (1990) Cell 63, 1349-1356) for ELFT (ELAM-1 ligand fucosyltransferase) which codes for a (1-3) fucosyltransferase, the developed the following oligonucleotide primer in order to amplify a DNA fragment which contains the open reading frame of the ELFT cDNA by means of the PCR technique: 5'-CAGCGCTGCCTGTTCGCGCCAT-3 '(ELFT-1B) and 5-GGAGATGCACAGTAGAGGATCA-3' (ELFT -2 B). ELFT-1B acts as a primer at base pairs 38-59 of the published sequence, and ELFT-2B at bp 1347-1326 in the antisense strand. A 1.3 kb fragment is amplified with these primers using the Perkin-Elmer Cetus Taq polymerase kit. The fragment is amplified from fresh HL60 cDNA in the presence of 5% DMSO and 2 mM MgCb, the cycle being as follows:

95 ° C 5 min

25 x (1 min 95 ° C, 1 min 60 ° C, 1.5 min 72 ° C)

10 x (1 min 95 ° C, 1 min 60 ° C, 1.5 min + 15 sec / cycle 72 ° C).

Agarose gel electrophoresis shows a distinct 1.3 kb band, which, when digested with Smal or Apal, shows the pattern predicted by the published sequence. The 1.3 kb band is purified using a gene clean kit (Bio 101) and subcloned into the vector pCR1000 (Invitrogen). A single clone with the correct 1.3 kb insert is selected and designated as BRB.ELFT / pCR1000-13. The FTcDNA is inserted into the vector so that it is oriented in opposite directions with respect to the 17 promoter. The open reading frame of the FTcDNA is completely sequenced and is identical to the published sequence (SEQ ID No. 5)

Example 21:

Construction of plasmids for inducible and constitutive expression of soluble FTfArqfip-Arcunsi in yeast

Soluble FT (Arg62-Arg405) is expressed by the FT cDNA starting at nucleotide position 241 (Nrul restriction site), lacking the region coding for the cytoplasmic tail and membrane-encompassing domain (see Sequence ID No. 5 ).

Plasmid BRB.ELFT / pCR1000-13 is digested with HindIII which cuts in the multiple cloning region 3 'of the FT cDNA insert. The cohesive ends are converted to blunt ends in a reaction with Klenow DNA polymerase. Xhol linkers (5 'CCTCGAGG 3', Biolabs) are treated with kinase, annealed and ligated to the blunt ends of the plasmid using a 100-fold molar excess of linkers. Excess linkers are removed by isopropanol precipitation of the DNA and this is further digested with Xhol and Nrul (cut at nucleotide position 240 of the

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FT cDNA according to sequence ID no. 5). The 1.1 kb Nrul-Xhol fragment (a) contains the FT-cDNA sequence without the region coding for the cytoplasmic tail and the membrane-encompassing domain up to amino acid 61.

The plasmids p31 RIT12 and p3 1 / PH05 (-173) RIT (see Example 2) are digested with Sali and Xhol, respectively. The 0.9 kb and 0.5 kb fragments are isolated and cut with Hgal. The resulting cohesive ends are filled in with Klenow DNA polymerase. The blunt ends so produced match the 3 'end of the coding sequence for the yeast invertase signal sequence. Subsequent cutting with BamHI yields a BamHI blunt end fragment (b) with 596 bp, which contains the inducible PH05 promoter and the invertase signal sequence with its own ATG or a BamHI blunt end fragment (c) with 234 bp with the short constitutive PH05M 73) promoter and the invertase signal sequence.

Plasmid p31 RIT12 is linearized with the restriction endonuclease Sali. Partial digestion with HindIII in the presence of ethidium bromide gives a 1 kb SalI-HindIII fragment which contains the 276 bp Sail-BamHI-pBR322 sequence, the 534 bp promoter of the acidic phosphatase from yeast PH05. contains the yeast invertase signal sequence (encoding 19 amino acids) and the PH05 transcription terminator. The 1 kb SalI-HindIII fragment of p31RIT12 is used in yeast E. coli shuttle vector pJDB207 (Beggs, J.D. in: Molecular Genetics in yeast, Alfred Benzon Symposium 16, Copenhagen, 1981, pages 383-389), which had previously been cut with Sali and Hindlll. The resulting plasmid with the 1 kb insert is called pJDB207 / PH05-RIT12.

Plasmid pJDB207 / PH05-RIT12 is digested with BamHI and Xhol, and the large 6.8 kb BamHI-Xhol fragment (d) is isolated. This fragment contains all sequences of the pJDB207 vector and the PH05 transcription terminator.

The BamHI blunt end fragment (b) with 596 bp, the 1.1.kb Nrul-Xhol fragment (a) and the 6.8 kb Xhol-BamHI vector fragment (d) become blunt under standard conditions for the ligation Ends ligated. Competent E. c.oli HB101 cells are transformed with aliquots of the ligation mixture. Plasmid DNA from ampicillin-resistant colonies is analyzed by restriction digestion. A single clone with the correct expression plasmid is called pJDB207 / PH05-l-FT.

Ligation of the DNA fragments (c), (a) and (d) gives the expression plasmid pJDB207 / PH05 (-173) -I-FT. The expression cassettes of these plasmids contain the coding sequence of the invertase signal sequence, which is fused in frame with that of the soluble a (1 ~ 3) fucosyltransferase, which is expressed under the control of the inducible PH05 or constitutive PH05M 73) promoter. The expression cassettes are cloned into the yeast U coli shuttle vector pJDB207 between the BamHI and HindIII restriction sites.

The nucleotide sequence at the fusion site between the invertase signal sequence and the cDNA coding for soluble FT is confirmed by DNA sequencing on a double-strand plasmid DNA, using the primer 5 'AGTCGAGGTTAGTATGGC 3', which is from position -77 to -60 Nucleotide sequence of the PH05 and the PH05 (-1731 promoter. The correct connection looks like this:

DNA

Sequence:

5 '

AAA

ATA

TCT

GCA

CGA

CCG

GTG

3 '

3 '

TTT

DID

AGA

G TG

GCT

GGC

CAC

5 '

protein

Lys

Ile

Ser

Ala

Arg

Per

Val

16

19th

62

inv ss FT sequence

Interface for signal endopeptidase

Example 22:

Construction of plasmids for the inducible and constitutive expression of membrane-bound FT in yeast

These constructions use the coding sequence of the FT cDNA with its own ATG. However, the nucleotide sequence immediately upstream of the ATG is relatively un23 due to its high G-C content

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favorable for expression in yeast. This region was replaced by an A-T-rich sequence using PCR methods. At the same time, an EcoRI restriction site is introduced at the new nucleotide positions -4 to -9.

Table 6: PCR primers

Primer sequence (5 'to 3') "^ corresponds

FT cDNA nucleotide

FT1 cgaga a 11 cat aATGGGGGCACCGTGGGGC 58-75

FT2 ccqctcgagGAGCGCGGCTTCACCGCTCG 1285-1266

1) Capital letters mean nucleotides from the FT, lower case letters are additional new sequences, restriction sites are underlined, and "start" and "stop" codons are printed in bold.

Using standard PCR conditions, the FT cDNA with the primers FT1 and FT2 (see Table 6) in 30 cycles of DNA synthesis (Taq DNA polymerase, Perkin-Elmer, 5 U / nl one minute at 72 ° C.), Denaturation (10 seconds at 93 ° C) and attachments (40 seconds at 60 ° C) amplified. The resulting 1.25 kb DNA fragment is purified by phenol extraction and ethanol precipitation, and then digested with EcoRI, Xhol and Nrul. The 191 bp EcoRI-Nrul fragment (e) is isolated on a preparative 4% Nusieve 3: 1 agarose gel (FMC BioProducts, Rockland, ME, USA) in Tris-Borate buffer pH 8.3, eluted from the gel and with ethanol like. Fragment (e) contains the 5 'part of the FT gene coding for amino acids 1 to 61. The DNA has a 5 extension with an EcoRI site as in the PCR primer FT1.

The plasmids p31RIT12 and p31 / PH05 (-173) RIT are digested with EcoRI and Xhol, respectively. The large vector fragments (f and g) are isolated, eluted and purified on a preparative 0.8% agarose gel. The 4.1 kb Xhol-EcoRI fragment (f) contains the vector based on pBR322, the 534 bp PH05 promoter (3 'EcoRI site) and the 131 bp PH05 transcription terminator (5' Xhol site). The 3.7 kb Xhol-EcoRI fragment (g) differs only in the short constitutive 172 bp PH05M73i promoter (3 'EcoRI site) instead of the full PH05 promoter.

The 191 bp EcoRI-Nrul fragment (e), the 1.1 kb Nrul-Xhol fragment (a) and the 4.1 kb Xhol-EcoRI fragment (f) are ligated. Competent E. coli HB101 cells are transformed with 1 nl aliquot of the ligation mixture. The plasmid DNA of ampicillin resistant colonies is analyzed. Single clone plasmid DNA is referred to as p31 R / PH05-ssFT.

Ligation of DNA fragments (e), (a) and (g) results in plasmid p31R / PH05 (-173) -ssFT. These plasmids contain the coding sequence of the membrane-bound FT under the control of the inducible PH05 or constitutive PH05M 731 promoter.

Example 23:

Cloning of the FT extra cassette in pDP34:

The plasmids p31 R / PH05-ssFT and p31 R / PH05 (-173) -ssFT are digested with HindIII which cuts at 3 'of the PH05 transcription terminator. After a reaction with Klenow DNA polymerase, the DNA is digested with Sali. The 2.3 kb and 1.9 kb Sall blunt end fragments are isolated.

The plasmids pJDB207 / PH05-l-FT and pJDB207 / PH05 (-173) -l-FT are mixed with Hindill in the presence of 0.1 mg / ml ethidium bromide (to cut at an additional Hindlll site in the invertase signal sequence to avoid) partially digested and then treated with Klenow DNA polymerase and saline as above. A 2.1 kb or 1.8 kb fragment is isolated.

Each of the 4 DNA fragments is ligated with the blunt-ended 11.8 kb Sali vector fragment of pDP34 (see Example 3). After transformation of competent E. coli HB 101 cells and analysis of the plasmid DNA of individual transformants, 4 correct expression plasmids are identified as pDP34 / PH05-1-FT; pDP34 / PH05 (-173) -I-FT; pDP34R / PH05-ssFT and pDP34R / PH05 (-173) -ssFT.

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The S. cerevisiae strains BT150 and H449 are each transformed with 5 μg of the 4 expression plasmids (above) analogously to Example 4. Individual transformed yeast cell colonies are selected and labeled as follows:

Saccharomvces cerevisiae

Saccharomvces cerevisiae BT150 / pDP34 / PH05-l-FT:

Saccharomvces cerevisiae BT 1 50 / dDP34 / PHQ5M 73Ì-I-FT:

Saccharomvces cerevisiae BT150 / pDP34R / PH05-ssFT:

Saccharomvces cerevisiae BT 1 50 / dD P34R / PH05M 73) -ssFT:

Saccharomvces cerevisiae H449 / pDP34 / PH05-l-FT:

Saccharomvces cerevisiae H449 / pDP34 / PH05C-1731-l-FT:

Saccharomvces cerevisiae H449 / pDP34R / PH05-ssFT:

Saccharomvces cerevisiae H449 / pDP34R / PH05M 731-ssFT

The fermentation and production of cell extracts is carried out according to Example 5. With a test analogous to that of Goetz et al. FT activity is described in the crude extracts from BT 150 / pDP34R / PH05-ssFT, BT150 / pDP34R / PH05 (-173) -ssFT, H449 / pDP34R / PH05-ssFT and H449 / pDP34R / PH05 (-173) -ssFT and in the culture broths of H449 / pDP34 / PH05-l-FT, H449 / pDP34 / PH05 (-173H-FT, BT 150 / pDP34 / PH05-l-FT and BT150 / pDP34 / PH05 (-173) -l -FT found.

Deposit of the microorganisms

The following microorganism strains were deposited with the German Collection of Microorganisms (DSM), Mascheroder Weg 16, D-3300 Braunschweig (the filing dates and entry numbers are given):

Escherichia coli JM109 / pDP34: March 14, 1988; DSM 4473 Escherichia coli HB101 / p30: October 23, 1987; DSM 4297 Escherichia coli HB101 / p31R: December 19, 1988; DSM 5116 Saccharomvces cerevisiae H 449: February 18, 1988; DSM 4413 Saccharomvces cerevisiae BT 150: May 23, 1991; DSM 6530

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SEQUENCE LOG

REO ID no. 1

SEQUENCE TYPE: Nucleotide with the corresponding protein

SEQUENCE LENGTH: 1265 bp

STRENGTH: double

TOPOLOGY: linear

MOLECULE TYPE: recombinant

DIRECT EXPERIMENTAL ORIGIN: Plasmid p4AD113 from E. coli DH5a / p4AD 113

CHARACTERISTICS: from 6 to 1200 bp from 1 to 6 bp from 497 to 504 bp from 1227 to 1232 bp from 1236 to 1241 bp from 1243 to 1248 bp

CDNA sequence encoding HeLa cell galactosyl transferase

EcoRI site

Notl position

EcoRI site

EcoRV office

Bglll site

PROPERTIES: EcoRI-HindIII fragment from plasmid p4AD113 with HeLa cell cDNA coding for complete galactosyltransferase (EC2.4.1.22)

GAATTC ATG AGG CTT CGG GAG CCG CTC CTG AGC GGC AGC Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser

5 10

39

GCC GCG ATG CCA GGC GCG TCC CTA CAG CGG GCC TGC CGC 78 Ala Ala Met Pro Gly Ala Ser Leu Gin Arg Ala Cys Arg 15 20

CTG CTC GTG GCC GTC TGC GCT CTG CAC CTT GGC GTC ACC Leu Leu Val Ala Val Cys Ala Leu His Leu Gly Val Thr 25 30 35

117

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CTC GTT TAC TAC CTG GCT GGC CGC GAC CTG AGC CGC CTG 156 Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser Arg Leu 40 45 50

CCC CAA CTG GTC GGA GTC TCC ACA CCG CTG CAG GGC GGC 195 Pro Gin Leu Val Gly Val -Ser Thr Pro Leu Gin Gly Gly

55 60

TCG AAC AGT GCC GCC GCC ATC GGG CAG TCC TCC GGG GAG 234 Ser Asn Ser Ala Ala Ala Ile Gly Gin Ser Ser Gly Glu 20 65 70 75

CTC CGG ACC GGA GGG GCC CGG CCG CCG CCT CCT CTA GGC 273 Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly 80 85

GCC TCC TCC CAG CCG CGC CCG GGT GGC GAC TCC AGC CCA 312 Ala Ser Ser Gin Pro Arg Pro Gly Gly Asp Ser Ser Pro 90 95 100

GTC GTG GAT TCT GGC CCT GGC CCC GCT AGC AAC TTG ACC 351 Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr.

105 110 115

TCG GTC CCA GTG CCC CAC ACC ACC GCA CTG TCG CTG CCC 3 90 Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro

120 125

GCC TGC CCT GAG GAG TCC CCG CTG CTT GTG GGC CCC ATG 42 9 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 55 120 135 140

CTG ATT GAG TTT AAC ATG CCT GTG GAC CTG GAG CTC GTG 468 Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val 145 150

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GCA AAG CAG AAC CCA AAT GTG AAG ATG GGC GGC CGC TAT 507 Ala Lys Gin Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 5 155 160 165

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GCC CCC AGG GAC TGC GTC TCT CCT CAC AAG GTG GCC ATC 546 Ala Pro Arg Asp Cys Val-Ser Pro His Lys Val Ala Ile 170 175 180

ATC ATT CCA TTC CGC AAC CGG CAG GAG CAC CTC AAG TAC 585 Ile Ile Pro Phe Arg Asn Arg Gin Glu His Leu Lys Tyr

185 190

TGG CTA TAT TAT TTG CAC CCA GTC CTG CAG CGC CAG CAG 624 25 Trp Leu Tyr Tyr Leu His Pro Val Leu Gin Arg Gin Gin 195 200 205

30 CTG GAC TAT GGC ATC TAT GTT ATC AAC CAG GCG GGA GAC 663 Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gin Ala Gly Asp 210 215

ACT ATA TTC AAT CGT GCT AAG CTC CTC AAT GTT GGC TTT 7 02 Thr Ile Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 220 225 230

CAA GAA GCC TTG AAG GAC TAT GAC TAC ACC TGC TTT GTG 741 Gin Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys Phe Val 235 240 245

TTT AGT GAC GTG GAC CTC ATT CCA ATG AAT GAC CAT AAT 780 Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn 55 250 255

GCG TAC AGG TGT TTT TCA CAG CCA CGG CAC ATT TCC GTT 819 Ala Tyr Arg Cys Phe Ser Gin Pro Arg His Ile Ser Val 260 265 270

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GCA ATG GAT AAG TTT GGA TTC AGC CTA CCT TAT GTT CAG 858 Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gin 275 280

TAT TTT GGA GGT GTC TCT GCT CTA AGT AAA CAA CAG TTT 897 Tyr Phe Gly Gly Val Ser -Ala Leu Ser Lys Gin Gin Phe 285 290 295

CTA ACC ATC AAT GGA TTT CCT AAT AAT TAT TGG GGC TGG 93 6 Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp 300 305 310

GGA GGA GAA GAT GAT GAC ATT TTT AAC AGA TTA GTT TTT 975 Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe

315 320

AGA GGC ATG TCT ATA TCT CGC CCA AAT GCT GTG GTC GGG 1014 Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 35 325 330 335

AGG TGT CGC ATG ATC CGC CAC TCA AGA GAC AAG AAA AAT 1053 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn 340 345

GAA CCC AAT CCT CAG AGG TTT GAC CGA ATT GCA CAC ACA 1092 Glu Pro Asn Pro Gin Arg Phe Asp Arg Ile Ala His Thr 350 355 360

AAG GAG ACA ATG CTC TCT GAT GGT TTG AAC TCA CTC ACC 1131 55 Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr 365 370 375

60 TAC CAG GTG CTG GAT GTA CAG AGA TAC CCA TTG TAT ACC 117 0 Tyr Gin Val Leu Asp Val Gin Arg Tyr Pro Leu Tyr Thr

380 385

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CAA ATC ACA GTG GAC ATC GGG ACA CCG AGC TAGGACTTTT 1210 Gin Ile Thr Val Asp Ile Gly Thr Pro Ser 390 395

GGTACAGGTA AAGACTGAAT TCATCGATAT CTAGATCTCG 1250

AGCTCGCGAA AGCTT 1265

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RFO ID no. 2nd

SEQUENCE TYPE: Protein SEQUENCE LENGTH: 357 amino acids

MOLECULE TYPE: C-terminal fragment of complete HeLa cell galactosyltransferase PROPERTIES: Soluble galactosyltransferase (EC 2.4.1.22) from HeLa cells

Leu Ala Gly Arg Asp Leu Ser Arg Leu

5

Pro Gin Leu Val Gly Val Ser Thr Pro Leu Gin Gly Gly 10 15 20

Ser Asn Ser Ala Ala Ala Ile Gly Gin Ser Ser Gly Glu 25 30 35

Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly

40 45

Ala Ser Ser Gin Pro Arg Pro Gly Gly Asp Ser Ser Pro 50 55 60

Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr 65 70

Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 75 80 85

Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 90 95 100

Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val

105 110

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Ala Lys Gin Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 115 120 125

Ala Pro Arg Asp Cys Val Ser Pro His Lys Val Ala Ile 130 • 135

Ile Ile Pro Phe Arg Asn Arg Gin Glu His Leu Lys iyr 140 145 150

Trp Leu Tyr Tyr Leu His Pro Val Leu Gin Arg Gin Gin 155 160 165

Leu Asp Tyr Gly Ile iyr Val Ile Asn Gin Ala Gly Asp

170 175

Thr Ile Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 180 185 190

Gin Glu Ala Leu Lys Asp iyr Asp Tyr Thr Cys Phe Val 195 200

Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn 205 210 215

Ala Tyr Arg Cys Phe Ser Gin Pro Arg His Ile Ser Val 220 225 230

Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gin

235 240

Tyr Phe Gly Gly Val Ser Ala Leu Ser Lys Gin Gin Phe 245 250 255

Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp 260 265

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Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe 270 275 280

Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 285 • 290 295

Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn

300 305

Glu Pro Asn Pro Gin Arg Phe Asp Arg Ile Ala His Thr 310 315 320

Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr 325 330

Tyr Gin Val Leu Asp Val Gin Arg Tyr Pro Leu Tyr Thr 335 340 345

Gin Ile Thr Val Asp Ile Gly Thr Pro Ser 350 355

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SEQ ID no. 3rd

SEQUENCE TYPE: Nucleotide with the corresponding protein

SEQUENCE LENGTH: 1246 bp

STRENGTH: double

TOPOLOGY: linear

MOLECULE TYPE: recombinant

DIRECT EXPERIMENTAL ORIGIN: Plasmid pSIA2 from E. coli DH5a / lpSIA2

CHARACTERISTICS: from 15 to 1232 bp from 1 to 6 bp from 6 to 11 bp from 144 to 149 bp from 1241 to 1246 bp

HepG2 cell sialyltransferase encoding cDNA sequence

Pstl position

EcoRI site

EcoRI site

BamHI site

PROPERTIES: PstI-BamHI fragment from plasmid pSIA2 with HepG2 cDNA coding for complete siaiyitransferase (EC 2.4.99.1)

CTGCAGAATT CAAA ATG ATT CAC ACC AAC CTG AAG AAA

Met Ile His Thr Asn Leu Lys Lys

5

38

AAG TTC AGC TGC TGC GTC CTG GTC TTT CTT CTG TTT GCA Lys Phe Ser Cys Cys Val Leu Val Phe Leu Leu Phe Ala 10 15 20

77

GTC ATC TGT GTG TGG AAG GAA AAG AAG AAA GGG AGT TAC Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly Ser Tyr 25 30

116

TAT GAT TCC TTT AAA TTG CAA ACC AAG GAA TTC CAG GTG Tyr Asp Ser Phe Lys Leu Gin Thr Lys Glu Phe Gin Val 35 40 45

155

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194

233

272

311

350

389

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467

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TTA AAG AGT CTG GGG AAA TTG GCC ATG GGG TCT GAT TCC Leu Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser 50 55 60

CAG TCT GTA TCC TCA AGC -AGC ACC CAG GAC CCC CAC AGG Gin Ser Val Ser Ser Ser Ser Thr Gin Asp Pro His Arg

65 70

GGC CGC CAG ACC CTC GGC AGT CTC AGA GGC CTA GCC AAG Gly Arg Gin Thr Leu Gly Ser Leu Arg Gly Leu Ala Lys 75 80 85

GCC AAA CCA GAG GCC TCC TTC CAG GTG TGG AAC AAG GAC Ala Lys Pro Glu Ala Ser Phe Gin Val Trp Asn Lys Asp 90 95

AGC TCT TCC AAA AAC CTT ATC CCT AGG CTG CAA AAG ATC Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gin Lys Ile 100 105 110

TGG AAG AAT TAC CTA AGC ATG AAC AAG TAC AAA GTG TCC Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser 115 120 125

TAC AAG GGG CCA GGA CCA GGC ATC AAG TTC AGT GCA GAG Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu

130 135

GCC CTG CGC TGC CAC CTC CGG GAC CAT GTG AAT GTA TCC Ala Leu Arg Cys His Leu Arg Asp His Val Asn Val Ser 140 145 150

ATG GTA GAG GTC ACA GAT TTT CCC TTC AAT ACC TCT GAA Met Val Glu Val Thr Asp Phe Pro Phe Asn Thr Ser Glu 155 160

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TGG GAG GGT TAT CTG CCC AAG GAG AGC ATT AGG ACC AAG 545 Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr Lys 165 170 175

GCT GGG CCT TGG GGC AGG -TGT GCT GTT GTG TCG TCA GCG 584 Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala 180 185 190

GGA TCT CTG AAG TCC TCC CAA CTA GGC AGA GAA ATC GAT 623 Gly Ser Leu Lys Ser Ser Gin Leu Gly Arg Glu Ile Asp 20 195 2 00

GAT CAT GAC GCA GTC CTG AGG TTT AAT GGG GCA CCC ACA 662 Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala Pro Thr 205 210 215

GCC AAC TTC CAA CAA GAT GTG GGC ACA AAA ACT ACC ATT 701 Ala Asn Phe Gin Gin Asp Val Gly Thr Lys Thr Thr Ile 220 225

CGC CTG ATG AAC TCT CAG TTG GTT ACC ACA GAG AAG CGC 740 40 Arg Leu Met Asn Ser Gin Leu Val Thr Thr Glu Lys Arg 230 235 240

45 TTC CTC AAA GAC AGT TTG TAC AAT GAA GGA ATC CTA ATT 779 Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile 245 250 255

GTA TGG GAC CCA TCT GTA TAC CAC TCA GAT ATC CCA AAG 818 Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys

260 265

TGG TAC CAG AAT CCG GAT TAT AAT TTC TTT AAC AAC TAC 857 Trp Tyr Gin Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr 270 275 280

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AAG ACT K Lys Thr

O

10 ATC CTC Ile Leu 295

15

CTT CAA Leu Gin

20th

CCA TCC

25th

Per ser

30th

CTG TGT Leu Cys 35 335

AAG CGC 40 Lys Arg

45

TTC GAT Phe Asp

360

50

CTC TAT 55 Leu iyr

60 ACA GAT Thr Asp

65

TAT CGT AAG Tyr Arg Lys 285

AAG CCC CAG Lys Pro Gin

GAA ATC TCC Glu Ile Ser 310

TCT GGG ATG Ser Gly Met 325

GAC CAG GTG Asp Gin Val

AAG ACT GAC Lys Thr Asp 350

AGT GCC TGC Ser Ala Cys

GAG AAG AAT Glu Lys Asn 375

GAG GAC ATC Glu Asp Ile 390

CTG CAC CCC Leu His Pro

ATG -CCT TGG Met Pro Trp 300

CCA GAA GAG Pro Glu Glu 315

CTT GGT ATC Leu Gly Ile

GAT ATT TAT Asp Ile Tyr 340

GTG TGC TAC Val Cys Tyr

ACG ATG GGT Thr Met Gly 365

TTG GTG AAG Leu Val Lys 380

TAC CTG CTT Tyr Leu Leu

AAT CAG CCC Asn Gin Pro 290

GAG CTA TGG Glu Leu Trp 305

ATT CAG CCA Ile Gin Pro

ATC ATC ATG Ile Ile Met 330

GAG TTC CTC Glu Phe Leu

TAC TAC CAG iyr Tyr Gin 355

GCC TAC CAC Ala Tyr His 370

CAT CTC AAC His Leu Asn

GGA AAA GCC Gly Lys Ala 395

TTT TAC 896 Phe Tyr

GAC ATT 935 Asp Ile

AAC CCC 974 Asn Pro 320

ATG ACG 1013 Met Thr

CCA TCC 1052 Pro Ser 345

AAG TTC 1091 Lys Phe

CCG CTG 1130 Pro Leu

CAG GGC 1169 Gin Gly 385

ACA CTG 12 08 Thr Leu

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CCT GGC TTC CGG ACC ATT CAC TGC TAAGCACAGG ATCC 1246

Pro Gly Phe Arg Thr Ile His Cys 400 405

SEQ ID NIR. 4th

SEQUENCE TYPE: Protein SEQUENCE LENGTH: 368 amino acids

MOLECULE TYPE: C-terminal fragment of complete siaiyitransferase PROPERTIES: Soluble siaiyitransferase (EC 2.4.99.1) from human HepG2 cells

Lys Leu Gin Thr Lys Glu Phe Gin Val

5

Leu Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser 10 15 20

Gin Ser Val Ser Ser Ser Ser Thr Gin Asp Pro His Arg 25 30 35

Gly Arg Gin Thr Leu Gly Ser Leu Arg Gly Leu Ala Lys

40 45

Ala Lys Pro Glu Ala Ser Phe Gin Val Trp Asn Lys Asp 50 55 60

Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gin Lys Ile 65 70

Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser 75 80 85

Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu 90 95 100

Ala Leu Arg Cys His Leu Arg Asp His Val Asn Val Ser

105 110

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Met Val Glu Val Thr Asp Phe Pro Phe Asn Thr Ser Glu 115 120 125

Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr Lys 130 '135

Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala 140 145 150

Gly Ser Leu Lys Ser Ser Gin Leu Gly Arg Glu Ile Asp 155 160 165

Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala Pro Thr

170 175

Ala Asn Phe Gin Gin Asp Val Gly Thr Lys Thr Thr Ile 180 185 190

Arg Leu Met Asn Ser Gin Leu Val Thr Thr Glu Lys Arg 195 200

Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile 205 210 215

Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys 220 225 230

Trp Tyr Gin Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr

235 240

Lys Thr Tyr Arg Lys Leu His Pro Asn Gin Pro Phe Tyr 245 250 255

Ile Leu Lys Pro Gin Met Pro Trp Glu Leu Trp Asp Ile 260 265

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Leu Gin Glu Ile Ser Pro 270 275

Per Ser Ser Gly Met Leu 285

Leu Cys Asp Gin Val Asp

300

Lys Arg Lys Thr Asp Val 310

Phe Asp Ser Ala Cys Thr 325

Glu Glu Ile Gin Pro Asn Pro

280

Gly Ile Ile Ile Met Met Thr 290 295

Ile Tyr Glu Phe Leu Pro Ser 305

Cys Tyr Tyr Tyr Gin Lys Phe 315 320

Met Gly Ala Tyr His Pro Leu 330

Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gin Gly 335 340 345

Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu 350 355 360

Pro Gly Phe Arg Thr Ile His Cys

365

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SFQ ID NO. 5

SEQUENCE TYPE: Nucleotide with the corresponding protein

SEQUENCE LENGTH: 1400 bp

STRENGTH: double

TOPOLOGY: linear

MOLECULE TYPE: recombinant

DIRECT EXPERIMENTAL ORIGIN: BRB.ELFT / pCR1000-13

CHARACTERISTICS: From 58 to 1272 bp cDNA sequence encoding human ct (1-3) fucosyltransferase from 238 to 243 bp Nrul site. CHARACTERISTICS: For complete cDNA encoding <x (1-3) fucosyltransferase

CGCTCCTCCA CGCCTGCGGA CGCGTGGCGA GCGGAGGCAG CGCTGCCTGT 50

TCGCGCC ATG GGG GCA CCG TGG GGC TCG CCG ACG GCG GCG 90

Met Gly Ala Pro Trp Gly Ser Pro Thr Ala Ala

5 10

GCG GGC GGG CGG CGC GGG TGG CGC CGA GGC CGG GGG CTG 129

Ala Gly Gly Arg Arg Gly Trp Arg Arg Gly Arg Gly Leu 15 20

CCA TGG ACC GTC TGT GTG CTG GCG GCC GCC GGC TTG ACG 168

Per Trp Thr Val Cys Val Leu Ala Ala Ala Gly Leu Thr 25 30 35

TGT ACG GCG CTG ATC ACC TAC GCT TGC TGG GGG CAG CTG 207

Cys Thr Ala Leu Ile Thr Tyr Ala Cys Trp Gly Gin Leu 40 45 50

CCG CCG CTG CCC TGG GCG TCG CCA ACC CCG TCG CGA CCG 246

Pro Pro Leu Pro Trp Ala Ser Pro Thr Pro Ser Arg Pro

55 60

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285

324

363

402

441

480

519

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GTG GGC GTG CTG CTG TGG TGG GAG CCC TTC GGG GGG CGC

Val Gly Val Leu Leu Trp Trp Glu Pro Phe Gly Gly Arg

65 70 75

GAT AGC GCC CCG AGG CCG CCC CCT GAC TGC CGG CTG CGC

Asp Ser Ala Pro Arg Pro Pro Pro Asp Cys Arg Leu Arg

80 85

TTC AAC ATC AGC GGC TGC CGC CTG CTC ACC GAC CGC GCG

Phe Asn Ile Ser Gly Cys Arg Leu Leu Thr Asp Arg Ala

90 95 100

TCC TAC GGA GAG GCT CAG GCC GTG CTT TTC CAC CAC CGC

Ser Tyr Gly Glu Ala Gin Ala Val Leu Phe His His Arg

105 110 115

GAC CTC GTG AAG GGG CCC CCC GAC TGG CCC CCG CCC TGG

Asp Leu Val Lys Gly Pro Pro Asp Trp Pro Pro Pro Trp

120 125

GGC ATC CAG GCG CAC ACT GCC GAG GAG GTG GAT CTG CGC

Gly Ile Gin Ala His Thr Ala Glu Glu Val Asp Leu Arg

130 135 140

GTG TTG GAC TAC GAG GAG GCA GCG GCG GCG GCA GAA GCC

Val Leu Asp Tyr Glu Glu Ala Ala Ala Ala Ala Glu Ala

145 150

CTG GCG ACC TCC AGC CCC AGG CCC CCG GGC CAG CGC TGG

Leu Ala Thr Ser Ser Pro Arg Pro Pro Gly Gin Arg Trp

155 160 165

GTT TGG ATG AAC TTC GAG TCG CCC TCG CAC TCC CCG GGG Val Trp Met Asn Phe Glu Ser Pro Ser His Ser Pro Gly 170 175 180

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CTG CGA AGC CTG GCA AGT AAC CTC TTC AAC TGG ACG CTC 636

Leu Arg Ser Leu Ala Ser Asn Leu Phe Asn Trp Thr Leu

185 190

TCC TAC CGG GCG GAC TCG GAC GTC TTT GTG CCT TAT GGC 675

Ser Tyr Arg Ala Asp Ser Asp Val Phe Val Pro Tyr Gly 195 200 205

TAC CTC TAC CCC AGA AGC CAC CCC GGC GAC CCG CCC TCA 714

Tyr Leu Tyr Pro Arg Ser His Pro Gly Asp Pro Pro Ser 210 215

GGC CTG GCC CCG CCA CTG TCC AGG AAA CAG GGG CTG GTG 7 53

Gly Lea Ala Pro Pro Leu Ser Arg Lys Gin Gly Leu Val 220 225 230

GCA TGG GTG GTG AGC CAC TGG GAC GAG CGC CAG GCC CGG 792

Ala Trp Val Val Ser His Trp Asp Glu Arg Gin Ala Arg 235 240 245

GTC CGC TAC TAC CAC CAA CTG AGC CAA CAT GTG ACC GTG 831

Val Arg Tyr Tyr His Gin Leu Ser Gin His Val Thr Val 40 250 255

GAC GTG TTC GGC CGG GGC GGG CCG GGG CAG CCG GTG CCC 87 0

Asp Val Phe Gly Arg Gly Gly Pro Gly Gin Pro Val Pro 260 265 270

GAA ATT GGG CTC CTG CAC ACA GTG GCC CGC TAC AAG TTC 909

Glu Ile Gly Leu Leu His Thr Val Ala Arg Tyr Lys Phe 275 280

TAC CTG GCT TTC GAG AAC TCG CAG CAC CTG GAT TAT ATC 948

Tyr Leu Ala Phe Glu Asn Ser Gin His Leu Asp Tyr Ile 285 290 295

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ACC GAG AAG CTC TGG CGC AAC GCG TTG CTC GCT GGG GCG 987

Thr Glu Lys Leu Trp Arg Asn Ala Leu Leu Ala Gly Ala 300 305 310

GTG CCG GTG GTG CTG GGC CCA GAC CGT GCC AAC TAC GAG 102 6

Val Pro Val Val Leu Gly Pro Asp Arg Ala Asn Tyr Glu 315 320

CGC TTT GTG CCC CGC GGC GCC TTC ATC CAC GTG GAC GAC 1065

Arg Phe Val Pro Arg Gly Ala Phe Ile His Val Asp Asp 325 330 335

TTC CCA AGT GCC TCC TCC CTG GCC TCG TAC CTG CTT TTC 1104

Phe Pro Ser Ala Ser Ser Leu Ala Ser Tyr Leu Leu Phe 340 345

CTC GAC CGC AAC CCC GCG GTC TAT CGC CGC TAC TTC CAC 1143

Leu Asp Arg Asn Pro Ala Val Tyr Arg Arg Tyr Phe His 350 355 360

TGG CGC CGG AGC TAC GCT GTC CAC ATC ACC TCC TTC TGG 1182 Trp Arg Arg Ser Tyr Ala Val His Ile Thr Ser Phe Trp 365 370 375

GAC GAG CCT TGG TGC CGG GTG TGC CAG GCT GTA CAG AGG 1221

Asp Glu Pro Trp Cys Arg Val Cys Gin Ala Val Gin Arg 380 385

GCT GGG GAC CGG CCC AAG AGC ATA CGG AAC TTG GCC AGC 1260

Ala Gly Asp Arg Pro Lys Ser Ile Arg Asn Leu Ala Ser 390 395 400

TGG TTC GAG CGG TGAAGCCGCG CTCCCCTGGA AGCGACCCAG 1302

Trp Phe Glu Arg 405

GGGAGGCCAA GTTGTCAGCT TTTTGATCCT CTACTGTGCA TCTCCTTGAC 1352

TGCCGCATCA TGGGAGTAAG TTCTTCAAAC ACCCATTTTT GCTCTATG 1400

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CH 685 057 A5

Claims (20)

Claims 1. A process for the preparation of a membrane-bound mammalian glycosyltransferase from the group consisting of a galactosyltransferase, a siaiyitransferase and a fucosyltransferase, or membrane-bound or soluble variants thereof, with the proviso that these variants are enzymatically active, characterized in that a yeast strain is cultivated, which is transformed with a hybrid vector which contains an expression cassette comprising a promoter and a DNA sequence coding for said glycosyltransferase or its variant, said DNA being controlled by said promoter, and the enzymatic activity is obtained. 2. The method according to claim 1, characterized in that the glycosyltransferase is of human origin. 3. The method for producing a variant according to claim 1, characterized in that the variant differs from the corresponding form with the full length by the absence of the cytoplasmic tail, the signal anchor and, if appropriate, a smaller part of the stem region. 4. The method for the production of a variant according to claim 3, characterized in that a yeast strain is cultivated, the expression cassette comprising a promoter, which is functionally linked to a first, a signal peptide coding DNA sequence, which is in the correct reading frame with a second DNA sequence coding for the variant mentioned, which is controlled by the promoter mentioned, and which contains the enzymatic activity. 5. The method according to claim 1, characterized in that the glycosyltransferase is a galactosyltransferase. 6. The method according to claim 5, characterized in that the galactosyl transferase UDP-galactose: β-galactoside-a (1-3) galactosyl transferase (EC 2.4.1.151) or UDP-galactose: β-N-acetylglucosamin-β (1 -4) galactosyltransferase (EC 2.4.1.22). 7. The method according to claim 5, characterized in that the galactosyltransferase the in SEQ ID no. 1 amino acid sequence shown. 8. The method according to claim 3, characterized in that the galactosyltransferase the in SEQ ID no. 2 amino acid sequence shown. 9. The method according to claim 1, characterized in that the glycosyltransferase is a siaiyitransferase. 10. The method according to claim 9, characterized in that the siaiyitransferase is CMP-NeuAc-β-galactoside-a (2-6) sialyltransferase (EC 2.4.99.1). 11. The method according to claim 9, characterized in that the siaiyitransferase the in SEQ ID no. 3 amino acid sequence shown. 12. The method according to claim 9, characterized in that the siaiyitransferase is referred to as ST (Lys27-Cys406) and from amino acids 27 to 406 of the SEQ ID no. 3 listed amino acid sequence. 13. The method according to claim 9, characterized in that the siaiyitransferase the in SEQ ID no. 4 amino acid sequence shown. 14. The method according to claim 1, characterized in that the glycosyltransferase is a fucosyltransferase. 15. The method according to claim 14, characterized in that the fucosyltransferase GDP-fucose: β-galactoside-a (1-2) fucosyltransferase (EC 2.4.1.69) and GDP-fucose: N-acetylglucosamine-a (1 -3/4 ) is fucosyltransferase (EC 2.4.1.65). 16. The method according to claim 14, characterized in that the fucosyltransferase the SEQ ID no. 5 amino acid sequence shown. 17. The method according to claim 14, characterized in that the fucosyl transferase is referred to as FT (Arg62-Arg405) and from amino acids 62 to 405 of the SEQ ID no. 5 amino acid sequence shown consists. 18. A yeast hybrid vector for a yeast strain for carrying out the method according to claim 1, characterized in that it has an expression cassette comprising a yeast promoter and one for a membrane-bound mammalian glycosyltransferase from the group consisting of a galactosyltransferase, a siaiyitransferase and a fucosyltransferase, or one thereof contains DNA sequences encoding membrane-bound or soluble enzymatic active variants, this DNA sequence being controlled by the promoter mentioned. 19. A yeast strain transformed with a hybrid vector according to claim 18 for carrying out the method according to claim 1. 20. A yeast strain according to claim 19, characterized in that the yeast strain is Saccharomvces cerevisiae BT 150, DSM 6530. 45