DE4217616A1 - IMPROVED METHOD FOR THE PRODUCTION OF GLYCOSYL TRANSFERASES - Google Patents

Abstract

The use of transformed yeast cells to produce membrane-bound mammalian glycosyltransferases is disclosed. The glycosyltransferase is selected from galactosyltransferases, sialyltransferases and fucosyltransferases, or variants thereof.

Description

The invention relates to the field of recombinant DNA technology and adjusts improved process for the production of glycosyltransferases by means of transformed Yeast strains ready.

Glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide sugar, to a specific acceptor sugar below Formation of a glycosidic bond. Depending on the type of sugar transferred These enzymes are divided into families, such. Galactosyltransferases, Sialyltransferases and fucosultransferases. As membrane-bound proteins, the occur mainly in the Golgi apparatus, the Glycosyltransferasen have a common domain structure consisting of a short amino-terminal cytoplasmic Tail, a signal anchor domain and an extended stem region, to which followed by a large carboxyl-terminal catalytic domain. The Signal anchor domain has both the function of a non-cleavable signal peptide and that of an anchor extending across the membrane and directs the catalytic Domain of glycosyltransferase within the lumen of the Golgi apparatus. Of the luminal stem region, also called spacer region, is believed to be the Function of a flexible chain, the catalytic domain has the glycosylation of Carbohydrate groups of membrane-bound and soluble proteins of the "Secretory Pathway" which pass through the Golgi apparatus. It was also discovered that the Stammabschnitt performs the function of a retention signal to the enzymes to the Golgi membrane bound to keep (PCT Application No. 91/06 635). Soluble forms Glycosyltransferases are found in milk, serum and others Body fluids. It is believed that these soluble glycosyltransferases by proteolytic release of the corresponding membrane bound forms of the Enzymes are formed by endogenous proteases, presumably by 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, causing the glycosyltransferases to a valuable tool for the modification or synthesis of glycoproteins, Glycolipids and oligosaccharides. Unlike chemical methods the time-consuming introduction of protective groups is superfluous.

Since glycosyltransferases occur in nature in very small quantities, their Isolation of natural products and their subsequent purification difficult. It was therefore worked on their production by means of the recombinant DNA method. For example were galactosyltransferases in cells of E. coli (PCT 90/07 000) and ovaries of the Chinese hamster (CHO) (Smith, D.F., et al. [1990], J. Biol. Chem., 265, 6225-34), sialyltransferases were expressed in CHO cells (Lee, E.U. [1990], Diss. Abstr. Int. B., 50, 3453-4) and COS-1 cells (Paulsen, J.C., et al., [1988] J. Cell Biol., 107, 10A). and fucosyltransferases have been expressed in COS-1 cells (Goelz, S.E., et al. [1990], Cell, 63, 1349-1356; Larsen, R.D., et al. [1990], Proc. Natl. 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 the desired Contaminate product is complicated, so there is a need for improved Process that facilitates the economic production of glycosyltransferases in large quantities Enable scale.

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

The present invention provides a process for the production of biologically active Glycosyltransferases prepared by the recombinant DNA technique 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, ready, said method thereby characterized in that a yeast strain is cultured with a hybrid vector, the an expression cassette consisting of a promoter and one for said  Glycosyltransferase or their variant coding DNA sequence contains, these DNA is controlled by said promoter, transformed, and the enzymatic activity is obtained.

In a first embodiment, the invention provides a method for production a membrane-bound glycosyltransferase from the group consisting of a Galactosyltransferase, a sialyltransferase and a fucosyltransferase or one of their variants, ready, said method thereby characterized in that a yeast strain is cultured with a hybrid vector, the an expression cassette comprising a promoter, one for the said Glycosyltransferase or its variant coding DNA sequence, said DNA of controlled promoter, and a DNA sequence with Yeast transcription termination signals, containing, transformed, and the enzymatic activity is obtained.

In a second embodiment, the invention relates to a method for the production a membrane bound glycosyltransferase from the group consisting of a Galactosyltransferase, a sialyltransferase and a fucosyltransferase or one of their variants, said method thereby characterized in that a yeast strain is cultured with a hybrid vector, the an expression cassette comprising a yeast promoter functional with a first, a signal peptide encoding DNA sequence is connected in the correct reading frame with a second coding for said glycosyltransferase or its variant DNA sequence, and a DNA sequence with yeast transcription termination signals, was transformed, and the enzymatic activity was recovered becomes.

The term "glycosyltransferase" whenever it is in the preceding or following Text is used, it should be understood that the family of galactosyltransferases, the family of sialyltransferases and the family of fucosyltransferases. The called glycosyltransferases are naturally occurring enzymes found in mammals, z. Cattle, mice, rats and humans. preferred Glycosyltransferases are naturally occurring human glycosyltransferases in their full length, including those enzymes that in the following text with their EC numbers are designated.  

The membrane bound galactosyltransferases and their variants, which are named after the Inventive processes catalyze the transfer of a Galactose residues from an activated donor, usually a nucleotide-activated one Donor, such as Uridine diphosphate galactose (UDP-Gal) on a carbohydrate moiety.

Examples of membrane-bound galactosyltransferases are UDP-galactose: β-galactoside-α (1-3) galactosyltransferase (EC 2.4.1.151), for galactose as Acceptor substrate to form an α (1-3) bond, and UDP-galactose: β-N-acetylglucosamine-β (1-4) -galactosyltransferase (EC 2.4.1.22), the galactose on Transfers N-acetylglucosamine (GlcNAc) to form a β (1-4) bond included in each variant. In the presence of α-lactalbumin, the β (1-4) -galactosyltransferase also glucose as the acceptor substrate, causing the Lactose synthesis is catalyzed.

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

The membrane bound sialyltransferases and their variants, which after the Processes of the invention are available which catalyze the transfer of Sialic acids (for example N-acetylneuraminic acid [NeuAc]) from an activated Donor, usually a cytidine monophosphatialic acid (CMP-SA), to one Kohlehydratakzeptorrest. An example of a membrane-bound sialyltransferase, the obtainable by the inventive process, CMP-NeuAc is: β-galactoside-α (2-6) -sialyltransferase (EC 2.4.99.1) containing the NeuAc-α (2-6) Gal-β (1-4) GlcNAc sequence forms in many N-linked Carbohydrate groups can be found.

The most preferred membrane-bound sialyltransferase is the enzyme with the Amino acid sequence listed in the sequence listing under SEQ ID NO. 3 is given.

The membrane bound fucosyltransferases and their variants, which after the inventive methods catalyze the transfer of a Fucoserests from an activated donor, usually a nucleotide-activated donor such. B. Guanosine diphosphate fucose (GDP-Fuc), on a carbohydrate group. Examples of such Fucosyltransferases are

GDP-fucose: β-galactoside-α (1-2) -fucosyltransferase (EC 2.4.1.69) and
GDP-fucose: N-acetylamine-α (1-3 / 4) -fucosyltransferase (EC 2.4.1.65).

The most preferred membrane-bound fucosyltransferase is the enzyme with the Amino acid sequence listed in the sequence listing under SEQ ID NO. 5 is given.

The term variants are used in the present application both membrane-bound as well as soluble variants of naturally occurring to understand membrane-bound glycosyltransferases, with the proviso that these Variants are enzymatically active. Preference is given to humans occurring Variants.

For example, the term "variants" should be understood to be naturally occurring involves membrane-bound variants of membrane-bound glycosyltransferases, that can be found within a certain kind, such as B. a variant of a Galactosyltransferase, which is different from the enzyme with SEQ ID NO. 1 indicated Amino acid sequence in so far as it lacks the serine in position 11 and in positions 31 and 32 valine and tyrosine in place of alanine respectively Leucine occur. Such a variant may be of a related gene of the same Genes or encoded by an allelic variant of a particular gene. The The term "variants" also includes glycosyltransferases derived from an in vitro mutated DNA as long as it encoded the said DNA Protein has the enzymatic activity of the native glycosyltransferase. Such Modifications may be in an addition, an exchange, and / or a deletion of Amino acids exist, in the latter case, shortened variants arise.

Preferred variants which are prepared by the method according to the invention are shortened variants, in particular soluble variants, d. H. Variants that are not are membrane bound. Shortened variants are for example soluble forms of membrane-bound glycosyltransferases and their membrane-bound variants, z. B. those above variants, which of a transformed in the inventive Process used yeast strain can be secreted. According to the In the present invention, these soluble enzymes are the preferred ones 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 sialyltransferase and a fucosyltransferase, this being A method characterized in that a yeast strain is cultured with a Hybrid vector comprising an expression cassette comprising a promoter, one with this Promoter functionally linked DNA sequence encoding a signal peptide, and the in the correct reading frame with a second coding for said variant DNA sequence, and a DNA sequence containing yeast Transkriptionsterminationssignale contains, was transformed, and that said variant is isolated.

The soluble form of a glycosyltransferase is by definition a shortened variant, which differ from the corresponding full length form, d. H. the membrane-bound form that naturally occurs in the endoplasmic reticulum or in the Golgi complex, due to the absence of the cytoplasmic tail, the Signal anchor and, if appropriate, a part of the trunk region is different. The term "Part of the parent region" is defined in the context of the present text as that it designates a smaller part of the N-terminal side of the stem region and off up to 12 amino acids. In other words, that means that the soluble variants, which are produced according to the present invention, from the substantially consist of the entire stem region and the catalytic domain.

The soluble variants are enzymatically active enzymes that differ from the respective ones Forms with the full length differ in that an NH₂-terminal peptide consisting of 26 to 61 amino acids is absent, with the proviso that in those forms, where part of the stem region is missing, only the smaller part of it defined above Region is missing. Preferred soluble variants are those derived from the membrane-bound glycosyltransferases are available for EC numbers and, in addition, soluble variants derived from the membrane-bound Fucosyltransferase with SEQ ID NO. 5 are available.

Preferred soluble variants of the galactosyltransferases differ from the respective forms with the full length in that they have a NH₂-terminal peptide is missing, which consists of 37 to 55, in particular 41 to 44 amino acids. The most preferred soluble variant prepared according to the inventive process is the enzyme having the amino acid sequence described under SEQ ID NO. 2 in the sequence listing is cited.  

Preferred soluble variants of sialyltransferases are absent compared to the forms the full length NH₂-terminal peptide consisting of 26 to 38 amino acids. The most preferred soluble variant according to the inventive method is the enzyme with the amino acid sequence shown in the Sequence Listing under SEQ ID NO. 4 is given. Also preferred is that with ST (Lys₂₇-Cys₄₀₆) designated soluble variant consisting of amino acids 27 to 406 of SEQ ID NO. 3 specified amino acid sequence.

Preferred soluble variants of the fucosyltransferases differ from the respective enzymes of full length by giving them an NH₂-terminal peptide is missing, which consists of 56 to 67, in particular 56 to 61 amino acids. Especially preferred is the FT (Arg₆₂-Arg₄₀₅) designated soluble variant of the Amino acids 62 to 405 of SEQ ID NO. 5 quoted amino acid sequence.

The yeast host strains and the components of the hybrid vectors are the ones below specified.

The transformed yeast strains are cultured by methods known to those skilled in the art are known.

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

A number of carbon sources can be used. Examples more preferred Carbon sources are assimilable carbohydrates such. Glucose, maltose, mannitol, Fructose or lactose, or an acetate such as sodium acetate, either alone or in suitable mixtures can be used. To the suitable nitrogen sources For example, amino acids such as casamino acids, peptides and proteins and their belong Degradation products such as tryptone, peptone or meat extracts, yeast extract, Malt extract, corn steep liquor, as well as ammonium salts such as ammonium chloride, Ammonium sulfate or ammonium nitrate, either alone or in suitable Mixtures can be used. To the inorganic salts that used can be, for example, sodium, potassium, magnesium and Calcium sulphates, chlorides, phosphates and carbonates. The nutrient medium may additionally also contain growth-promoting substances. To the growth-promoting substances  For example, trace elements such as iron, zinc, manganese u. Ä., or individual Amino acids.

Because of the incompatibility between the endogenous two-micron DNA and Hybrid vectors with their replicon show yeast cells containing such hybrid vectors transformed, a tendency to lose it. Such yeast cells must be under be grown selective conditions, d. H. Conditions under which the Expression of a plasmid-encoded gene is required for growth. Most currently used selective and in the hybrid vectors of the invention occurring (see below) markers are genes that are enzymes for the amino acid or Code for purine biosynthesis. Therefore, synthetic minimal media in which the respective amino acid or purine base is absent. It is, however possible to use genes conferring resistance to a suitable biocide (eg. a gene conferring resistance to the aminoglycoside G418). Yeast cells with Vectors containing genes for antibiotic resistance have been transformed bred complex media containing the respective antibiotic, resulting in higher Growth rates and greater cell density can be achieved.

Hybrid vectors containing the complete two-micron DNA (including a functional DNA) Replication start site) remain stable within Saccharomyces cerevisiae strains lacking endogenous two micron plasmids (so-called cir ° strains) so that they are grown under non-selective growth conditions can, d. H. in a complex medium.

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

The breeding takes place by means of usual techniques. The breeding conditions like Temperature, pH of the medium and fermentation time are chosen so that a Maximum heterologous protein is produced. A selected yeast strain will  preferably 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. B. about pH 5, at least 1 to 3 days bred, preferably as long as satisfactory protein yields are obtained.

After its expression in yeast, the membrane-bound glycosyltransferase or its Variant either enriched within the cells or secreted into the culture medium and isolated by conventional means.

For example, the first step is usually that the cells through Centrifuging be separated from the culture liquid. Has the membrane-bound glycosyltransferase or its variant within the cells Enriched, the protein must be released from the cell interior by cell disruption become. Yeast cells can be prepared in various ways known to those skilled in the art, be unlocked: z. B. by applying mechanical forces such as shaking with Glass beads, by ultrasonic vibration, osmotic shock and / or by enzymatic Digestion of the cell wall. Is the desired membrane-bound glycosyltransferase or their variant associated or bound to one membrane fraction may be another Enrichment z. B. be achieved in that the cell extract of a differential Centrifugation is subjected, and the respective fraction optionally followed with a detergent such. B. Triton is treated. To those for cleaning the "Crude" glycosyltransferase or its variant suitable methods include the usual ones chromatographic methods such as affinity chromatography, for example with a suitable substrate, with antibodies or concanavalin A, Ion exchange chromatography, gel filtration, partition chromatography, HPLC, Electrophoresis, precipitation steps such as ammonium sulfate precipitation, and other methods, in particular the methods known from the literature.

Is the glycosyltransferase or its variant of the yeast cell in the can be secreted periplasmic space, a simplified isolation protocol The protein is used without cell lysis by enzymatic removal of the Cell wall or by chemicals, eg. Thiol reagents or EDTA, which Induce cell wall damage and thus release the produced glycosyltransferase enable, won. Is the glycosyltransferase or variant in the Secreted culture fluid, it can be obtained directly from it and by means of be cleaned above methods.  

For the detection of glycosyltransferase activity known from the literature be used. Galactosyltransferase activity may e.g. B. be determined by that the amount of radioactively labeled galactose that is converted into a suitable acceptor molecule how a glycoprotein or a free sugar residue is incorporated is measured. On analogous way sialyltransferase activity can be tested by z. B. Sialic acid is incorporated into suitable substrates, and fucosyltransferase activity can be tested by the transfer of fucose to a suitable acceptor.

The transformed yeast host cells of the invention may be administered using recombinant DNA techniques according to the following steps:

• Preparation of a hybrid vector comprising a yeast promoter and a membrane-bound one Glycosyltransferase or its variant coding DNA sequence, whereby this DNA sequence is 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 containing a Yeast promoter and one for the membrane-bound glycosyltransferase or a Variant of which encodes DNA sequence, said DNA sequence of controlled by said promoter.

In a first embodiment, the yeast hybrid vector according to the invention contains a Expression cassette containing a yeast promoter, one for a membrane-bound Glycosyltransferase or a variant of their DNA-encoding DNA sequence, these DNA sequence is controlled by said promoter, and a DNA sequence, the Contains yeast transcription termination signals.

In a second embodiment, the yeast hybrid vector according to the invention contains a Expression cassette containing a yeast promoter that is functional with a first Connected DNA sequence that encodes a signal peptide, and in the right Reading frame is linked to a second DNA sequence that is responsible for a membrane-bound glycosyltransferase or a variant thereof encoded, and a DNA sequence containing yeast transcription termination signals.  

The yeast promoter is a regulated (inducible) or constitutive promoter that preferably from a highly expressed yeast gene, in particular a Saccharomyces cerevisiae gene is derived. So can the promoters of the TRPI gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the Yeast mating pheromone genes encoding the α or α factor, or a promoter from a gene coding for a glycolytic enzyme, such as the promoter of the enolase, Glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (PGK) -, Hexokinase, pyruvate decarboxylase, phosphofructokinase, Glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, Triosephosphate isomerase, phosphoglucose isomerase or glucokinase genes become. Furthermore, it is possible to use hybrid promoters containing "upstream activation Sequences "(UAS) of a yeast gene and" downstream promoter elements "with a functional "TATA box" of another yeast gene, for example one 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 its functional fragments at nucleotides between positions -550 and -180, in particular at nucleotide 540, 263 or -198, and at nucleotide 5 of the GAP gene ends. Another preferred promoter of the regulated type is the PH05 promoter. A preferred constitutive promoter is a truncated one Acid phosphatase PH05 promoter to which the "upstream regulatory elements" (UAS) as well as the PH05 (-173) promoter element found at nucleotide 173 of the PH05 gene begins and ends at nucleotide-9 of this gene.

The signal sequence encoding DNA sequence is preferably derived from a yeast gene encoding a polypeptide which is normally secreted. Other signal sequences of heterologous proteins that are normally secreted can also be selected. Yeast signal sequences are, for example, the signal and prepro sequences of the yeast invertase gene, a gene-factor, pheromone peptidase (KEX1) gene, "killer toxin" gene and the repressible acid phosphatase (PH05) - Gene, and the glucoamylase signal sequence from Aspergillus awamori. Alternatively, combined signal sequences can be constructed by ligating a portion of the signal sequence (if any) of the gene naturally linked to the promoter used (for example PH05) to a portion of the signal sequence of another heterologous protein. Preferred combinations are those that permit precise cleavage between the signal sequence and the glycosyltransferase amino acid sequence. Additional sequences, such as pro or spacer sequences, with or without special processing signals, may also be included in the designs to allow accurate processing of precursor molecules. However, it is also possible to prepare fusion proteins with internal processing signals that allow for maturation in vivo or in vitro. For example, the processing signals contain Lys-Arg 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.

If a full-length glycosyltransferase or one of its membrane-bound Variants is expressed in yeast, the preferred yeast hybrid vector contains a Expression cassette containing a yeast promoter, one for said glycosyltransferase or variant coding DNA sequence controlled by said promoter, and a DNA sequence containing yeast transcription termination signals.

If a soluble variant of a glycosyltransferase is expressed in yeast, the Preferred yeast hybrid vector is an expression cassette containing a yeast promoter, the is operably linked to a first DNA sequence encoding a signal peptide and which is connected in the correct reading frame with a second DNA sequence which is responsible for the variant coded, and a DNA sequence, the yeast transcription termination signals contains.

DNA encoding a membrane-bound glycosyltransferase or one of its variants can be prepared by means of methods known in the art and contains genomic DNA, e.g. B. DNA derived from a genomic DNA library of Mammals, e.g. Rat, mouse and bovine cells or human cells has been. If necessary, the introns present in the genomic DNA coding for the Enzyme encodes, occurs, removes. One for a membrane bound Glycosyltransferase or a variant of their coding DNA also includes cDNA consisting of a mammalian cDNA library isolated or prepared from the corresponding mRNA can be. The cDNA library can be prepared from cells of different tissues, e.g. B. Placental cells or liver cells. The cDNA is transcribed via the mRNA conventional methods such as polymerase chain reaction (PCR).

For example, the isolation of poly (A) ⁺RNA from mammalian cells, e.g. B. HeLa cells, and the subsequent synthesis of the first strand of cDNA according to the Known standard methods. From this synthesized DNA template  starting from PCR, the target sequence, d. H. the glycosyltransferase DNA or one of its fragments can be amplified while amplifying the excess present unwanted sequences is minimized. For this purpose, the Sequence of a small nucleotide portion be known on both sides of the target sequence. With These flanking sequences become two synthetic single-stranded primer oligonucleotides whose sequence is chosen so that the bases to the respective flanking sequence are complementary. PCR begins with the denaturation of the mRNA-DNA hybrid strand, followed by the addition of the primer to the Target sequence flanking sequences. By adding a DNA polymerase and Deoxynucleoside triphosphates are formed into two pieces of double-stranded DNA, wherein each starts at the primer and spans the target sequence, the latter being copied becomes. Each of the newly synthesized products can be used as a template for the attachment of Primers and for extensions (in the next cycle) can be used, causing this leads to an exponential increase in discrete-length double-stranded fragments.

In addition, a DNA encoding a membrane-bound glycosyltransferase or one of their variants is encoded, enzymatically or chemically synthesized. 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 are encoded by a DNA mutant. Among a mutated DNA is also a silent mutant to understand in the one or more nucleotides by other nucleotides be replaced, wherein the new codons for the same (n) amino acid (s) encode. Such a mutated sequence can also be a degenerate DNA sequence. The latter are insofar as the genetic code degenerates, as an unlimited number of Nucleotides can be replaced by other nucleotides, without any change the initially encoded amino acid sequence means. Such degenerate DNA sequences may be useful as they have different restriction sites and / or frequencies of certain codons derived from a specific host for the optimal expression of a glycosyltransferase are preferred. Such DNA sequences preferably have codons, as they are preferably used by yeast.

A DNA mutant can also be obtained by using a naturally occurring genomic DNA or a cDNA according to methods known in the art in vitro is mutated. For example, from one for each membrane bound Glycosyltransferase-encoding full-length DNA is a partial DNA coding for a  soluble form of a glycosyltransferase encoded using Restriction enzymes are cut out. For this purpose, it is beneficial if an appropriate restriction site is available.

A preferred DNA sequence containing yeast transcriptional termination signals is the 3 'flanking sequence of a yeast gene, the correct transcriptional termination signal and contains the polyadenylation. Suitable 3 'flanking sequences are e.g. B. the sequences of the yeast gene naturally associated with the promoter used are connected. The preferred flanking sequence is that of the yeast PH05 gene.

The yeast promoter, the optional DNA sequence that is responsible for the Signal peptide encodes the DNA sequence responsible for a membrane-bound glycosyltransferase or one of their variants, and the DNA sequence encoding the yeast transcription termination signals contains are functionally in a tandem arrangement connected, d. they are juxtaposed in such a way that their normal Functions are retained. They are arranged so that the promoter is a right one Expression of the DNA sequence containing a membrane-bound glycosyltransferase or a their Varinaten coded causes (possibly with an upstream signal frequency), that the transcription termination signals ensure proper termination of transcription and cause the polyadenylation, and that the optionally present signal sequence in right reading frame associated with the above-mentioned DNA sequence in such a manner is that the last codon of the signal sequence directly with the first codon of said DNA sequence is linked and the protein is secreted. If the promoter and the Signal sequence derived from different genes, the promoter is preferably on the signal sequence at a position between the mRNA's onset and the ATG of the attached to the promoter naturally associated gene. The signal sequence should be hers own own ATG for the translation initiation. These sequences can by means of synthetic oligodeoxynucleotide linker with the recognition sequence of an endonuclease be joined together.

Vectors suitable for yeast replication and expression contain one Yeast replication start site. Hybrid vectors that have a yeast replication start site contain, for example, the chromosomal autonomously replicating segment (ARS), remain extrachromosomal in the yeast cell after transformation and are during mitosis replicates autonomously. Hybrid vectors containing sequences associated with the Yeast 2μ plasmid DNA are homologous may also be used. Such  Hybrid vectors are integrated by recombination into 2μ plasmids already in the Cell exist 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 replication start site for a bacterial host, especially Escherichia coli.

As far as the selective gene markers for yeast are concerned, all marker genes The selection for transformants is based on the phenotype Allow expression of the marker gene. Examples of suitable markers for yeast are those expressing resistance to antibiotics or, in the case of auxotrophic Yeast mutants, genes that compensate for deficiencies of the host. The genes in question confer, for example, resistance to the antibiotics G418, hygromycin or Bleomycin, or mediate prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2 or TRP1 gene.

Since the amplification of the hybrid vectors carried out in a convenient way in E. coli it is advantageous to have a genetic marker for E. coli and an E. coli replication start site. These can be obtained from E. coli plasmids such. B. pBR322, or a pUC plasmid, for example pUC18 or pUC19, which has 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 known by experts Methods, for example, characterized in that the expression cassette comprising a Yeast promoter and one coding for a glycosyltransferase, or a variant DNA sequence controlled by said promoter, or more Components of the expression cassette, containing the DNA fragments, the selective genetic markers for yeast and for a bacterial host and replication origins for yeast and for a bacterial host, in the predetermined order get connected.  

Yeast strains and their transformation

Suitable yeast host organisms are strains of the genus Saccharomyces, in particular Strains of Saccharomyces cerevisiae. The yeast strains mentioned include Yeast strains optionally freed of endogenous two micron plasmids and / or optionally no yeast peptidase activity (s), e.g. Peptidase yscα-, have yscA, yscB, yscY and / or yscS activity.

The invention further relates to a yeast strain which is hybridized with a vector containing an expression cassette comprising a yeast promoter and a coding for a membrane-bound glycosyltransferase or one of its variants Contains DNA sequence, said DNA is controlled by said promoter.

In a first embodiment, the yeast strain according to the invention with a A hybrid vector comprising an expression cassette comprising a yeast promoter, a DNA sequence coding for a membrane-bound glycosyltransferase or one of its Variants are coded, whereby this DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transformation termination signals, transformed.

In a second embodiment, the yeast strain of the invention with a Hybrid vector comprising an expression cassette comprising a yeast promoter, which is operably linked to a first DNA sequence coding for a signal peptide which is linked in the correct reading frame with a second DNA sequence, the encodes a membrane-bound glycosyltransferase or one of its variants, and a DNA sequence containing yeast transcription termination signals transformed.

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

The membrane-bound prepared by the process according to the invention Glycosyltransferases and their variants can be used in a manner known per se be, for. For the synthesis and / or modification of glycoproteins, Oligosaccharides and glycolipids (US Pat. No. 4,925,796, EP application 4 14 171).  

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

In the examples, the following abbreviations are used:

GT galactosyltransferase (EC 2.4.1.22);
PCR polymerase chain reaction;
ST sialyltransferase (EC 2.4.99.1) variants;
FT = fucosyltransferase.

Example 1 Cloning of galactosyltransferase (GT) cDNA from HeLa cells

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

1.1 Preparation of poly (A) ⁺RNA from HeLa cells

For RNA preparation, HeLa cells are grown on 5 plates (23 x 23 cm) in one Single cell culture grown. RNA gets out of the cultured cells quickly and effectively by extraction with guanidine HCl as described by MacDonald, R.J., et al. (Meth. Enzymol. [1987], 152, 226-227). In general, the yields about 0.6 to 1 mg total RNA per plate of confluent cells. The Poly (A) ⁺RNA is purified by affinity chromatography on oligo (dT) -cellulose after im Enriched Maniatis manual (Sambrook, J., Fritsch, E.F., and Maniatis, T. [1989], Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA), with 4 mg total RNA at one 400 ul column are applied. 3% of the applied RNA is enriched as Poly (A) ⁺RNA recovered, precipitated with three times the volume of ethanol and aliquoted at -70 ° C until use.

1.2 Synthesis of First Strand cDNA for PCR

Poly (A) ⁺RNA (mRNA) is reverse transcribed in DNA with Moloney murine leukemia virus RNase H⁻Reverse transcriptase (M-MLV H⁻RT) (BRL). The preparation of the 20 μl reaction mixture is followed by a small variation according to the protocol provided by BRL: 1 μg HeLa cell poly (A) ⁺RNA and 500 ng oligo (dt) _12-18 (Pharmacia) in 11.5 μl of sterile H₂O are heated at 70 ° C for 10 minutes and then cooled quickly on ice. Then 4 μl reaction buffer (BRL; 250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl₂), 2 μl 0.1 M dithiothreitol, 1 μl mixed dNTP (10 mM dATP, dCTP, dGTP, TTP , Pharmacia), 0.5 μl (17.5 U) RNAguard (RNase inhibitor from Pharmacia) and 1 μl (200 U) M-MLVH⁻RT. The reaction is carried out at 42 ° C and stopped after one hour by heating the tube for 10 minutes at 95 ° C.

To check the effectiveness of the reaction, an aliquot of the mixture (5 μl) in the presence of 2 μCi of α-32 P dCTP. By measuring the built-in dCTP the amount of synthesized cDNA is calculated. The yield in the synthesis of first strand is routinely between 5 and 15%.

1.3 Polymerase Chain Reaction

The oligodeoxynucleotide primers used for the PCR are analyzed in vitro according to the Phosphoramidite method (M.H. Caruthers in Chemical and Enzymatic Synthesis of Gene Fragments, H.G. Gassen and A. Lang, ed. Verlag Chemie, Weinheim, FRG) in an Applied Biosystems Synthesizer, Model 380B. They are in Table 1 cited.

Table 1

PCR primers

The following are standard PCR conditions for a 30 μl incubation: 1 μl the reverse transcriptase reaction (see Example 1.2), the approximately 5 ng first-strand cDNA contains, in each case 15 pmol of the relevant primer, in each case 200 .mu.mol of 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 60th (Biomed) under the following conditions: 0.5 minutes denaturation at 95 ° C, 1 minute annealing at 56 ° C and 1 minute 15 seconds extension at 72 ° C, over a total of 20-25 cycles. In the last cycle the primer extension lasts at 72 ° C For 5 minutes.

For sequencing and subcloning, the HeLa GT cDNA becomes overlapping in two Pieces amplified using different primer combinations:

• (1) Fragment P1-P4: Primers P1 and P4 are used for the amplification of a 0.55 kb DNA fragment located above nucleotide positions 7-556 in HeLa GT cDNA extends (SEQ ID NO: 1).
• (2) Fragment P3-P2d: Primers P3 and P2d are used for the amplification of a 0.77- kb fragment extending over nucleotide positions 457-1232 (SEQ ID NO: 1).

To avoid errors during the amplification, 4 independent PCR per Fragment performed. Primer P1up (KpnI) is used together with primer P4 to to determine the DNA sequence followed by the "start" codon.

After PCR amplification, fragment P1-P4 is restricted with the restriction enzymes EcoRI and HindIII digested, digestion on a 1.2% agarose gel analyzed from the gel eluted using GENECLEAN (BIO 101) and in the vector pUC18 (Pharmacia), with digested with the same enzymes, subcloned. Fragment P3-P2d is labeled with SacI and EcoRI digested, the digest is analyzed on a 1.2% gel, eluted and in Subcloned pUC18, which was digested with SacI and EcoRI. The resulting subclones are pUC18 / P1-P4 or pUC18 / P3-P2d. For the subcloning, the ligation and the Transformation of E. coli strain DH5α is performed according to standard protocols as in Example 2 described, proceed. Mini preparations of the plasmids pUC18 / P1-P4 or pUC18 / P3-P2d are denatured for dideoxy sequencing Double-stranded DNA was used with the T7 polymerase sequencing kit (Pharmacia). to Sequencing overlapping fragments obtained from both DNA strands by digestion with different restriction enzymes were prepared, M13 / pUC Sequencing primer and reverse sequencing primer (Pharmacia) used. Further subcloning of restriction fragments of the GT gene is in-depth Sequencing of overlapping fragments of both strands required. The sequence of  Fragments amplified by independent PCR show that the Amplification error is less than 1 in 3000 nucleotides. The complete Nucleotide sequence of the HeLa cell GT cDNA described in SEQ ID NO. 1 is to 99.2% homologous to that of the human placenta (Genbank entry no. M22921). It there are three differences:

• (a) Three additional base pairs in nucleotide positions 37-39 (SEQ ID NO: 1), the give an additional amino acid (Ser) in the N-terminal region of the protein;
• (B) bp 98 to 101 are "CTCT" instead of "TCTG" in the human sequence Placenta, giving two conservative amino acid substitutions (AlaLeu instead of ValTyr) at amino acid positions 31 and 32 in the membrane-comprising domain of GT Consequence;
• (c) the nucleotide at position 1047 changes from "A" to "G" without this Changes in the amino acid sequence causes.

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

The complete HeLa cell GT cDNA (SEQ ID NO: 1) is expressed as 1.2 kb EcoRI-EcoRI Restriction fragment cloned into plasmid pIC-7, wherein pIC-7 is a derivative of pUC8 with additional restriction sites in the multiple cloning site (Marsh, J.L., Erfle, M., and Wykes, E.J. [1984], Gene, 32, 481-485), yielding the vector p4AD113 results. To construct the GT expression cassette, the EcoRI restriction site becomes (bp 1227) at the 3 'end of the cDNA sequence as follows: vector p4AD113 is first linearized by digestion with EcoRV and then with alkaline phosphatase treated. In addition, 1 ug of the linearized plasmid DNA with 0.25 U EcoRI 1 Partially digested at 37 ° C for 1 hour. After electrophoresis on agarose gel is removed from the gel isolated by GENECLEAN (Bio 101) a fragment that is the size of the linearized Plasmid (3.95 kb) corresponds. The supernatant EcoRI end is with Klenow polymerase as described in the Maniatis manual (supra). After phenolization and precipitation with ethanol, the plasmid is re-ligated and for the Transformation of E. coli DH5α (Gibco / BRL) used. Of six transformants Plasmid miniprep preparations are prepared. The resulting plasmids are purified by means of Restriction analysis for the lack of EcoRI and EcoRV restriction sites at the 3 'end the HeLa GT cDNA checked. The plasmid designated p4AE113 is used for the selected following experiments, since its DNA sequence is identical to that of plasmid p4AD113 is with the exception that bp 1232-1238 with the  EcoRI-EcoRV restriction sites are eliminated.

example 2 Construction of expression cassettes for complete GT

For heterologous expression in Saccharomyces cerevisiae, the complete HeLa-GT cDNA sequence (SEQ ID NO: 1) is combined with yeast transcriptional control signals to achieve efficient initiation and termination of transcription. The promoter and terminator sequences are derived from the yeast acid phosphatase gene (PH05) (EP 1 00 561). The complete PH05 promoter is regulated by the supply of inorganic phosphate in the culture medium. High P _i concentrations lead to repression of the promoter, while a low P _i content induces. However, a short 173 bp PH05 promoter fragment can be used which lacks all regulatory elements and therefore behaves like a constitutive promoter.

2.1 Construction of a Phosphate-Inducible Expression Cassette

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

(a) Complete HeLa GT cDNA sequence

Vector p4AE113 with the complete GT cDNA sequence is combined with the Restriction enzymes EcoRI and BglII digested. The DNA fragments become separated electrophoretically on a 1% agarose gel. A 1.2 kb DNA fragment, the contains the complete cDNA sequence for HeLa-GT, is absorbed by the gel by adsorption "Glasmilk" isolated using the GENECLEAN kit (B10 101). On this fragment is the "ATG" start codon for the protein synthesis of GT directly behind the interface for EcoRI, while following the "TAG" stop codon 32 bp at whose Composition of the 3 'untranslated region of the HeLa GT and the multiple Cloning site of the vector with the BglII restriction site are involved.

(b) Vector for amplification in E. coli

The amplification vector, plasmid p31R (see EP 1 00 561), 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 such as isolated from the gel described above. This DNA fragment contains the big one SalI-HindIII vector fragment of the pBR322 derivative and a 337 bp PH05 Transcription termination sequence instead of the HindIII-BamHI sequence of pBR322.  

(c) Sequence for the inducible PH05 promoter

That containing the regulatory elements (UASp) for phosphate induction PH05 promoter fragment is from plasmid p31R (see EP 1 00 561) by digestion with the restriction enzymes SalI and EcoRI isolated. The 0.8 kb SalI-EcoRI DNA fragment contains the 276 bp SalI-BamHI pBR322 sequence and the 534 bp BamHI-EcoRI PH05 promoter fragment with the EcoRI linker (5'-GAATTC-3 '), which is in position -8 of the PH05 promoter sequence was introduced.

(d) Construction of plasmid pGTA 1132

The three DNA fragments (a) to (c) are ligated in a 12 μl ligation mixture: 100 ng DNA fragment (a) and each 30 ng of fragments (b) and (c) at 18 ° C 18 For hours with 0.3 U T4 DNA ligase (Boehringer) in the supplied ligase buffer (66 mM Tris-HCl pH 7.5, 1 mM dithioerythritol, 5 mM MgCl₂, 1 mM ATP). With Half of the ligation mixture becomes competent cells of E. coli strain DH5α (Gibco / BRL). For the production of competent cells and for the Transformation is done according to the standard protocol, as in the Maniatis manual (see above), proceeded. The cells are selective, at 75 μg / ml Ampicillin supplemented LB medium and incubated at 37 ° C. One receives approx. 120 transformants. Become from six independent transformants Plasmid minipreparations according to the modified alkaline lysis protocol of Birnboim, H.C., and Doly, J., as noted in the Maniatis manual (see above), carried out. The isolated plasmids are analyzed by restriction analysis with 4 various enzymes (EcoRI, PstI, HindIII, SalI and combinations). All six plasmids show the expected restriction fragments. One of the clones becomes selected and referred to as pGTA 1132. Plasmid pGTA 1132 contains the Expression cassette with the complete HeLa-GT cDNA, under the control of the phosphate-regulated PH05 promoter, and the PH05 transcription termination sequence. This expression cassette can be prepared from pGTA-1132 as a 2.35 kb salI HindIII fragment are excised and is used as a DNA fragment (1A) designated.

2.2 Construction of a constitutive expression cassette

For the construction of an expression cassette with a constitutive, unregulated Promoter becomes a 5'-end truncated PH05 promoter fragment without Used phosphate regulatory elements isolated from plasmid p31 / PH05 (-173) RIT  becomes.

(a) Construction of plasmid p31 / PH05 (-173) RIT

Plasmid p31RIT12 (EP 2 88 435) contains the complete regulated PH05 promoter (with one at nucleotide position -8 on a 534 bp BamHI-EcoRI fragment introduced EcoRI site), to which the coding sequence for the Yeast invertase signal sequence (72 bp EcoRI-XhoI) and the PH05 transcription termination signal (135bp XhoI-HindIII) cloned into Tandem arrangement between BamHI and HindIII of the pBR322-derived vector, followed.

The constitutive PH05 (-173) promoter element from plasmid pJDB207 / PH05 (-173) -YHIR (EP 3 40 170) comprises the nucleotide sequence of the yeast PH05 promoter from the Nucleotide position -9 to -173 (BstEII restriction site), but has no "upstream" regulatory sequences (UASp). The PH05 (-173) promoter behaves like a constitutive promoter. This example describes the replacement of the regulated one PH05 promoter in plasmid p31RIT12 by the short, constitutive PH05 (-173) promoter element to obtain plasmid p31 / PH05 (-173) RIT.

Plasmids p31RIT12 (EP 2 88 435) and pJDB207 / PH05 (-173) -YHIR (EP 3 40 170) are digested with the restriction endonucleases SalI and EcoRI. The respective 3.6 kb and 0.4 kb SalI-EcoRI fragments are isolated on a 0.8% agarose gel eluted the gel, precipitated with ethanol and in H₂O in a concentration of 0.1 pmol / ul resuspended. The two DNA fragments are ligated and aliquots of the Ligation mix of 1 μl is used for the transformation of competent E. coli HB101 (ATCC) cells used. Ampicillin-resistant colonies are individually in with ampicillin (100 μg / ml) supplemented LB medium. Plasmid DNA is after the method of Holmes, D.S., et al. (Anal. Biochem. [1981], 144, 193) and by means of Restriction digestion with SalI and EcoRI analyzed. The plasmid of a clone with the Right restriction fragments are referred to as p31 / PH05 (-173) RIT.

(b) Construction of plasmid pGTB1135

Plasmid p31 / PH05 (-173) RIT is digested with the restriction enzymes EcoRI and SalI. After separation on a 1% agarose gel, a 0.45 kb SalI-EcoRI fragment is obtained isolated from the gel using GENECLEAN (BIO 101). This fragment contains the 276-bp SalI-BamHI sequence of PBR322 and the 173bp BamHI (BstEII) -EcoRI fragment of  constitutive PH05 promoter. The 0.45 kb SalI-EcoRI fragment is mixed with the 1.2 kb EcoRI-BglII-GT cDNA (fragment (a)) to become the 3.5 kb BamHI-SalI vector part for amplification in E. coli with that in Example 2.1. described PH05 terminator (Fragment (b)) ligated. Ligation and transformation of E. coli strain DH5α be as above, to obtain 58 transformants. Plasmids are off isolated and through six independent colonies by minipreparations Restriction analysis characterized. All six plasmids show the expected Fragmentation pattern. A correct clone is referred to as pGTB 1135 and for Further cloning experiments were used to construct the expression cassette for HeLa-GT under the control of the constitutive PH05 (-173) promoter fragment. This expression cassette can be prepared from vector pGTB 1135 as a 2 kb SalI-HindIII fragment and is referred to as DNA fragment (1B).

example 3 Construction of expression vectors pDPGTA8 and pDPGTB5

The yeast vector used for heterologous expression is the episomal vector pDP34 (11.8 kb), which contains a yeast E. coli shuttle vector with the ampicillin resistance marker for E. coli and the yeast selective markers URA3 and dLEU2. Vector pDP34 (see EP 3 40 170) is digested with the restriction enzyme BamHI. The linearized Vector is isolated by means of GENECLEAN, and the overhanging ends by means of Klenow polymerase treatment as described in the Maniatis Handbook (see above), refilled. The reaction is after 30 minutes by heating at 65 ° C for 20 minutes stopped together with 10 mM EDTA. After ethanol precipitation, the plasmid is treated with SalI digested and digestion by gel electrophoresis on a 0.8% agarose gel separated. The (BamHI) -tuning, SalI cut vector pDP34 is analyzed by means of of the GENECLEAN kit as an 11.8 kb DNA fragment isolated from the gel.

The plasmids pGTA 1132 and pGTB 1135 are analogous to the vector preparation digested with HindIII. The protruding ends of the linearized plasmids are Klenow polymerase treatment and then a SalI digestion subjected to,

(2A) a 2.35 kb (HindIII) dumbbell SalI fragment with the phosphate-regulated expression cassette or
(2B) a 2.0 kb (HindIII) truncated SalI fragment is created with the constitutive expression cassette.

Ligation of blunt-ended SalI pDP34 vector portion with fragment 2A or fragment 2B and the transformation of competent cells of E. coli strain DH5α will be like carried out in Example 2 above, with 80 ng of the vector part and 40 ng Fragment 2A and 2B, respectively. 58 or 24 transformants are obtained. From each transformation, six plasmids are prepared and by means of Restriction analysis characterized. 2 plasmids show that for the construction with the Regulated expression cassette expected restriction pattern (fragment 2A). one the clones are chosen and designated pDPGTA8.

A plasmid exhibits this for construction with the constitutive expression cassette (Fragment 2B) expected restriction pattern and is referred to as pDPGTB5.

example 4 Transformation of S. cerevisiae strain BT 150

CsCl-purified DNA of the expression vectors pDPGTA8 and pDPGTB5 according to the protocol of R. Treisman in the Maniatis manual (see above). Each of the protease-deficient S. cerevisiae strains BT 150 (MaTα, his4, leu2, ura3, pra1, prb1, prc1, cps1) and H 449 (MATa, prb1, cps1, ura3Δ5, leu 2-3, 2-112, cir °) with in each case 5 μg of the plasmids pDPGTA8, pDPTGB5 and pDP34 (control without Expression cassette) according to the lithium acetate transformation method (Ito, H., et al., See above). Ura⁺ transformants are isolated and screened for GT activity (see above) used. Single transformed yeast cells will be selected and labeled as follows:

Saccharomyces cerevisiae BT 150 / pDPGTA8
Saccharomyces cerevisiae BT 150 / pDPGTB5
Saccharomyces cerevisiae BT 150 / pDP34

Saccharomyces cerevisiae H 449 / pDPGTA8
Saccharomyces cerevisiae H 449 / pDPGTB5
Saccharomyces cerevisiae H 449 / pDP34

example 5 Enzyme activity of complete, yeast expressed GT 5.1 Preparation of Cell Extracts

Cells of the transformed Saccharomyces cerevisiae strains BT 150 and H 449 each under uracil selection in histidine and leucine enriched yeast minimal media (Difco) bred. The growth rate of the cells will in no case by the  Introduction of an expression vector affected. Exponentially growing cells (at one OD₅₇₈ of 0.5) or stationary cells are harvested by centrifugation, once at 5 mM Tris-HCl buffer, pH 7.4 (buffer 1) and in a concentration corresponding to 0.1-0.2 OD₅₇₈ suspended in buffer 1. The cells are mechanically through 4 minutes of vigorous shaking on a vortex blender with glass balls (0.45-0.5 mm Diameter) broken, with cooling in between. The crude extracts are used directly for the determination of enzyme activity.

By differential centrifugation of the extract, a fractionation of the Cell components achieved. The extract is first centrifuged for 5 minutes at 450 g; the The supernatant obtained is then centrifuged for 45 minutes at 20,000 g.

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

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

5.2 Protein Test

The protein concentration is determined by the use of a BCA protein assay kit (Pierce) certainly.

5.3 Test for GT activity

GT activity can be monitored using radiochemical methods using either Ovalbumin, a glycoprotein having exclusively GlcNAc as acceptor, or of free GlcNAc as the acceptor substrate. Cell extracts (with 1-2 cell-induced ODs₅₇₈) are incubated for 45 or 60 minutes at 37 ° C in 100 μl Incubation mixture with 100 mM Tris-HCl, pH 7.4, 50 nCi UDP-1⁴C-gal (325 mCi / mmol), 80 nmol UDP-Gal, 1 μmol MnCl₂, 1% Triton X-100 and 1 mg ovalbumin or 2 μmol GlcNAc as acceptor. When a glycoprotein acceptor substrate is used, the reaction is stopped by acid precipitation of the protein, and the The amount of ¹⁴C-galactose incorporated into the ovalbumin is determined by means of Liquid scintillation counting (Berger, E.G., et al., 1978, Eur. J. Biochem. 213-222). If GlcNAc is the acceptor substrate, the reaction is terminated by addition of 0.4 ml  ice-cold H₂O stopped, and the not recorded UDP-¹⁴C-galactose is of the ¹⁴C products on an anion exchange column (AG X1-8, BioRad), such as described separately (Masibay, A.S., and Qasba, P.K., [1989], Proc. Natl. Acad Sci., USA 86, 5733-5737). Tests with and without acceptor molecules are carried out to test the To assess extent of hydrolysis of UDP-gal by nucleotide pyrophosphatases. The Results are shown in Table 2.

Table 2

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

The GT activity of cultures switched to inducible conditions (minimal P _i -containing medium) is approximately equal to the activity of cultures in minimal media constitutively expressing GT (Table 2). As expected, no enzyme activity is found in cells that are transformed only with the vector.

During fractionation, most of the GT activity is found (70-90%, see table 3) and the highest specific activity always at high acceleration obtained pellet (20,000 g). The enzyme activity can be increased by an addition of 1-2% Triton X-100 for enzyme testing. Both results suggest that that recombinant GT is membrane-bound in yeast cells as well as in HeLa cells is present.  

Table 3

Distribution of GT activity during fractionation of BT 150 / pDPGT5 cells

example 6 Purification of the recombinant GT

To release the membrane-bound enzyme, the 20,000 g pellet fraction of BT 150 / pDPGTB5 cells for 10 minutes at room temperature with 1% (w / v) Triton X-100 treated 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 becomes 0.2 μm filtered, and a GlcNAc-p-aminophenyl Sepharose column (Berger, E.G., et al. [1976], Experientia 32, 690-691) is charged with the filtrate, the bed volume being 5 ml and the flow rate is 0.2 ml / min at 4 ° C. The enzyme is treated with 50 mM Tris-HCl, pH 7.4, 5 mM GlcNAc, 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 combined and added to 2 x 1 L of 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100 dialyzed. The purified 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 when a yeast promoter is functionally linked to a first DNA sequence coding for encodes the signal sequence of the yeast invertase gene SUC2 and this in the correct reading frame is linked to a cDNA encoding a soluble GT.

(a) HeLa GT cDNA partial sequence

GT cDNA is released from plasmid p4AD113 (Example 1) by EcoRI digestion. The 1.2 kb fragment with complete GT cDNA is isolated and mixed with MvnI (Boehringer) partially digested, wherein the GT sequence in positions 43, 55, 140 and 288 of the in SEQ ID no. 1 nucleotide sequence shown. When 0.2 μg of the EcoRI-EcoRI fragments are digested with 0.75 μ MvnI for one hour, the  GT cDNA was restricted to nucleotide position 134 only once, with a 1,1- kb-MvnI-EcoRI fragment is formed.

(b) Vector for amplification in E. coli

Plasmid pUC18 (Pharmacia) is mixed with BamHI and EcoRI at the multiple Cloning site cut. The plasmid is then treated with alkaline Phosphatase, as described in the Maniatis manual (supra), a Subjected to agarose gel electrophoresis and from the gel as a 2.7 kb DNA fragment isolated.

(c) The constitutive PH05 (-173) promoter and the SUC2 signal sequence

The vector p31 / PH05 (-173) -RIT (Example 2) is labeled with the restriction enzymes BamHI and XhoI digested. A 0.25 kb BamHI-XhoI fragment containing the constitutive PH05 (-173) promoter and the adjacent sequence responsible for the invertase signal sequence (inv ss) is isolated. The fragment is then repeated with HgaI (BioLabs) cut. The HgaI recognition sequence is located on the DNA antisense strand. The restriction enzyme cuts "upstream" of the recognition sequence to those In such a way that the 5-protruding end of the antisense strand adjoins the end of the coding Sequence of the invertase signal sequence matches. That with BamHI and HgaI cut 0.24 kb DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence.

(d) adapter

Fragment (a) is linked to fragment (c) by means of an adapter sequence consisting of equimolar amounts of the synthetic oligonucleotides (microsynth) 5'CTGCACTG GCTGGCCG3 'and 5'CGGCCAGCCAG3' for the complementary strand becomes. The oligonucleotides are annealed to each other by first at 95 ° C are heated and then slowly cooled to 20 ° C. The attached adapter will stored in frozen condition.

(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 encoding the signal peptide, and Adapter (d) used in a molar ratio of 1: 2: 2: 30-100. The ligation will in 12 ul ligase buffer (66 mM Tris-HCl, pH 7.5, 1 mM dithioerythritol, 5 mM MgCl₂, 1 mM ATP) for 18 hours at 16 ° C. The ligation mixture is used for the  Transformation of competent E. coli strain DH5α cells (see above) independent transformants are carried out plasmid minipreparations. The isolated plasmids are prepared by restriction analysis with four different enzymes (BamHI, PstI, EcoRI, XhoI, also in combination). A single clone with the expected restriction pattern is referred to as psGT.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide, with the cDNA coding for soluble GT is confirmed for plasmid psGT in that the T7 sequencing kit (Pharmacia) and primer 5'AGTCGAGGTTAGTATGGC3 ', which is available in Position -77 in the constitutive PH05 (-173) promoter begins to be used.

The soluble Gt expression cassette containing the PH05 (-173) constitutive promoter, the for invertase signal peptide-encoding DNA sequence and the partial GT cDNA from the Plasmid psGT can be in the form of a 1.35 kb SalI (BamHI) EcoRI fragment be cut out. The expression cassette is still missing the PH05 terminator sequences added in the subsequent cloning step.

example 8 Construction of the expression vector pDPGTS

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

(a) vector part

Plasmid pDP34 is digested and pretreated as described in Example 3, with a blunt SalI vector fragment of 11.8 kb is formed.  

(b) Expression cassette

Plasmid psGT is first digested with SalI (at the multiple cloning site) linearized and then partially digested with EcoRI. A 1.35 kb DNA fragment containing the constitutive PH05 (-173) promoter, which encodes the yeast invertase signal peptide DNA sequence and the partial GT cDNA is isolated.

(c) PH05 terminator sequence

The PH05 terminator sequence is isolated from plasmid p31, which as described in EP 1 00 561 described from plasmid p30 is constructed. After digestion with the restriction enzyme HindIII become the protruding ends by Klenow polymerase treatment, such as described above, filled. The plasmid is then digested with EcoRI and inserted truncated EcoRI fragment of 0.39 kb with the PH05 terminator sequences isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells E. coli strain DH5α are carried out as described in Example 2. From the obtained transformants are isolated plasmids and by restriction analysis characterized. The plasmid of a clone with the correct restriction fragments becomes referred to as pDPGTS.

example 9 Expression of soluble GT in yeast

Analogously to Example 4, CsCl-purified DNA of the expression vector pDPGTS used for the transformation of S. cerevisiae strains BT 150 and H 449. Ura⁺ transformants are isolated and screened for GT activity. In each case one transformant is selected and each as Saccharomyces cerevisiae BT 150 / pDPGTS and Saccharomyces cerevisiae H 449 / pDPGTS. at Carrying out the test described in Example 5, there is GT activity in the Culture broths of both transformants.

example 10 Cloning of sialyltransferase (ST) cDNA from human HepG2 cells

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

Table 5

PCR primers

HepG2-ST cDNA can be prepared by using primers SIA1 and SIA3 as a 1.2 kb DNA fragment be amplified. The PCR is as described for GT cDNA under easy modified cycle conditions: 0.5 minutes denaturation at 95 ° C, 1 minute 15 seconds annealing at 56 ° C and 1 minute 30 seconds extension 72 ° C, over a total of 25 to 35 cycles. The last cycle takes the Primer extension at 72 ° C for 5 minutes.

After PCR amplification, the 1.2 kb fragment is digested with the restriction enzymes BamHI and PstI digested, the digest is analyzed on a 1.2% agarose gel, eluted from the gel and subcloned into the vector pUC18. The resulting subclone is called pSIA2.

example 11 Construction of the constitutive ST expression cassette

For constitutive heterologous expression, ST cDNA is used 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 an internal restriction site for the enzyme EcoRI contains (at bp 144), a 1.2 kb fragment with the complete St cDNA (SEQ ID NO: 3) prepared by partial digestion with EcoRI, with 1 μg of DNA and 0.25 U EcoRI (1 h, 37 ° C). After gel electrophoresis, the 1.2 kb EcoRI-BamHI fragment with the complete ST cDNA (SEQ ID NO: 3) isolated. On This DNA fragment contains the "ATG" start codon for the translation of ST into near the EcoRI restriction site. Three adenosine phosphates (see PCR primer SIA1)  provide an "A" in the bp position 12, which is in the consensus sequence to find "ATG" from highly expressed genes in yeast (Hamilton, R., et al. [1987], Nucleic Acids Res. 14, 5125-5149). To the stop codon "TAA" and to 5 bp of the 3 'untranslated Gene region joins the BamHI site.

The 1.2 kb EcoRI-BamHI-ST cDNA fragment is mixed with the 0.45 kb SalI-EcoRI fragment with the constitutive PH05 (-173) promoter [Example 2.2 (b)] and with a 3.5 kb BamHI-SalI vector portion for amplification in E. coli containing the PH05 terminator sequence contains [cf. Example 2.1, fragment (b)] ligated. Ligation and Transformation of the E. coli strain DH5α, as detailed in Example 2.1, carried out. One clone that has the expected restriction pattern is called pST2 designated.

Vector pST2 contains the expression cassette for HepG2-ST under the control of constitutive PH05 (-173) promoter in the form of a 2.0 kb SalI-HindIII fragment, which is referred to as DNA fragment (1C).

example 12 Expression of ST in yeast

Vector pDP34 (compare EP 3 40 170) is digested with the restriction enzyme BamHI. The linearized vector is isolated with GENECLEAN, and the protruding ends become by Klenow polymerase treatment as in Maniatis manual (see above) filled up. The reaction is after 30 minutes by heating for 20 minutes stopped at 65 ° C in the presence of 10 mM EDTA. After ethanol precipitation, the Plasmid digested with SAlI and gel electrophoresis on a 0.8% agarose gel subjected. The (BamHI) -stuffed, SalI cut vector pDP34 is used with isolated from the GENECLEAN kit in the form of a 11.8 kb DNA fragment.

Plasmid pST2 is digested with the restriction enzyme HindIII and analogously to Preparation of the vector by Klenow polymerase treatment at the HindIII site filled up, and analogous to the preparation of the vector. The product comes with SalI digested with a 2.0 kb (HindIII) dumbbell SalI fragment containing the constitutive ST expression cassette is formed (2C).

The ligation of 80 ng of the pDP34 vector with 40 ng fragment 2C and the Transformation of competent cells of E. coli strain DH5α becomes as in Example 2 described carried out. A clone that has the expected restriction pattern becomes  selected and referred to as pDPST5.

To transform yeast, CsCl purified DNA of the expression vector pDPST5 according to the standard method given in the Maniatis manual (see above) manufactured. S. cerevisiae strains BT 150 and H 449 are each given 5 μg Plasmid DNA transformed by the lithium acetate transformation method (Ito, H., et al, see above). Ura⁺ transformants are selected and screened Subjected to ST activity. In each case a positive transformant is selected and as Saccharomyces cerevisiae BT 150 / pDPST5 and Saccharomyces cerevisiae H 449 / pDPST5.

example 13 Enzyme activity of ST completely expressed in yeast 13.1 Preparation of Cell Extracts

Saccharomyces cerevisiae BT 150 / pDPST5 cells are cultured under uracil selection Cultured histidine and leucine enriched minimal yeast media. Exponentially growing cells (at an OD₅₇₈ of 0.5) or stationary cells are going through Centrifuged, washed once with 50 mM imidazole buffer, pH 7.0 (buffer 1) and resuspended in buffer 1 at a concentration corresponding to 0.1-0.2 OD₅₇₈. The cells are mechanically shaken vigorously for 4 minutes with glass beads (0.45-0.5 mm diameter) broken on a vortex mixer, with in between is cooled.

The ST activity in the crude extracts can be measured by the test described below become.

13.2 Test for ST activity

The ST activity can be determined by measuring the amount of radiolabelled Sialic acid transferred from CMP sialic acid to a glycoprotein acceptor, is measured. Upon completion of the reaction by acid precipitation, the precipitate becomes filtered through glass fiber filters (Whatman GFA), washed thoroughly with ice-cold ethanol and by liquid scintillation counting (Hesford et al. [1984], Glycoconjugate J. 1, 141-153) were tested for radioactivity. The cell extracts are in a 45 minutes long Incubation mixture tested, the 37 ul cell extract, giving about 0.5 mg of protein and 3 μl imidazole buffer, 50 mmol / l, pH 7.0, 50 nmol CMP-N-acetylneuraminic acid (Sigma) with an addition of CMP-3 H-N-acetylneuraminic acid (Amersham) to finally obtain a specific activity of 7.3 Ci / mol, and 75 μg  Asialo-fetuin (prepared by 60 minutes acid hydrolysis with 0.1 M H₂SO₄ at 80 ° C. and subsequent neutralization, dialysis and lyophilization).

ST activity is found in S. cerevisiae BT 150 / pDPST5- and H 449 / pDPST5 cells prepared crude extracts.

example 14 Construction of a soluble ST expression cassette (Lys₃₉-Cys₄₀₆)

The term ST (Lys₃₉-Cys₄₀₆) soluble ST is an N-terminal truncated 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 isolated.

(b) Vector for amplification in E. coli

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

(c) The constitutive PH05 (-173) promoter and the SUC2 signal sequence

The vector p31 / pH05 (-173) -RIT (Example 2) is labeled with the restriction enzymes BamHI and XhoI digested. A 0.25 kb BamHI-XhoI fragment with the constitutive PH05 (-173) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is isolated. The fragment is then repeated with HgaI (BioLabs) cut. The HgaI recognition sequence is found on the DNA antisense strand. The Restriction enzyme cuts above the recognition sequence in such a way that the 5-projecting end of the antisense strand with the end of the coding sequence of Invertase signal sequence matches. The 0.24 kb cut with BamHI and HgaI DNA fragment contains the constitutive linked to the yeast invertase signal sequence PH05 (-173) -Promotersequenz.

(d) adapter

Fragment (a) is attached to fragment (c) via an adapter sequence consisting of equimolar amounts of the synthetic oligonucleotides 5'CTGCAAAATTGCAAACCAAGG3 'and  5'AATTCCTTGGTTTGCAATTT3 'is produced in the case of the complementary strand. The oligonucleotides are annealed to each other by first heating to 95 ° C and then slowly cooled to 20 ° C. The attached adapter is in kept frozen state.

(e) Construction of a plasmid psST

For ligation, the linearized vector (b), the ST cDNA fragment (a), fragment (c) with the promoter and the signal peptide coding sequence and adapter (d) used in a molar ratio of 1: 2: 2: 30-100. Ligation takes place over 18 Hours in 12 μl ligase buffer (66 mM Tris-HCl, pH 7.5, 1 mM dithioerythritol, 5 mM MgCl₂, 1 mM ATP) at 16 ° C. With the ligation mixture are competent cells of the E. coli strain DH5α as described above. Plasmid minipreps are made from 24 independent transformants. A single clone following Characterization by means of restriction analysis with four different enzymes (BamHI, PstI, EcoRI, XhoI, also in combination) the expected restriction pattern is reported as psST.

The correct sequence at the fusion site of the coding for the invertase signal peptide Sequence with the coding for soluble ST cDNA (Lys₃₉-Cys₄₀₆) is for plasmid psST using the T7 sequencing kit (Pharmacia) and primer 5'ACGAGGTTAATGGC3 ', the at position -77 in the constitutive PH05 (-173) promoter begins, confirmed.

The expression cassette for soluble ST with the constitutive PH05 (-173) promoter, the for invertase signal peptide encoding DNA sequence and the partial ST cDNA  Plasmid psST may be in the form of a 1.35 kb SalI (BamHI) EcoRI fragment be cut out. The expression cassette is still missing in the subsequent cloning step to add PH05 terminator sequences.

example 15 Construction of the expression vector pDPSTS

For the construction of the expression vector for soluble ST (Lys₃₉-Cys₄₀₆), the assembled following fragments:

(a) vector part

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

(b) Expression cassette

Plasmid psSt is first digested with SalI (at the multiple cloning site) linearized and then partially digested with EcoRI. A 1.35 kb DNA fragment containing the constitutive Ph05 (-173) promoter, one coding for the yeast invertase signal peptide DNA sequence and partial ST cDNA is isolated.

(c) PH05 terminator sequence

The PH05 terminator sequence is isolated from plasmid p31, which is derived from plasmid p30 in EP 1 00 561. After digestion with the restriction enzyme HindIII become the protruding ends by Klenow polymerase treatment filled out above. The plasmid is then digested with EcoRI, and a blunt ended EcoRI fragment of 0.39 kb with the PH05 terminator sequences isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells E. coli strain DH5α are carried out as described in Example 2. plasmids are isolated from the obtained transformants and by restriction analysis characterized. The plasmid of a clone with the correct restriction fragments becomes referred to as pDPSTS.

example 16 Expression of soluble ST in yeast

Analogously to Example 4, S. cerevisiae strains BT 150 and H 449 by means of CsCl purified DNA of the expression vector pDPSTS transformed. Ura⁺ transformants are isolated and subjected to screening for ST activity. One each  Transformant is selected and as Saccharomyces cerevisiae BT 150 / pDPSTS and Saccharomyces cerevisiae H 449 / pDPSTS. With that in Example 13 described test finds ST activity in the culture broths of both transformants.

example 17 Construction of an expression cassette for soluble ST (Lys₂₇-Cys₄₀₆

The soluble St termed ST (Lys₂₇-Cys₄₀₆) is an N-terminal truncated variant, which comprises the entire stem region and the catalytic domain. It consists of 380 Amino acids, that is the amino acids 27 to 406 of SEQ ID NO. 3 specified Amino acid sequence.

(a) HepG2 ST cDNA partial sequence

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

(b) vector for amplification in U. coli

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

(c) The constitutive PH05 (-173) promoter and the SUC2 signal sequence

The vector p31 / PH05 (-173) -RIT (Example 2) is labeled with the restriction enzymes BamHI and XhoI digested. A 0.25 kb BamHI-XhoI fragment with the constitutive PH05 (-173) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is isolated. The fragment is then repeated with HgaI (BioLabs) cut. The HgaI recognition sequence is found on the DNA antisense strand. The Restriction enzyme cuts "upstream" of the recognition sequence in such a way that the 5-projecting end of the Antisensestranges with the end of the coding sequence of Invertase signal sequence matches. The 0.24 kb cut with BamHI and HgaI DNA fragment contains the bound to the yeast invertase signal sequence PH05 (-173) -Promotersequenz.

(d) adapter

Fragment (a) is attached to fragment (c) via an adapter sequence consisting of equimolar amounts of the synthetic oligonucleotides
5'CTGCAAAGGAAAAGAAGAAAGGGAGTTACTATGATTCCTTTAAATTGCAAACCAAGG3 '
and
5'AATTCCTTGTTGCAATTTAAAGGAATCATAGTAACTCCCTTTCTTCTTTTCCTT3 '
in the case of the complementary strand. The oligonucleotides are annealed to each other by first heating to 95 ° C and then slowly cooling to 20 ° C. The attached adapter is kept in the frozen state.

(e) Construction of the plasmid psST1

For ligation, the linearized vector (b), the ST cDNA fragment (a), fragment (c) with the promoter and the signal peptide coding sequence and adapter (d) used in a molar ratio of 1: 2: 2: 30-100. Ligation takes place over 18 Hours in 12 μl of ligase buffer (66 mM Tris-HCl, pH 7.5, 1 mM dithioerythritol, 5 mM MgCl₂, 1 mM ATP) at 16 ° C. With the ligation mixture are competent cells of the U. coli strain DH5α as described above. Plasmid minipreps are made from 24 independent transformants. A single clone following Characterization by means of restriction analysis with four different enzymes (BamHI, PstI, EcoRI, XhoI, also in combination) the expected restriction pattern is reported as designated psST1.

The correct sequence at the fusion site of the coding for the invertase signal peptide Sequence with soluble ST coding cDNA (Lys₂₇-Cys₄₀₆) is for plasmid psST1 using the T7 sequencing kit (Pharmacia) and primer 5'AGTCGAGTAGTATGGC3 ', which starts at position -77 in the constitutive PH05 (-173) promoter, confirmed.

The expression cassette for soluble ST (Lys₂₇-Cys₄₀₆) with the constitutive PH05 (-173) promoter, the DNA sequence coding for invertase signal peptide and the  partial ST cDNA from plasmid psST1 may be in the form of a 1.35 kb SalI (BamHI) - EcoRI fragments are excised. The expression cassette is still missing the PH05 terminator sequences to be added in the subsequent cloning step.

example 18 Construction of the expression vector pDPSTS1

For the construction of the expression vector for soluble ST (Lys₂₇-Cys₄₀₆), the assembled following fragments:

(a) vector part

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

(b) Expression cassette

Plasmid psST1 is first digested with SalI (at the multiple cloning site) linearized and then partially digested with EcoRI. A 1.35 kb DNA fragment containing the constitutive PH05 (-173) promoter, one coding for the yeast invertase signal peptide DNA sequence and partial ST cDNA is isolated.

(c) PH05 terminator sequence

The PH05 terminator sequence is isolated from plasmid p31 which, starting from Plasmid p30 as constructed in EP 1 00 561. After digestion with the Restriction enzyme HindIII by means of the protruding ends Klenow polymerase treatment as described above. The plasmid then becomes digested with EcoRI, and a blunt ended EcoRI fragment of 0.39 kb with the PH05 terminator sequences are isolated.

The ligation of fragments (a), (b) and (c) and the transformation of competent cells of U. coli strain DH5α are carried out as described in Example 2. From the obtained transformants, plasmids are isolated and by restriction analysis characterized. The plasmid of a clone with the correct restriction fragments becomes referred to as pDPSTS1.

example 19 Expression of soluble ST (Lys₂₇-Cys₄₀₆) in yeast

Analogously to Example 4, S. cerevisiae strains BT 150 and H 449 by means of CsCl purified DNA of the expression vector pDPSTS1 transformed. Ura⁺ transformants are isolated and subjected to screening for ST activity. One each  Transformant is selected and named as Saccharomyces cerevisiae BT 150 / pDPSTS1 and Saccharomyces cerevisiae H 449 / pDPSTS1. With that in Example 13 ST analysis is found in the culture broths of both transformants.

example 20 Cloning the FT cDNA from the human HL 60 cell line

Based on the published cDNA sequence (Goelz, S.E., et al. [1990], Cell 63, 1349-1356) for ELFT (ELAM-1 ligand fucosyltransferase) useful for α (1-3) fucosyltransferase, the following are oligonucleotide primers designed to contain a DNA fragment containing the open reading frame of the ELFT cDNA, PCR-amplification: 5'CAGCGCTGCCTGTTCGCGCCAT3 ' (ELFT-1B) and 5'GGAGATGCACAGTAGAGGATCA3 '(ELFT-2B). 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. With 15393 00070 552 001000280000000200012000285911528200040 0002004217616 00004 15274 these primers is a 1.3 kb Fragment amplified using the Perkin Elmer Cetus Taq polymerase kit. The fragment is prepared from fresh HL60 cDNA in the presence of 5% DMSO and 2mM MgCl₂ amplified, the cycle being as follows:

95 ° C 5 min,
25 × (1 min 95 ° C, 1 min 60 ° C, 1.5 min 72 ° C),
10 × (1 min 95 ° C, 1 min 60 ° C, 1.5 min + 15 sec / cycle 72 ° C).

In agarose gel electrophoresis, a clear 1.3 kb band appears, which is at Digest with SmaI or ApaI the pattern predicted from the published sequence having. The 1.3 kb band is purified by means of a Gene-clean kit (Bio 101) and incubated in subcloned the vector pCR1000 (Invitrogen). A single clone with the correct 1.3 kb Insert is selected and named BRB.ELFT / pCR1000-13. The FT cDNA is in the vector inserted so that it is oriented in opposite directions with respect to the T7 promoter. The open reading frame of the FT cDNA is completely sequenced and is compatible with the published sequence identical (SEQ ID NO: 5).

example 21 Construction of plasmids for inducible and constitutive expression soluble FT (Arg₆₂-Arg₄₀₅) in yeast

Soluble FT (Arg₆₂-Arg₄₀₅) is obtained from the FT cDNA encoding at nucleotide position 241 (NruI). Restriction site) begins to be expressed using the cytoplasmic tail and the region encompassing the membrane encompassing domain is missing (see Sequence ID No. 5).

Plasmid BRB.ELFT / pCR1000-13 is digested with HindIII, which in the multiple  Cloning region 3 'of the FT cDNA insert. The cohesive ends are in a reaction with Klenow DNA polymerase into blunt ends. XhoI linkers (5'CCTCGAGG3 ', Biolabs) are treated with kinase, annealed and ligated to the blunt ends of the plasmid, with a 100-fold molar excess of Linker is used. Excess linkers are removed by isopropanol precipitation of DNA is removed, and this is further digested with XhoI and NruI (cut to Nucleotide position 240 of the FT cDNA according to Sequence ID no. 5). The 1.1 kb NruI-XhoI fragment (a) contains the FT cDNA sequence without those for the cytoplasmic tail and the membrane-encompassing domain coding region bis to amino acid 61.

Plasmids p31RIT12 and p31 / PH05 (-173) RIT (see Example 2) are each labeled with SalI and XhoI digested. The 0.9 kb and 0.5 kb fragments are isolated and digested with HgaI cut. The resulting cohesive ends are blotted with Klenow DNA polymerase refilled. The blunt ends thus produced are in agreement with the 3 'end of the coding sequence for the yeast invertase signal sequence. then Cutting with BamHI yields a BamHI "blunt end" fragment (b) of 596 bp the inducible PH05 promoter and the invertase signal sequence with their own ATG or a BamHI "blunt end" fragment (c) with 234 bp with the short constitutive PH05 (-173) promoter and the invertase signal sequence.

Plasmid p31RIT12 is linearized with the restriction endonuclease SalI. Partial digestion with HindIII in the presence of ethidium bromide yields a 1 kb SalI-HindIII fragment which the 276 bp SalI-BamHI pBR322 sequence, the 534 bp acid phosphatase promoter of yeast PH05, the yeast invertase signal sequence (which codes for 19 amino acids) and contains the PH05 transcription terminator. The 1 kb SalI-HindIII fragment of p31RIT12 is added to the yeast E. coli shuttle vector pJDB207 (Beggs, J.D., Molecular Genetics in yeast, Alfred Benzon Symposium 16, Copenhagen, 1981, pages 383-389) cloned previously cut with SalI and HindIII. The resulting plasmid with the 1 kb insert is referred to as pJDB207 / PH05-RIT12.

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

The BamHI "blunt end" fragment (b) at 596 bp, the 1.1 kb NruI-XhoI fragment (a) and  the 6.8 kb XhoI-BamHI vector fragment (d) under standard conditions for the Ligation blunt ends ligated. Competent E. coli HB101 cells are aliquoted Parts of the ligation mixture transformed. Plasmid DNA from ampicillin-resistant Colonies are analyzed by restriction digestion. A single clone with the right one Expression plasmid is referred to as pJDB207 / PH05-I-FT.

The ligation of the DNA fragments (c), (a) and (d) yields the expression plasmid pJDB207 / PH05 (-173) -I-FT. The expression cassettes of these plasmids contain the Coding sequence of the invertase signal sequence in the reading frame with that of the soluble α (1-3) fucosyltransferase, which is under the control of the inducible PH05 or constitutive PH05 (-173) promoter. The Expression cassettes are used in the yeast U. coli shuttle vector pJDB207 between the Cloned BamHI and HindIII restriction sites.

The nucleotide sequence at the fusion site between the invertase signal sequence and that for soluble FT coding cDNA is prepared by DNA sequencing on a double-stranded Plasmid DNA confirmed using the primer 5'AGTCGAGGTTAGTATGGC3 ' which, from position -77 to -60, the nucleotide sequence of the PH05 and the PH05 (-173) promoter. The correct connection looks like this:

example 22 Construction of plasmids for inducible and constitutive expression of membrane-bound FT in yeast

These constructs use the coding sequence of the FT cDNA with their own ATG. The nucleotide sequence immediately upstream of the ATG is, however, because of its high G-C content is relatively unfavorable for expression in yeast. This region was replaced by a  A-T-rich sequence replaced by PCR methods. At the same time, a EcoRI restriction site introduced at the new nucleotide positions -4 to -9.

Table 6

PCR primers

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

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

The 191 bp EcoRI-NruI fragment (e), the 1.1 kb NruI-XhoI fragment (a) and the 4.1 kb  XhoI-EcoRI fragment (f) are ligated. Competent E. coli HB101 cells are included 1 μl aliquot of the ligation mixture transformed. The plasmid DNA ampicillin-resistant colonies is analyzed. Plasmid DNA of a single clone is called p31R / PH05-ssFT.

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

example 23 Cloning of the FT expression cassette into pDP34

Plasmids p31R / PH05-ssFT and p31R / PH05 (-173) -ssFT are digested with HindIII, which cuts at the 3 'of the PH05 transcriptional terminator. After a reaction with Klenow DNA polymerase digests the DNA with SalI. The 2.3 kb and 1.9 kb SalI "blunt end" fragments are each isolated.

Plasmids pJDB207 / PH05-I-FT and pJDB207 / PH05 (-173) -I-FT are digested with HindIII in the presence of 0.1 mg / ml ethidium bromide (to cut on an additional To avoid HindIII site in the invertase signal sequence) and then digested treated with Klenow DNA polymerase and SalI as above. A 2.1 kb or 1.8 kb Fragment is isolated.

Each of the 4 DNA fragments is blunt-ended with the 11.8 kb SalI vector fragment of pDP34 (see Example 3). After transformation competent E. coli HB101 cells and analysis of single transformant plasmid DNA become 4 correct expression plasmids as pDP34 / PH05-I-FT; pDP34 / PH05 (-173) -I-FT; pDP34R / PH05-ssFT and pDP34R / PH05 (-173) -ssFT.

The S. cerevisiae strains BT150 and H449 are each 5 μg of the 4th Expression plasmids (top) transformed analogously to Example 4. Single transformed Yeast cell colonies are selected and designated as follows:

Saccharomyces cerevisiae
BT150 / pDP34 / PH05-I-FT;
BT150 / pDP34 / PH05 (-173) -I-FT;
BT150 / pDP34R / PH05 SSFT;
BT150 / pDP34R / PH05 (-173) -ssFT;
H449 / pDP34 / PH05-I-FT;
H449 / pDP34 / PH05 (-173) -I-FT;
H449 / pDP34R / PH05 SSFT;
H449 / pDP34R / PH05 (-173) -ssFT.

The fermentation and preparation of cell extracts is carried out according to Example 5. With a test analogous to that of Goetz et al. (above) describes FT activity in the crude extracts from BT150 / pDP34R / PH05-ssFT, BT150 / pDP34R / PH05 (-173) -sFT, H449 / pDP34R / PH05-ssFT and H449 / pDP34R / PH05 (-173) -ssFT and in culture broths from H449 / pDP34 / PH05-I-FT, H449 / pDP34 / PH05 (-173) -I-FT, BT150 / pDP34 / PH05-I-FT and BT150 / pDP34 / PH05 (-173) -I-FT.  

Deposit of microorganisms

The following microorganism strains were used in the German Collection of Microorganisms (DSM), Mascher or Weg 16, D-3300 Braunschweig, deposited (specified are the deposit data and entry numbers):

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
Saccharomyces cerevisiae H 449: February 18, 1988; DSM 4413
Saccharomyces cerevisiae BT 150: May 23, 1991; DSM 6530

sequence Listing

Seq ID no. 1

Sequence type: nucleotide with corresponding protein
Sequence length: 1265 bp
Strängigkeit: double
Topology: linear
Molecule type: recombinant
Immediate experimental origin: plasmid p4AD113 from E. coli DH5α / p4AD113

Features: from 6 to 1200 bp CDNA sequence coding for HeLa cell galactosyltransferase from 1 to 6 bp EcoRI site from 497 to 504 bp I site from 1227 to 1232 bp EcoRI site from 1236 to 1241 bp EcoRV site from 1243 to 1248 bp BglII site

properties

EcoRI-HindIII fragment from plasmid p4AD113 with complete Galactosyltransferase encoding HeLa cell cDNA (EC 2.4.1.22)

Seq ID no. 2

Sequence type: protein
Sequence length: 357 amino acids
Type of molecule: C-terminal fragment of complete HeLa cell galactosyltransferase

properties

Soluble galactosyltransferase (EC 2.4.1.22) from HeLa cells

Seq ID no. 3

Sequence type: nucleotide with corresponding protein
Sequence length: 1246 bp
Strängigkeit: double
Topology: linear
Molecule type: recombinant
Immediate experimental origin: plasmid pSIA2 from E. coli DH5α / pSIA2

Features: from 15 to 1232 bp CDNA sequence encoding HepG2 cell sialyltransferase from 1 to 6 bp PstI site from 6 to 11 bp EcoRI site from 144 to 149 bp EcoRI site from 1241 to 1246 bp BamHI site

properties

PstI-BamHI fragment from plasmid pSIA2 with complete sialyltransferase coding HepG2 cDNA (EC 2.4.99.1)

Seq ID no. 4

Sequence type: protein
Sequence length: 368 amino acids
Type of molecule: C-terminal fragment of complete sialyltransferase

properties

Soluble sialyltransferase (EC 2.4.99.1) from human HepG2 cells

Seq ID no. 5

Sequence type: nucleotide with corresponding protein
Sequence length: 1400 bp
Strängigkeit: double
Topology: linear
Molecule type: recombinant
Immediate experimental origin: BRB.ELFT / pCR1000-13

Characteristics:
From 58 to 1272 bp cDNA coding for human α (1-3) fucosyltransferase
from 238 to 243 bp NruI site

properties

CDNA encoding complete α (1-3) fucosyltransferase

Claims (19)

A process for the production of a membrane-bound mammalian glycosyltransferase from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a variant thereof, characterized in that a yeast strain is cultured with a hybrid vector comprising an expression cassette comprising a promoter and a contains DNA sequence encoding said glycosyltransferase or its variant, which DNA is controlled by said promoter, transformed, and the enzymatic activity is recovered. 2. The method according to claim 1, characterized in that the glycosyltransferase of human origin. 3. A process for the preparation of a variant according to claim 1, characterized in that that the variant is due to the absence of the cytoplasmic tail, the Signal anchor and, if appropriate, a smaller part of the trunk region of the corresponding form with the full length. 4. A process for the production of a variant according to claim 3, characterized in that that a yeast strain is cultured, comprising an expression cassette comprising a Promoter functional with a first signal peptide encoding DNA sequence connected in the correct reading frame with a second, for the said variant linked to the coding DNA sequence controlled by said promoter, contains, and the enzymatic activity is recovered. 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 galactosyltransferase UDP-galactose: β-galactoside-α (1-3) galactosyltransferase (EC 2.4.1.151) or UDP-galactose: β-N-acetylglucosamine-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 has pictured amino acid sequence.   8. The method according to claim 3, characterized in that the galactosyltransferase the in Seq ID no. 2 has pictured amino acid sequence. 9. The method according to claim 1, characterized in that the glycosyltransferase a Sialyltransferase is. 10. The method according to claim 9, characterized in that the sialyltransferase CMP-NeuAc-β-galactoside-α (2-6) sialyltransferase (EC 2.4.99.1). 11. The method according to claim 9, characterized in that the sialyltransferase in Seq ID no. 3 has pictured amino acid sequence. 12. The method according to claim 9, characterized in that the sialyltransferase as ST (Lys₂₇-Cys₄₀₆) and from the amino acids 27 to 406 of Seq ID no. 3 listed amino acid sequence exists. 13. The method according to claim 9, characterized in that the sialyltransferase in Seq ID no. 4 has pictured amino acid sequence. 14. The method according to claim 1, characterized in that the glycosyltransferase a Fucosyltransferase is. 15. The method according to claim 14, characterized in that the fucosyltransferase GDP-fucose: β-galactoside-α (1-2) fucosyltransferase (EC 2.4.1.69) and GDP-fucose: N-Actylglucosamine α (1-3 / 4) fucosyltransferase (EC 2.4.1.65). 16. The method according to claim 14, characterized in that the fucosyltransferase the under Seq ID no. 5 has pictured amino acid sequence. 17. The method according to claim 14, characterized in that the fucosyltransferase as FT (Arg₆₂-Arg₄₀₅) and from the amino acids 62 to 405 of Seq ID no. 5 depicted amino acid sequence. 18. A yeast hybrid vector, characterized in that it comprises an expression cassette comprising a yeast promoter and one for a membrane-bound Mammalian glycosyltransferase selected from the group consisting of a galactosyltransferase,  a sialyltransferase and a fucosyltransferase, or a Variants encoding DNA sequence contains, said DNA sequence of said Promoter is controlled. 19. A yeast strain transformed with a hybrid vector according to claim 18.