IE69059B1 - Yeast as host for expression of heterologous glycosyl transferase enzymes - Google Patents

Abstract

4-18658/A/BEG Improved process for the production of glycosyltransferases of the disclosure The invention relates to the field of recombinant DNA technology and provides a novel method for the production of glycosyltransferases by use of transformed yeast strains. [CA2070057A1]

Description

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

Glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide sugar, to a specific acceptor sugar thus forming a glycosidic linkage. Based on the type of sugar transferred, these enzymes are grouped into families, e.g. galactosyltransferases, sialyltransferases and fucosyltransferases. Being resident membrane proteins primarily located in the Golgi apparatus, the glycosyltransferases share a common domain structure consisting of a short amino-terminal cytoplasmic tail, a signal-anchor domain, and an extended stem region which is followed by a large carboxy-terminal catalytic domain. The signal-anchor domain acts as both uncleavable signal peptide and as membrane spanning region and orients the catalytic domain of the glycosyltransferase within the lumen of the Golgi apparatus. The luminal stem or spacer region is supposed to serve as a flexible tether, allowing the catalytic domain to glycosylate carbohydrate 15 groups of membrane-bound and soluble proteins of the secretory pathway enroute through the Golgi apparatus. Furthermore, the stem portion was discovered to function as retention signal to keep the enzyme bound to the Golgi membrane (FCT Application No. 91/06635). Soluble forms of glycosyltransferases are found in milk, serum and other body fluids. These soluble glycosyltransferases are supposed to result from proteolytic release from the 20 corresponding membrane-bound forms of the enzymes by endogenous proteases, presumably by cleavage between the catalytic domain and the transmembrane domain.

Enzymatic synthesis of carbohydrate structures has the advantage of a high stereoselectivity and iegioselectivity, rendering the glycosyltransferases a valuable tool for the modification or synthesis of glycoproteins, glycolipids and oligosaccharides. In contrast to chemical methods the time-consuming introduction of protective groups is superfluous.

As glycosyltransferases are naturally occurring in very low amounts, isolation from natural sources and subsequent purification are difficult Therefore, production using -2recombinant DNA technology has been worked on. For example, galactosyltransferases have been expressed in E. coli (PCT 90/07000) and Chinese hamster ovary (CHO) cells (Smith, D.F. etal. (1990) J. Biol. Chem. 265,6225-34), sialyltransferases have been 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 produced 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 ceUs (Potvin, B. (1990) J. Biol. Chem. 265,1615-1622). Considering the facts that heterologous expression in prokaryotes has the disadvantage of providing unglycosylated products, glycosyl10 transferases, however, being glycoproteins, and that glycosyltransferase production by use of mammalian hosts is very expensive as well as complicated due to the presence of many endogenous glycosyltransferases which would contaminate the desired product, there is a need for improved methods which render possible the economic production of glycosyltransferases on a large 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 by a recombinant DNA technology using a yeast vector expression system.

More specifically, the present invention provides a process far the production of a membrane-bound mammalian glycosyltransferase selected from the group consisting of a galactosyltransferase, a sialyltransferase and a fuoosyltransferase, or a membrane-bound or a soluble variant thereof, respectively, consisting of essentially the whole stem region and the catalytic domain said process comprising culturing a yeast strain which has been transformed with a hybrid vector, ootqprising an expression cassette comprising a promoter and a ENA sequence coding far said glycosyltransferase or variant which ENA is controlled by said promoter, and recovering the enzymatic activity.

In a first embodiment, the invention relates to a process for the production of a membrane-bound glycosyltransferase selected from the group consisting of a galactosyl3 0 transferase, a sialyltransferase and a fucosyltransferase, or a variant thereof, respectively, said process comprising culturing a yeast strain which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter, a DNA sequence coding for said glycosyltransferase or variant which DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transcription termination signals, and -3recovering the enzymatic activity.

In a second embodiment, the invention relates to a process for the production of a membrane-bound glycosyltransferase selected from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a variant thereof, respectively, 5 said process comprising culturing a yeast strain which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence coding for said glycosyltransferase or variant, and a DNA sequence containing yeast transcription termination signals, and recovering the enzymatic activity.

The term glycosyltransferase whenever used hereinbefore or hereinafter is intended to embrace the family of galactosyltransferases, the family of sialyltransferases and the family of fucosyltransferases. Said glycosyltransferases are naturally occurring enzymes of mammalian, e.g. bovine, murine, rat and human origin. Preferred are naturally occurring human full-length glycosyltransferases including those enzymes identified 15 hereinafter by their EC-numbers.

The membrane-bound galactosyltransferases and their variants obtainable according to the inventive process catalyse the transfer of a galactose residue from an activated donor, usually a nucleotide activated donor such as uridine diphosphate galactose (UDP-Gal), to a carbohydrate group.

Examples of membrane-bound galactosyltransferases are UDP-Galactose: β-galactoside a(l-3)-galactosyltransferase (EC 2.4.1.151) which uses galactose as acceptor substrate forming an a(l-3)-linkage and UDP-Galactose: β-Ν-acetylglucosamine β(1-4)^8ΐ3<Ή^1ΐηη$ί6Γ3$6 (EC 2.4.1.22) which transfers galactose to N-acetylglucosamine (GlcNAc) forming a p(l-4)-linkage, including variants thereof, respectively. In the presence of α-lactalbumin, said P(l-4)-gaIac tosyl transferase also accepts glucose as an acceptor substrate, thus catalysing the synthesis of lactose.

The most preferred membrane-bound galactosyltransferase is the enzyme having the amino acid sequence depicted in the sequence listing with the SEO ID NO. L The membrane-bound sialyltransferases and their variants obtainable according to the 30 process of the invention catalyse the transfer of sialic acids (for example N-acetyl -4neuraminic acid (NeuAc)) from an activated donor, usually a cytidine monophosphate sialic acid (CMP-SA) to a carbohydrate acceptor residue. An example of a membrane-bound sialyltransferase obtainable according to the inventive method is the CMP-NeuAc: β-galactoside a(2-6)-sialyltransferase (EC 2.4.99.1) which forms the NeuAc-a(2-6)Gal-3(l-4)GlcNAc-sequence common to many N-linked carbohydrate groups.

The most preferred membrane-bound sialyltransferase is the enzyme having the amino acid sequence depicted in the sequence listing with the SEQ ID NO. 3.

The membrane-bound fucosyltransferases and their variants obtainable according to the 10 process of the invention catalyse the transfer of a fucose residue from an activated donor, usually a nucleotide-activated donor, such as guanosine diphosphate fucose (GDP-Fuc), to a carbohydrate group. Examples of such fucosyltransferases are GDP-Fucose:p-galactoside a(l-2)-fucosyltransfera$e (EC 2.4.1.69) and GDP-Fucose:N-acetylglucosamine α( 1 -3/4)-fucosyltransferase (EC 2.4.1.65).

The most preferred membrane-bound fucosyltransferase is the enzyme having the amino acid sequence depicted in the sequence listing with SEQ ID NO. 5.

The term variants as used herein is intended to embrace both membrane-bound and soluble variants of the naturally occurring membrane-bound glycosyltransferases of mammalian origin with the provision that these variants are enzymatically active.

Preferred are variants of human origin.

For example, the term variants is intended to include naturally occurring membrane-bound variants of membrane-bound glycosyltransferases found within a particular species, e.g. a variant of a galactosyltransferase which differs from the enzyme having the amino acid sequence with the SEQ ID NO. 1 in that it lacks serine in position 11 and has the amino acids valine and tyrosine instead of alanine and leucine in positions 31 and 32, respectively. Such a variant may be encoded by a related gene of the same gene family or by an allelic variant of a particular gene. The term variants also embraces glycosyltransferases produced from a DNA which has been subjected to in vitro mutagenesis, with the provision that the protein encoded by said DNA has the enzymatic activity of the native glycosyltransferase. Such modifications may consist in an addition, exchange and/or deletion of amino acids, the latter resulting in shortened variants. -5Prefeired variants prepared according to the process of the invention are shortened variants, particularly soluble variants, i.e. variants which are not membrane-bound. Shortened variants include for example soluble forms of membrane-bound glycosyltransferases and their membrane-bound variants, e.g. those variants mentioned above, which are secretable by a transformed yeast strain used in the process according to the invention. According to the present invention, these soluble enzymes are the preferred truncated variants.

The invention also relates to a process for the production of a soluble variant of a membrane-bound glycosyltransferase selected from the group consisting of a galactosyl10 transferase, a sialyltransferase and a fucosyltransferase, said process comprising culturing a yeast strain which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence coding for said variant, and a DNA sequence containing yeast transcription termination signals, and isolating said variant By definition the soluble form of a glycosyltransferase is a shortened variant differing from the corresponding full-length, i.e. the membrane-bound form naturally located in the endoplasmic reticulum or the Golgi complex, by lack of the cytoplasmic tail, the signal-anchor and, optionally, part of the stem region. The term part of the stem region 20 as used herein is defined to be a minor part of the N-terminal side of the stem region consisting of up to 12 amino acids. In other words, the soluble variants prepared according to the process of the present invention consist of essentially the whole stem region and the catalytic domain.

The soluble variants are enzymatically active enzymes differing from the corresponding 25 full-length forms by the absence of an NH2-terminal peptide consisting of 26 to 67, particularly 26 to 61, amino acid residues, with the provision that those forms lacking part of the stem region only lack the above-defined minor part therof. Preferred are soluble variants obtainable from the membrane-bound glycosyltransferases identified hereinbefore by their EC-numberS and additionally soluble variants Obtainable from the membrane-bound fucosyltransferase with SEQ. ID NO. 5.

Preferred soluble variants of galactosyltransferases are distinct from the corresponding -6full-length forms in that they lack an NH2-terminal peptide consisting of 37 to 55. particularly 41 to 44, amino acids. The most preferred soluble variant prepared according to the inventive process is the enzyme having the amino acid sequence depicted in the sequence listing with the SEQ ID NO. 2.

Preferred soluble variants of sialyltransferases miss an NH2-terminal peptide consisting of 26 to 38 amino acids compared to the full length form. The most preferred soluble variant prepared according to the inventive process is the enzyme having the amino acid sequence depicted in the sequence listing with the SEQ ID NO. 4. Likewise preferred is the soluble variant designated ST(Lys27-Cys406) consisting of the amino acids 27 to 406 of the amino acid sequence listed in SEQ ID NO. 3.

Preferred soluble variants of fucosyltransferases differ from the corresponding full-length enzymes in that they lack an NH2-terminal peptide consisting of 56 to 67. particularly 56 to 61, amino acids. Especially preferred is the soluble variant designated FT(Arg62-Arg405) consisting of the amino acids 62 to 405 of the amino acid sequence listed in SEQ ID NO. 5.

The yeast host strains and the constituents of the hybrid vectors are those specified below.

The transformed yeast strains are cultured using methods known in the art Thus, the transformed yeast strains according to the invention are cultured in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts.

Various carbon sources are usable. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol, fructose or lactose, or an acetate such as sodium acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as casamino acids, peptides and proteins and their degradation products, such as tiyptone, peptone or meat extracts, furthermore yeast extract, malt extract, com steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or nitrate which can be used either alone or in suitable mixtures. Inorganic salts which may be used include, for example, sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally, the nutrient medium may also contain growth promoting substances. Substances which promote growth include, for example, trace elements, such as iron, zinc, manganese and -7the like, or individual amino acids.

Due to the incompatibility between the endogenous two-micron DNA and hybrid vectors carrying its replicon, yeast cells transformed with such hybrid vectors tend to lose the latter. Such yeast cells have to be grown under selective conditions, i.e. conditions which 5 require the expression of a plasmid-encoded gene for growth. Most selective markers currently in use and present in the hybrid vectors according to the invention (infra) are genes coding for enzymes of amino acid or purine biosynthesis. This makes it necessary to use synthetic minimal media deficient in the corresponding amino acid or purine base. However, genes conferring resistance to an appropriate biocide may be used as well [e.g. a o gene conferring resistance to the amino-glycoside G418]. Yeast cells transformed with vectors containing antibiotic resistance genes are grown in complex media containing the corresponding antibiotic whereby faster growth rates and higher cell densities are reached.

Hybrid vectors comprising the complete two-micron DNA (including a functional origin of replication) are stably maintained within strains of Saccharomvces cerevisiae which are devoid of endogenous two-micron plasmids (so-called cir° strains) so that the cultivation can be carried out under non-selective growth conditions, i.e. in a complex medium.

Yeast cells containing hybrid plasmids with a constitutive promoter express the DNA encoding a glycosyltransferase, or a variant thereof, controlled by said promoter without induction. However, if said DNA is under the control of a regulated promoter the composition of the growth medium has to be adapted in order to obtain maximum levels of mRNA transcripts, e.g. when using the PH05 promoter the growth medium must contain a low concentration of inorganic phosphate for derepression of this promoter.

The cultivation is carried out by employing conventional techniques. The culturing conditions, such as temperature, pH of the medium and fermentation time are selected in such a way that maximal levels of the heterologous protein are produced. A chosen yeast strain is preferably grown under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 25° to 35°C, preferably at about 28°C, at a pH value of from 4 to 7, for example at approximately pH 5, and for at least 1 to 3 days, preferably as long as satisfactory yields of protein are obtained, After expression in yeast the glycosyltransferase, or its variant, is either accumulated inside the cells or secreted into the culture medium and is isolated by conventional means. -8For example, the first step usually consists in separating the cells from the culture fluid by centrifugation. In case the glycosyltransferase, or its variant, has accumulated within the cells, the protein has to be liberated from the cell interior by cell disruption. Yeast cells can be disrupted in various ways well-known in the art: e.g. by exerting mechanical forces such as shaking with glass beads, by ultrasonic vibration, osmotic shock and/or by enzymatic digestion of the cell wall. In case the glycosyltransferase, or its variant, to be isolated is associated with or bound to a membranous fraction, further enrichment may be achieved for example by differential centrifugation of the cell extract and, optionally, subsequent treatment of the particular fraction with a detergent, such as Triton ( triton is a Trade-Mark). Methods suitable for the purification of the crude glycosyltransferase, or the variant thereof include standard chromatographic procedures such as affinity chromatography, for example with a suitable substrate, antibodies or Concanavalin A, ion exchange chromatography, gel filtration, partition chromatography, HPLC, electrophoresis, precipitation steps such as ammonium sulfate precipitation and other processes, especially those known from the literature.

In case the glycosyltransferase, or its variant, is secreted by the yeast cell into the periplasmic space, a simplified isolation protocol can be used: the protein is recovered without cell lysis by enzymatic removal of the cell wall or by chemical agents, e.g. thiol reagents or EDTA, which gives rise to cell wall damages permitting the produced glycosyltransferase to be released. In case the glycosyltransferase, or its variant, is secrected into the culture broth, it can be recovered directly therefrom and be purified using the methods specified above.

In order to detect glycosyltransferase activity assays known from the literature can be used. For example, galactosyltransfcrase activity can be measured by determing the amount of radioactively labelled galactose incorporated into a suitable acceptor molecule such as a glycoprotein or a free sugar residue. Analogously, sialyltranslerasc activity may be assayed e.g. by the incorporation of sialic acid into suitable substrates, and fucosyltransfcrasc activity can be assayed by the transfer of fucose to a suitable acceptor.

The transformed yeast host cells according to the invention can be prepared by recombinant DNA techniques comprising the steps of: - preparing a hybrid vector comprising a yeast promoter and a DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, which DNA sequence is -9controlled by said promoter, - transforming a yeast host strain with said hybrid vector, - and selecting transformed yeast cells from untransformed yeast cells.

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

In a first embodiment, the yeast hybrid vector according to the invention comprises an expression cassette comprising a yeast promoter, a DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, which DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transcription termination signals.

In a second embodiment, the yeast hybrid vector according to the invention comprises an expression cassette comprising a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding a membrane-bound glycosyltransferase, or a variant therof, and a DNA sequence containing yeast transcription termination signals..

The yeast promoter is a regulated or a constitutive promoter preferably derived from a highly expressed yeast gene, especially a Saccharomvces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI or ΑΡΗΠ gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or α-factor or a promoter derived from a gene encoding 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 can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05 - GAP hybrid promoter). A preferred promoter is the promoter of the GAP gene, - 10especially functional fragments thereof starting at nucleotides between positions -550 and -180, in particular at nucleotide -540, -263 or -198, and ending at nucleotide -5 of the GAP gene. Another preferred promoter of the regulated type is the PH05 promoter. As a constitutive promoter a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) is preferred as is the PH05 (-173) promoter element starting at nucleotide -173-and ending at nucleotide -9 of the PH05 gene.

The DNA sequence encoding a signal peptide (signal sequence) is preferably derived from a yeast gene coding for a polypeptide which is ordinarily secreted. Other signal sequences of heterologous proteins, which are ordinarily secreted can also be chosen.

Yeast signal sequences are, for example, the signal and prepro sequences of the yeast invertase, q-factor, pheromone peptidase (KEX1), killer toxin and repressible acid phosphatase (FH05) genes and the glucoamylase signal sequence from Aspergillus awamori. Alternatively, fused signal sequences may be constructed by ligating part of the signal sequence (if present) of the gene naturally linked to the promoter used (for example PH05). with part of the signal sequence of another heterologous protein. Those combinations are favoured which allow a precise cleavage between the signal sequence and the glycosyltransferase amino acid sequence. Additional sequences, such as pro- or spacersequences which may or may not cany specific processing signals can also be included in the constructions to facilitate accurate processing of precursor molecules. Alternatively, fused proteins can be generated containing internal processing signals which allow proper maturation in vivo or in vitro. For example, the processing signals contain Lys-Arg, which is recognized by a yeast endopeptidase located 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 a membrane-bound variant thereof, is expressed in yeast, the preferred yeast hybrid vector comprises an expression cassette comprising a yeast promoter, a DNA sequence encoding said glycosyltransferase or variant, which DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transcription termination signals. If the DNA encodes a membrane-bound enzyme there is no need for an additional signal sequence.

In case a soluble variant of a membrane-bound glycosyltransferase is expressed in yeast, the preferred yeast hybrid vector comprises an expression cassette comprising a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said variant and a DNA -11sequence containing yeast transcription termination signals.

DNA encoding a membrane-bound glycosyltransferase, or a variant thereof, can be prepared by methods known in the art and comprises genomic DNA, e.g. isolated from a mammalian genomic DNA library, e.g. from rat, murine, bovine or human cells. If necessary, the introns occurring in genomic DNA encoding the enzyme are deleted.

Furthermore, DNA encoding a membrane-bound glycosyltransferase, or a variant thereof, comprises cDNA which can be isolated from a mammalian cDNA library or produced from the corresponding mRNA. The cDNA library may be derived from cells from different tissues, e.g. placenta cells or liver cells. The preparation of cDNA via the mRNA route is achieved using conventional methods such as the polymerase chain reaction (PCR).

For example, isolation of poly(A)+RNA from mammalian cells, e.g. HeLa cells, and subsequent first strand cDNA synthesis are performed following standard procedures known in the art Starting from this synthesized DNA template, PCR can be used to amplify the targeted sequence, i.e. the glycosyltransferase DNA or a fragment thereof, while the amplification of the numerically overwhelming nontarget sequences is minimized. For this purpose, the sequence of a small stretch of nucleotides on each side of the target sequence must be known. These flanking sequences are used to design two synthetic single-stranded primer oligonucleotides the sequence of which is chosen so that each has basepair comple20 mentarity with its respective flanking sequence. PCR starts by denaturing of the mRNADNA hybrid strand, followed by annealing the primers to the sequences flanking the target Addition of a DNA polymerase and desoxynucleoside triphosphates causes two pieces of double-stranded DNA to form, each beginning at the primer and extending across the target sequence, thereby copying the latter. Each of the newly synthesized products can serve as templates for primer annealing and extensions (next cycle) thus leading to an exponential increase in double-stranded fragments of discrete length.

·· Furthermore, DNA encoding a membrane-bound glycosyltransferase, or a variant thereof, can be enzymatically or chemically synthesized. A variant of a membrane-bound glycosyltransferase having enzymatic activity and an amino acid sequence in which one or 30 more amino acids are deleted (DNA fragments) and/or exchanged with one or more other , w -----amino acids, is encoded by a mutant DNA. Furthermore, a mutant DNA is intended to include a silent mutant wherein one or more nucleotides are replaced with other nucleotides with the new codons coding for the same amino acid(s). Such a mutant - 12sequence is also a degenerated DNA sequence. Degenerated DNA sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated DNA sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host to obtain optimal expression of a glycosyltransferase or a variant thereof. Preferably, such DNA sequences have the yeast preferred codon usage.

A mutant DNA can also be obtained by in vitro mutation of a naturally occurring genomic DNA or a cDNA according to methods known in the art. For example, the partial DNA coding for a soluble form of a glycosyltransferase may be excised from the full-length DNA coding for the corresponding membrane-bound glycosyltransferase by using restriction enzymes. The availability of an appropriate restriction site is advantageous therefor.

A DNA sequence containing yeast transcription termination signals is preferably the 3* flanking sequence of a yeast gene which contains proper signals for transcription termination and polyadenylation. Suitable 3* flanking sequences are for example those of the yeast gene naturally linked to the promoter used. The preferred flanking sequence is that of the yeast PH05 gene.

The yeast promoter, the optional DNA sequence coding for the signal peptide, the DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, and the DNA sequence containing yeast transcription termination signals are operably linked in a tandem array, i.e. they are juxtaposed in such a manner that their normal functions are maintained. The array is such that the promoter effects proper expression of the DNA sequence encoding a membrane-bound glycosyltransferase, or a variant thereof, (optionally preceded by a signal sequence), the transcription termination signals effect proper termination of transcription and polyadenylation and the optional signal sequence is linked in the proper reading frame to the above-mentioned DNA sequence in such a manner that the last codon of the signal sequence is directly linked to the first codon of said DNA sequence and secretion of the protein occurs. If the promoter and the signal sequence are derived from different genes, the promoter is preferably joined to the signal sequence at a site between the major mRNA start and the ATG of the gene naturally linked to the promoter. The signal sequence should have its own ATG for translation initiation. The junction of these sequences may be effected by means of synthetic - 13oligodeoxynucleotide linkers carrying the recognition sequence of an endonuclease.

Vectors suitable for replication and expression in yeast contain a yeast replication origin. Hybrid vectors that contain a yeast replication origin, for example the chromosomal autonomously replicating segment (ars), are retained extrachromosomally within the yeast cell after transformation and are replicated autonomously during mitosis. Also, hybrid vectors that contain sequences homologous to the yeast 2μ plasmid DNA can be used. Such hybrid vectors are integrated by recombination in 2μ plasmids already present within the cell, or replicate autonomously.

Preferably, the hybrid vectors according to the invention include one or more, especially one or two, selective genetic markers for yeast and such a marker and an origin of replication for a bacterial host, especially Escherichia coli.

As to the selective gene markers for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those expressing antibiotic resistance or, in the case of auxotrophic yeast mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotics G418, hygromycin or bleomycin or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2. LYS2 orTRP1 gene.

As the amplification of the hybrid vectors is conveniently done in E. coli. an E coli genetic marker and an E coli replication origin are included advantageously. These can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid, for example pUC18 or pUC19, which contain both E coli replication origin and E coli genetic marker conferring resistance to antibiotics, such as ampicillin.

The hybrid vectors according to the invention are prepared by methods known in the art, for example by linking the expression cassette comprising a yeast promoter and a DNA sequence coding for a glycosyltransferase, or a variant thereof, which DNA sequence is controlled by^said promoter, or the several constituents of the expression cassette, and the DNA fragments containing selective genetic markers for yeast and for a bacterial host and origins of replication for yeast and for a bacterial host in the predetermined order.

The hybrid vectors of the invention are used for the transformation of the yeast strains - 14described below.

Yeast strains and transformation thereof Suitable yeast host organisms are strains of the genus Saccharomyces, especially strains of Saccharomyces cerevisiae. Said yeast strains include strains which, optionally, have been cured of endogenous two-micron plasmids and/or which optionally lack yeast peptidase activity(ies), e.g. peptidase ysca, yscA, yscB, yscY and/or yscS activity.

The invention concerns furthermore a yeast strain which has been transformed with a hybrid vector comprising an expression cassette comprising a yeast promoter and a DNA sequence coding for a membrane-bound glycosyltransferase, or a variant therof, which DNA is controlled by said promoter.

In a first embodiment, the yeast strain according to the invention has been transformed with a hybrid vector comprising an expression cassette comprising a yeast promoter, a DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, which DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transcription termination signals.

In a second embodiment, the yeast strain according to the invention has been transformed with a hybrid vector comprising an expression cassette consisting of a yeast promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding a membrane-bound glycosyltransferase, or a variant thereof, and a DNA sequence containing yeast transcription termination signals.

The yeast strains of the invention are used for the preparation of a membrane-bound glycosyltransferase or a variant thereof.

The transformation of yeast with the hybrid vectors according to the invention is accomplished by methods known in the art, for example according to the methods described by Hinnen et al. (Proc. Natl. Acad. Sci. USA (1978) 75,1929) and Ito et al.

(J. Bact. (1983) 153,163-168).

The membrane-bound glycosyltransferases, and the variants thereof, prepared by the process according to the invention can be used in a manner known per se, e.g. for the - 15synthesis and/or modification of glycoproteins, oligosaccharides and glycolipids (US Patent 4,925,796; EP Application 414 171).

The invention concerns especially the method for the production of membrane-bound glycosyltransferases, and variants therof, the hybrid vectors, the transformed yeast strains, and the glycosyltransferases obtainable according to the inventive process, as described in the Examples.

In the Examples, the following abbreviations ate used: GT = galactosyltransferase (EC 2.4.1.22), PCR = polymerase chain reaction; ST 0 sialyltransferase (EC 2.4.99.1); FT= fucosyltransferase.

Example 1: Cloning of the 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) by the polymerase chain reaction (PCR) method: 1.1 Preparation of poly(A)+RNA from HeLa cells For RNA preparation HeLa cells are grown in monolayer culture on 5 plates (23x23 cm). The rapid and efficient isolation of RNA from cultured cells is performed by extraction with guanidine-HCl as described by Mac Donald, R.J. et al- (Meth. Enzymol. (1987) 152, 226-227). Generally, yields are about 0.6 -1 mg total RNA per plate of confluent cells. Enrichment of poly(A)+RNA is achieved by affinity chromatography on oligo(dT)-cellulose according to the method described in the Maniatis manual (Sambrook, J., Fritsch, EF. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Habor, USA), applying 4 mg of total RNA on a 400 μΐ column. 3 % of the loaded RNA are recovered as enriched poly( A)+RNA which is stored in aliquots precipitated with 3 volumes of ethanol at -70°C until it is used. 1.2 First strand cDNA synthesis for PCR Poly(A)+RNA (mRNA) is reverse-transcribed into DNA by Moloney Murine Leukemia Virus RNase H‘ Reverse Transcriptase (M-MLV H' RT) (BRL). In setting up the 20 μΐ reaction mix, the protocol provided by BRL is followed with minor variations: I ug of HeLa cell poly(A)+RNA and 500 ng Oligo(dT)12.18 (Pharmacia) in 11.5 μ! sterile H2O are heated to 70°C for 10 min and then quickly chilled on ice. Then 4 μΐ reaction buffer -16provided by BRL (250 mM Tris-HCl pH 8.3,375 mM KC1,15 mM MgCl2), 2 μΐ 0.1 M dithiothreitol, 1 μΐ mixed dNTP (10 mM each dATP, dCTP, dGTP, TTP, Pharmacia), 0.5 μΐ (17.5 U) RNAguard (RNase Inhibitor of Pharmacia) and 1 μΐ (200 U)M-MLVH' RT are added. The reaction is carried out at 42°C and stopped after 1 h by heating the tube to 95°C for 10 min.

In order to check the efficiency of the reaction an aliquot of the mixture (5 μΐ) is incubated in the presence of 2 pCi a-32P dCTP. By measuring the incorporated dCTP, the amount of cDNA synthesized is calculated. The yield of first strand synthesis is routinely between 5 and 15 %. 1.3 Polymerase chain reaction The oligodeoxynucleotide primers used for PCR are synthesized in vitro by the phosphoramidite method (M.H. Caruthers, in Chemical and Enzymatic Synthesis of Gene Fragments, H.G. Gassen and A. Lang, eds., Verlag Chemie, Weinheim, FRG) on an Applied Biosystems Model 380B synthesizer. They are listed in Table 1.

Table 1: PCR-primers corresponding to primer sequence (5’ to 3’ )υ bpinGTcDNA2* Plup(Kpnl) cgcggtACCCTTCTTAAAGCGGCGGCGGGAAGATG (-26)- 3 Pl (EcoRI) gccgaattcATGAGGCllCGGGAGCCGCTCCTGAGCG 1 - 28 P3 (Sacl) CTGGAGCTCGTGGCAAAGCAGAACCC 448 - 473 P2d (EcoRI) gccgaaTTC AGTCTCTTATCCGTGTACCAAAACGC CTA 1222-1192 P4 (Hindlll) cccaagctTGGAATGATGATGGCCACCTTGTGAGG 546- 520 Capital letters represent sequences from GT, small tetters are additional sequences, sites for restriction enzymes are underlined. Codons for ‘start* and 'stop* of RNA translation are highlighted in boldface. 21 GT cDNA sequence from human placenta as published in GenBank (Accession Nr. M22921).

Standard PCR-conditions for a 30 μΐ incubation mixture are: 1 pi of the Reverse Transcriptase reaction (see Example 1.2), containing about 5 ng first strand cDNA, 15 pmol each of the relevant primers, 200 pmol each 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 - 17KC1,1.5 mM MgCl2,0.001 % gelatine) and 0.5 U AmpliTaq Polymerase (Perkin Elmer).

The amplification is performed in the Thermocycler 60 (Biomed) using the following / conditions: 0.5 min denaturing at 95°C, 1 min annealing at 56°C, and 1 min 15 sec extension at 72°C, for a total of 20 - 25 cycles. In the last cycle, primer extension at 72°C is carried out for 5 min.

For sequencing and subcloning, the HeLa GT cDNA is amplified in two overlapping pieces, using different primer combinations: (1) Fragment Pl -P4: Primers Pl and P4 are used to amplify a 0.55 kb DNA fragment covering nucleotide positions 7-556 in HeLa GT cDNA (SEQ ID NO. 1) (2) Fragment P3 - P2d: Primers P3 and P2d are used to amplify a 0.77 kb fragment covering nucleotide positions 457 -1232 (SEQ ID NO. 1).

In order to avoid errors during amplification four independent PCRs are carried out for each fragment. Also primer Plup (Kpnl) in combination with primer P4 is used to determine the DNA sequence followed by the ’start’ codon.

After PCR amplification, fragment Pl - P4 is digested with the restriction enzymes EcoRI and Hindm, analysed on a 1.2 % agarose gel, eluted from the gel by GENECLEAN (BIO 101) and subcloned into the vector pUC18 (Pharmacia), digested with the same. enzymes. Fragment P3 - P2d is digested with Sacl and EcoRI, analysed on a 1.2 % gel, eluted and subcloned into pUC18, digested with Sad and EcoRI. The resulting subclones are pUC18/Pl - P4 and pUC18/P3 - P2d, respectively. For subcloning, ligation and transformation ofE. coli strain DH5a, standard protocols are followed as described in Example 2. Minipreparations of Plasmids pUC18/Pl - P4 and pUC18/P3 - P2d are used for dideoxy-sequencing of denatured double-stranded DNA with the T7 polymerase Sequencing kit (Pharmacia). M13/pUC sequencing primers and reverse sequencing primers (Pharmacia) are applied to sequence overlapping fragments produced from both DNA strands by digestion with various restriction enzymes. Further subcloning of restriction fragments of the GT gene is necessary for extensive sequencing of overlapping fragments of-both strands. The sequence of fragments amplified by independent PCRs shows that the error of amnlification is less than 1 in 3000 nucleotides. The comolete * « nucleotide sequence of the HeLa cell GT cDNA which is presented in SEQ ID NO. 1 is 99.2 % homologous to that of human placenta (Genbank Accession No. M22921). Three differences are found: - 18(a) Three extra base pairs at nucleotide positions 37-39 (SEQ ID NO. 1) resulting in one extra amino acid (Ser) in the N-terminal region of the protein; (b) bp 98 to 101 are ’CTCT’ instead of ’TCTG’ in the sequence of human placenta, leading to two conservative amino acid substitutions (Ala Leu instead of ValTyr) at amino acid positions 31 and 32 in the membrane spanning domain of GT; (c) the nucleotide at position 1047 is changed from ’ A’ to ’G’ without ensuing a change in amino acid sequence.

The two overlapping DNA-fragments Pl - P4 and P3 - P2d encoding the HeLa GT cDNA are joined via the Notl restriction site at nucleotide position 498 which is present in both fragments.

The complete HeLa cell GT cDNA (SEQ ID No. 1) is cloned as a 1.2 kb EcoRI-EcoRI restriction fragment in plasmid plC-7, a derivative of pUC8 with additional restriction sites in the multicloning site (Marsh, J.L., Erfle, M. and Wykes, E.J. (1984) Gene 32, 481-485), resulting in vector p4ADl 13. For the purpose of creating the GT expression cassette the EcoRl restriction site (bp 1227) at the 3' end of the cDNA sequence is deleted as follows: vector p4ADl 13 is first linearized by digestion with EcoRV and then treated with alkaline phosphatase. Furthermore, 1 pg of the linearised plasmid DNA is partially digested with 0.25 U EcoRl for 1 h at 37°C. After agarose gel electrophoresis a fragment corresponding to the size of the linearized plasmid (3.95 kb) is isolated from the gel by GENECLEAN (Bio 101). The protruding EcoRl end is filled in with Klenow polymerase as described in.the Maniatis manual (supra). After phenolisation and ethanol precipitation the plasmid is religated and used to transform E. coli DH5a (Gibco/BRL). Minipreparation of plasmids are prepared from six transformants. The plasmids obtained are checked by restriction analysis for the absence of the EcoRl and EcoRV restriction sites at the 3* end of HeLa GT cDNA. The plasmid designated p4AEl 13 is chosen for the following experiments, its DNA sequence being identical to that of plasmid p4ADl 13, with the exception that bp 1232-1238 with the EcoRI-EcoRV restriction sites are deleted.

Example 2: Construction of expression cassettes for full length GT For heterologous expression in Saccharomyces cerevisiae the full length HeLa GT cDNA sequence (SEQ ID NO. 1) is fused to transcriptional control signals of yeast for efficient initiation and termination of transcription. The promoter and terminator sequences originate from the yeast acid phosphatase gene (PH05) (EP100561). The full-length PH05 promoter is regulated by the supply of inorganic phosphate in the culture medium. High Pi -19concentrations lead to promoter repression whereas low Pi acts by induction. Alternatively, a short, 173 bp PH05 promoter fragment is used, which is devoid of all regulatory elements and therefore behaves as a constitutive promoter. 2.1 Construction of a phosphate inducible expression cassette The GT cDNA sequence is combined with the yeast PH05 promoter and transcription terminator sequences as follows: (a) Full length HeLa GT cDNA sequence: Vector p4AE113 with the full length GT cDNA sequence is digested with the restriction enzymes EcoRI and Bglll. The DNA fragments are electrophoretically separated on a 1 % agarose gel. A 1.2 kb DNA fragment containing the complete cDNA sequence for HeLa GT is isolated from the gel by adsorption to glasmilk, using the GENECLEAN kit (BIO 101). On this fragment the ’ATG’ start codon for protein synthesis of GT is located directly behind the restriction site for EcoRI, whereas the stop codon ’TAG' is followed by 32 bp contributed by the 3’untranslated region of HeLa GT and the multiple cloning site of the vector with the Bglll restriction site. (b) Vector for amplification in E coli: The vector for amplification, plasmid p31R (cf. EP 100561), a derivative of pBR322, is digested with the restriction enzymes BamHI and Sail. The restriction fragments are separated on a 1 % agarose gel and a 3.5 kb vector fragment is isolated from the gel as described before. This DNA fragment contains the large Sail - HindlH vector fragment of the pBR322 derivative as well as a 337 bp PH05 transcription terminator sequence in place of the Hindm - BamHI sequence of pBR322. (c) Sequence for the inducible PH05 promoter The PH05 promoter fragment containing the regulatory elements (UASp) for phosphate induction is isolated from plasmid p31R (cf. EP 100561) by digestion with the restriction enzymes Sail and EcoRI. The 0.8 kb Sail - EcoRI DNA fragment comprises the 276bp Sail - BamHI pBR322 sequence and the 534 bp BamHI-EcoRI PH05 promoter fragment with the EcoRL linker (5’-GAATTC-3’) introduced at position -8 of the PH05 promoter sequence. (d) Construction of plasmid pGTA 1132 The three DNA fragments (a) to (c) are ligated in a 12 μΐ ligation mixture: 100 ng of DNA -20fragment (a) and 30 ng each of fragments (b) and (c) are ligated using 0.3 U T4 DNA ligase (Boehringer) in the supplied ligase buffer (66 mM Tris-HCl pH 7.5,1 mM dithioerythiitol, 5 mM MgCl2.1 mM ATP) at 15°C for 18 hours.

Half of the ligation mix is used to transform competent cells ofE. coli strain DH5a (Gibco/BRL). For preparing competent cells and for transformation, the standard protocol as given in the Maniatis manual (supra) is followed. The cells are plated on selective LB-medium, supplemented with 75 gg/ml ampicillin and incubated at 37°C. About 120 transformants are obtained. Minipreparations of plasmid are performed from six independent transformants by using the modified alkaline lysis protocol of Bimboim, H.C. and Doly, J. as described in the Maniatis manual (supra). The isolated plasmids are characterized by restriction analysis with four different enzymes (EcoRI, Pstl, Hindu, Sail, also in combination). All six plasmids show the expected restriction fragments. One of the clones is chosen and referred to as pGTA 1132. Plasmid pGTA 1132 contains the expression cassette with the full-length HeLaGT cDNA under the control of the phosphate regulated PH05 promoter, and the PH05 transcriptional terminator sequence. This expression cassette can be excised from pGTA 1132 as a 2.35 kb Sail - Hindu fragment, referred to as DNA fragment (1 A). 2.2. Construction of a constitutive expression cassette: For the construction of an expression cassette with a constitutive, nonregulated promoter, a 5’ truncated PH05 promoter fragment without phosphate regulatory elements is used, which is isolated from plasmid p31/PH05(-173)RIT. (a) Construction of plasmid p31ZPHO5(- 173)RIT Plasmid p31 RTT12 (EP 288435) comprises the full length, regulated PH05 promoter (with an EcoRI site introduced at nucleotide position -8 on a 534bp BamHI - EcoRI fragment, followed by the coding sequence for the yeast invertase signal sequence (72bp EcoRI Xhol) and the PH05 transcription termination signal (135bp Xhol - Hindlll) cloned in a tandem array between BamHI and Hindlll of the pBR322 derived vector.

The constitutive PH05(-173) promoter element from plasmid pJDB207/PH05(-173)-YHIR (EP 340170) comprises the nucleotide sequence of the yeast PH05 promoter from nucleotide position -9 to -173 (BstEII restriction site), but has no upstream regulatory sequences (UASp). The PH05M73) promoter, therefore, behaves like a constitutive promoter. This example describes the replacement of the regulated PH05 promoter in plasmid p31Rm2 by the short, constitutive PH05 (-173) promoter element in order to -21obtain plasmid P31/PH05 (-173) RIT.

Plasmids p31RIT12 (EP 288435) and pJDB207/PH05(-173)- YHIR (EP 340170) are digested with restriction endonucleases Sail and EcoRI. The respective 3.6 kb and 0.4 kb Sail - EcoRI fragments are isolated on a 0.8 % agarose gel, eluted from the gel, ethanol precipitated and-resuspended in H2‘O at a concentration of 0.1 pmoles/μΐ. Both DNA fragments are ligated and 1 μΐ aliquots of the ligation mix are used to transform E. coli HB101 (ATCC) competent cells. Ampicillin resistant colonies are grown individually in LB medium supplemented with ampicillin (100 pg/ml). Plasmid DNA is isolated according to the method of Holmes, D.S. et al. (Anal. Biochem. (1981) 144,193) and analysed by restriction digests with Sail and EcoRI. The plasmid of one clone with the correct restriction fragments is referred to as p31/PH05(-173)RIT. (b) Construction of plasmid pGTB 1135 Plasmid p3 l/PH05(-173)R1T is digested with the restriction enzymes EcoRI and Sail. After separation on a 1 % agarose gel, a 0.45 kb Sail - EcoRI fragment is isolated from the gel by GENECLEAN (BIO 101). This fragment contains the 276 bp Sall-BamHI sequence of pBR322 and the 173bp BamHI(BstEII)-EcoRI constitutive PH05 promoter fragment The 0.45 kb Sall-EcoRI fragment is ligated to the 1.2 kb EcoRI - Bglll GT cDNA (fragment (a)) and the 3.5 kb BamHI-Sall vector part for amplification in E. coli with the PH05 terminator (fragment (b)) described in Example 2.1. Ligation and transformation of E. coli strain DH5a are carried out as described above yielding 58 transformants. Plasmids are isolated from six independent colonies by minipreparations and characterized by restriction analysis. All six plasmids show the expected fragments. One correct clone is referred to as pGTB 1135 and used for further cloning experiments to provide the expression cassette for HeLa GT under the control of the constitutive PH05 (-173) promoter fragment This expression cassette can be excised from the vector pGTB 1135 as a 2 kb Sail - Hindlll fragment referred to as DNA fragment (IB).

Example 3: Construction of the expression vectors PDPGTA8 and pDPGTB5 The yeast vector used for heterologous expression is the episomal vector pDP34 (11.8 kb) which is a yeast - E. coli shuttle vector with the ampicillin resistance marker for E. coli and the UR A3 and dLEU2 veast selective markers. Vector nDP34 (cf F.P 3401701 is - » a · - - - \--- — — · - - -z — digested with the restriction enzyme BamHI. The linearized vector is isolated with GENECLEAN and the protruding ends are filled in by Klenow polymerase treatment as described in the Maniatis manual (supra). The reaction is stopped after 30 min by heating -22to 65°C for 20 min in the presence of 10 mM EDTA. After ethanol precipitation the plasmid is digested with Sail and subjected to gel electrophoresis on a 0.8 % agarose gel. The (BamHI) blunt end-Sall cut vector pDP34 is isolated as an 11.8 kb DNA fragment from the gel with the GENECLEAN kit In analogy to the vector preparation plasmids pGTA 1132 and pGTB 1135 are each digested with Hindm. The protruding ends of the linearized plasmids are filled in by Klenow polymerase treatment and subsequently subjected to Sail digestion, resulting in (2A) a 2.35 kb (Hindni)blunt end - Sail fragment with the phosphate regulated expression cassette, or (2B) a 2.0 kb (HindHI)blunt end - Sail fragment with the constitutive expression cassette.

Ligation of the blunt end-Sall pDP34 vector part with fragment 2A or fragment 2B and transformation of competent cells of E. coli strain DH5a is carried out as described in Example 2 using 80 iig of the vector part and 40 ng of fragment 2A or 2B, respectively. 58 and 24 transformants are obtained, respectively. From each transformation six plasmids are prepared and characterized by restriction analysis. For the construction with the regulated expression cassette (fragment 2A), two plasmids show the expected restriction pattern. One of the clones is chosen and designated pDPGTA8.

For the construction with the constitutive expression cassette (fragment 2B) one plasmid shows the expected restriction pattern, and is designated pDPGTB5.

Example 4: Transformation of S. cerevisiae strain BT 150 CsCl-purified DNA of the expression vectors pDPGTA8 and pDPGTB 5 is prepared following the protocol of R. Treisman in the Maniatis manual (supra). The protease deficient S. cerevisiae strains BT 150 (MATa, his4, leu2, ura3, pral, prbl, prcl, cpsl) and H 449 (MATa, prbl, cpsl, ura3A5, leu 2-3,2-112, cir°) are each transformed with 5 pg each of plasmids pDPGTA8, pDPGTB5 and pDP34 (control without expression cassette) according to the lithium-acetate transformation method (Ito, H. et al., supra). Ura+-transformants are isolated and screened for GT activity (infra). Single transformed yeast cells are selected and referred to as Saccharomvces cerevisiae BT 150/pDPGTA8 Saccharomvces cerevisiae BT 150/pDPGTB5 Saccharomvces cerevisiae BT 150/pDP34 -23Saccharomyces cerevisiae H 449/pDPGTA8 Saccharomyces cerevisiae H 449/pDPGTB5 Saccharomyces cerevisiae H 449/pDP34 Example 5: Enzyme activity of full-length GT expressed in yeast .1 Preparation of cell extracts Cells of transformed Saccharomyces cerevisiae strains BT 150 and H 449 are each grown under uracil selection in yeast minimal media (Difco) supplemented with histidine and leucine. The growth rate of the cells is not affected by the introduction of any of the expression vectors. Exponentially growing cells (at ODS7g of 0.5) or stationary cells are collected by centrifugation, washed once with 50 mM Tris-HCl buffer pH 7.4 (buffer 1) and resuspended in buffer 1 at a concentration corresponding to 0.1 - 0.2 ODS7g. Mechanical breakage of the cells is effected by vigorous shaking on a vortex mixer with glass beads (0.45 - 0.5 mm diameter) for 4 min with intermittent cooling. The crude extracts are used directly for determination of enzyme activity.

Fractionation of cellular components is achieved by differential centrifugation of the extract. First, the extract is centrifuged at 450 g for 5 min; secondly, the supernatant obtained is centrifuged at 20000 g for 45 min.

The supernatant is collected and the pellets are resuspended in buffer 1.

For phosphate induction of GT expression under the control of the inducible PH05 promoter, cells of transformants BT 150/pDPGTA8 and H 449/pDPGTA8 are massshifted to low phosphate minimal medium, respectively, (Meyhack, B. et al. Embo J. (1982) 1,675-680). The cell extracts are prepared as described above. .2 Protein assay The protein concentration is determined by use of the BCA-Protein Assay Kit (Pierce). Ί Accov fr»r fTT activitir mw* «/·*# 4 unyuj «vi w» λ mwm GT activity can be measured with radiochemical methods using either ovalbumin, a glycoprotein which solely exposes GlcNAc as acceptor site, or free GlcNAc as acceptor substrates. Cell extracts (of 1 - 2 ODs 57g of cells) are assayed for 45 or 60 min at 37°C in -24a 100 μΐ incubation mixture containing 100 mM Tris-HClpH 7.4,50 nCi UDP-14C-Gal (325 mCi/mmol), 80 nmol UDP-Gal, 1 gmol MnCl2,1 % Triton X-100 and 1 mg ovalbumin or 2 pmol GlcNAc as acceptor. In case a glycoprotein acceptor substrate is used, the reaction is terminated by acid precipition of the protein and the amount of 14C galactose incorporated into ovalbumin is determined by liquid scintillation counting (Berger, E.G. et al. (1978) Eur. J. Biochem. 90,213-222). For GlcNAc as acceptor substrate, the reaction is terminated by the addition of 0.4 ml ice cold H2O and the unused UDP-14C-galactose is separated from 14C products on an anion exchanged column (AG Xl-8, BioRad) as described (Masibay, A.S. and Qasaba, P.K. (1989) Proc. Natl. Acad. Sci. USA 86, 5733-5737). Assays are performed with and without acceptor molecules to assess the 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 different plasmids. plasmid GT specific activity (mU/mg protein) PH05 promoter high Pi low Pi pDP34 -- <0.01 n.d. pDPGTA8 phosphate regulated 0.1 0.6-1 pDPGTB5 constitutive 0.6 n.d. » 1) notdeteimined GT activity of cultures shifted to inducible conditions (lowPi minimal medium) is about the same as the activity of cultures in minimal media expressing GT constitutively (Table 2). As expected, no enzyme activity is found in cells transformed with the vector only.

During fractionation, most of the GT activity (70 - 90 %, see Table 3) and highest specific activity is always found in the high speed pellet (20000 g). Enzyme activity can be increased by the addition of 1 - 2 % Triton X-100 to the enzyme assay. Both findings suggest that recombinant GT is membrane bound in yeast cells, as it is in HeLa cells. -25Table 3: Distribution of GT activity during fractionation of BT 150/pDPGT5 cells Fraction GT activity cpm UDP-14C-Gal incorporated in GlcNAc % erode extract 19400 400 g pellet 1000 20000 g pellet 15800 20000 g supernatant 5000 4.6% 72.5% 22.9% total: 21800 100% Example 6: Purification of the recombinant GT In order to liberate the membrane-bound enzyme the 20000 g pellet fraction of BT 150/pDPGTB5 cells is treated with 1 % (w/v) Triton X-100 for 10 min at room temperature and equilibrated with 50 mM Tris HCl pH 7.4,25 mM MgCl2,0.5 mM UMP and 1 % (w/v) Triton X-100. The supernatant obtained after centrifugation is 0.2 gm filtered and passed over a GlcNAc-p-aminophenyl-Sepharose-column (Berger, E.G. et al. (1976) Experientia 32,690-691) with a bed volume of 5 ml at a flow rate of 0.2 ml/min at 4°C. The enzyme is eluted with 50 mM Tris-HCl pH 7.4,5 mM GlcNAc, 25 mM EDTA and 1 % Triton X-100. A single peak of enzyme activity is eluted within five fractions (size: ml). These fractions are pooled and dialyzed against 2x 11 of 50 mM Tris-HCl pH 7.4, 0.1 % Triton X-100. 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 if a yeast promoter is operably linked to a first DNA sequence encoding the signal sequence of the yeast invertase gen SUC2 linked in the proper reading frame to a cDNA encoding soluble GT. (a) Partial HeLa GT cDNA sequence GT cDNA is excised from plasmid p4ADl 13 (Example 1) by digestion with EcoRI. The 1.2 kb fragment containing the complete GT cDNA is isolated and partially digested with MvnI (Boehringer) cutting the GT sequence at positions 43,55,140 and 288 of the nucleotide sequence depicted in SEQ ID NO. 1. When digesting 0.2 gg of the EcoRIEcoRI fragment with 0.75 g MvnI for 1 h the GT cDNA is cut only once at nucleotide position 134 yielding a 1.1 kb MvnI-EcoRI fragment. -26(b) Vector for amplification in E. coli Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRl in the multiple cloning site. Then the plasmid is treated with alkaline phosphatase as described in the Maniatis manual (supra), subjected to agarose gel electrophoresis and isolated from the gel as a 2.7 kb DNA fragment (c) Constitutive PH05 (-173) promoter and SUC2 signal sequence The vector p31/PH05 (-173) RTT (Example 2) is digested with the restriction enzymes BamHI and XhoL A 0.25 kb BamHl-XhoI fragment with the constitutive PH05 (-173) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is isolated. Then the fragment is recut with Hgal (BioLabs). The Hgal recognition sequence is on the antisense DNA strand. The restriction enzyme cuts upstream of the recognition sequence in such a way that the 5 staggered end of the antisense strand coincides with the end of the coding sequence of the invertase signal sequence. The 0.24 kb BamHI and Hgal cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence. (d) Adaptor Fragment (a) is linked to fragment (c) by means of an adaptor sequence which is prepared from equimolar amounts of the synthetic oligonucleotides (Microsynth) 5'CT GCA CTG GCT GGC CG 3' and 5’CG GCC AGC CAG 3* for the complementary strand. The oligonucleotides are annealed to each other by first heating to 95°C and then slowly cooling to 20°C. The annealed adaptor is stored frozen. (e) Construction of plasmid psGT For ligation, linearized vector (b), the GTcDNA fragment (a), fragment (c) containing the promoter and the sequence encoding the signal peptide and the adaptor (d) are used in a molar ratio of 1:2:2: 30-100. Ligation is carried out in 12 μΐ of ligase buffer (66 mM Tris-HCl pH 7.5,1 mM dithioerythritol, 5 mM MgCl2,1 mM ATP) at 16°C for 18 hours. The ligation mix is used to transform competent cells of E. coli strain DH5a as described above. Minipreparations of plasmid are performed from 24 independent transformants.

The isolated plasmids are characterized by restriction analysis using four different enzymes (BamHI, Pstl, EcoRl, Xhol, also in combination). A single clone with the expected restriction pattern is referred to as psGT. -27The 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 by using the T7-Sequencing kit (Pharmacia) and primer 5'AGTCGAGGTTAGTATGGC 3' starting at position -77 in the constitutive PH05 (-173) promoter.

Sequence for MvnI DNA1J: 5' AAA ATA Tct gca ctg get ggc cgC GAC CTG AGC 3' 3' TTT TAT AGA CGT gac ega ccg gcG CAG GAC TCG 5' Protein: Lys He Ser Ala ι Leu Ala Gly Arg Asp Leu Ser 16 19 42 inv ss GT sequence cleavage site for signal endopeptidase Small letters represent the adaptor sequence.

The expression cassette for secreted GT containing the constitutive PH05 (-173) promoter, the DNA sequence encoding invertase signal peptide and the partial GT cDNA from plasmid psGT can be excised as a 1.35 kb Sail (BamHI) - EcoRI fragment. The expression cassette is still.lacking the PH05 terminator sequences to be added in the following cloning step.

Example 8: Construction of the expression vector pDPGTS For construction of the expression vector for soluble GT the following fragments are combined: (a) Vector part Plasmid pDP34 is digested and pretreated as described in Example 3 resulting in a 11.8 kb 25 blunt end - Sail vector fragment. (b) Expression-cassette Plasmid nsGT is first linearized by disestion with Sail (in the multiple clonins site) and then partially digested with EcoRI. A 1.35 kb DNA fragment is isolated containing the constitutive PH05 (-173) promotor, a DNA sequence encoding the yeast invertase signal peptide and the partial GT cDNA. -28(c) PH05 terminator sequence The PH05 terminator sequence is isolated from plasmid p31 which is constructed starting from plasmid p30 as described in EP 100561. After digestion with the restriction enzyme Hindin the protruding ends are filled up by Klenow polymerase treatment as described above. Then the plasmid is digested with EcoRI and a 0.39 kb blunt end - EcoRI fragment with the PH05 terminator sequences is isolated.

Ligation of fragments (a), (b) and (c) and transformation of competent cells of E. coli strain DH5oc is carried out as described in Example 2. From the obtained transformants plasmids are isolated and characterized by restriction analysis. The plasmid of one clone with the correct restriction fragments is referred to as pDPGTS.

Example 9: Expression of soluble GT in yeast In analogy to Example 4, CsCl-purified DNA of the expression vector pDPGTS is used to transform 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 referred to as Saccharomvces cerevisiae BT 150/pDPGTS and Saccharomyces cerevisiae 15 H 449/pDPGTS, respectively. Employing the assay described in Example 5 GT-activity is found in the culture broths of both transformants.

Example 10: Cloning of the sialvltransferase (ST) cDNA from human HepG2 cells ST cDNA is isolated from HepG2 cells by PCR in analogy to GT cDNA. Preparation of poly (A)+RNA and first strand cDNA synthesis are performed as described in Example 1.

The primers (Microsynth) listed in Table 5 are used for PCR. -29Table 5: PCR-primers corresponding to bp primer sequence (5’ to 3’)1J in ST cDNA1 2) PstVEcoRI SIA1 cgctgcagaattcaaaATGATTCACACCAACCTGAAGAAAAAGT 1 - 28 BamHl SIA3 cgcggatCCTGTGCTTAGCAGTGAATGGTCCGGAAGCC 1228 - 1198 1) Capital letters represent sequences from ST, small letters are additional sequences with sites for restriction enzymes (underlined). Codons for 'start* and 'stop* for protein synthesis are indicated in boldface. 2) ST cDNA sequence from human placenta (27) as published in EMBL Data Bank (Accession Nr.X17247). 1 θ' HepG2 ST cDNA can be amplified as one DNA fragment of 1.2 kb using the primers SIA1 and SIA3. PCR is performed as described for GT cDNA under slightly modified cycling conditions: 0.5 min denaturing at 95°C, 1 min. 15 sec annealing at 56°C, and 1 min 30 sec extension at 72°C, for a total of 25-35 cycles. In the last cycle, primer extension at 72°C is carried out for 5 min.

After PCR amplification, the 1.2 kb fragment is digested with the restriction enzymes BamHl and Pstl, analysed on a 1.2 % agarose gel, eluted from the gel and subcloned into the vector pUC 18. The resulting subclone is designated pSIA2.

Example 11: Construction of the constitutive ST expression cassette For constitutive heterologous expression, ST cDNA is ligated to the constitutive PH05 20 (-173) promoter fragment and PH05 terminator sequences.

Plasmid pSIA2 is first linearized by digestion with the restriction enzyme BamHl and subsequently partially digested with EcoRI. Since ST cDNA also contains an internal restriction site for the enzyme EcoRI (at bp 144), a 1.2 kb fragment with the complete ST cDNA (SEQ. ID NO. 3) is created by partial digestion with EcoRI using 1 pg of DNA and 0.25 U EcoRI (1 h, 37°C). After gel electrophoresis the 1.2 kb EcoRI-BamHI fragment comprising the complete ST cDNA (SEQ ID NO. 3) is isolated. On this DNA fragment the ’ATG’ start codon for translation of ST is located close to the EcoRI restriction site. Three adenosine phosphates (see PCR primer SIA 1) provide an Ά’ at bp position 12, which is found in the consensus sequence around the ’ATG’ from highly expressed genes in yeast (Hamilton, R. et al. (1987) Nucleic Acids Res. 14,5125-5149). The stop codon -30’TAA’ and 5 bp of the 3’ untranslated region of the gene are followed by the BamHI site.

The 1.2 kb EcoRI-BamHI ST cDNA fragment is ligated to the 0.45 kb Sall-EcoRI fragment containing the constitutive PH05 (-173) promoter (Example 2.2(b)) and a 3.5 kb BamHI-Sall vector part for amplification in E. coli containing the PH05 terminator sequence (cf. Example 2.1, fragment (b)). Ligation and transformation of E. coli strain DH5a is performed as detailed in Example 2.1. One clone showing the expected restriction pattern is designated pST2.

Vector pST2 comprises the expression cassette for HepG2 ST under the control of the constitutive PH05 (-173) promotor as a 2.0 kb Sall-Hindin fragment, referred to as DNA fragment (1C).

Example 12: Expression of ST in veast Vector pDP34 (cf. EP 340 170) is digested with the restriction enzyme BamHI. The linearized vector is isolated with GENECLEAN and the protruding ends are filled in by Klenow polymerase treatment as described in the Maniatis manual (supra). The reaction is stopped after 30 min by heating tO-65°C for 20 min in the presence of 10 mM EDTA. After ethanol precipitation the plasmid is digested with Sail and subjected to gel electrophoresis on a 0.8 % agarose gel. The (BamHI) blunt end-Sall cut vector pDP34 is isolated as an 11.8 kb DNA fragment with the GENECLEAN kit.

Plasmid pST2 is digested with the restriction enzyme Hindin and in analogy to the preparation of the vector part filled in at the Hindlll site by Klenow polymerase treatment The product is subjected to Sail digestion, resulting in a 2.0 kb (Hindlll) blunt end - Sail fragment comprising the constitutive ST expression cassette (2C).

Ligation of 80 ng of the pDP34 vector with 40 ng of fragment 2C and transformation of competent cells of E. coli strain DH5a is performed as described in Example 2. One clone showing the expected restriction pattern is chosen and referred to as pDPST5.

For transformation of yeast, CsCl purified DNA of the expression vector pDPST5 is prepared following the standard procedure given in the Maniatis manual (supra). S. cerevisiae strains BT 150 and H 449 are each is transformed with 5 gg of plasmid DNA according to the lithium-acetate transformation method (Ito, H. et al, supra). Ura+-transformants are selected and screened for ST activity. In each case, one positive -31 transformant is selected and referred to as Saccharomvces cerevisiae BT 150/pDPST5 and Saccharomyces cerevisiae H 449/pDPST5.

Example 13: Enzyme activity of full-length ST expressed in yeast 13.1 Preparation of cell extracts Cells of Saccharomvces cerevisiae BT 150/pDPST5 are grown under uracil selection in yeast minimal media supplemented with histidine and leucine. Exponentionally growing cells (at ODS78 of 0.5) or stationary cells are collected by centrifugation, 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 ODS78. Mechanical breakage of the cells is effected by vigorous shaking on a vortex mixer with glass beads (0.45-0.5 mm diameter) for 4 min with intermittent cooling*.

ST-activity can be measured in the crude extracts employing the assay described below. 13.2 Assay for ST activity ST activity can be determined by measuring the amount of radiolabeled sialic acid which is transferred from CMP-sialic acid to a glycoprotein acceptor. Al ter termination of the reaction by acid precipitation the precipitate is filtered using glass fiber filters (Whatman GFA - WHATMAN Is a Trade Mark), washed extensively with ice-cold ethanol and assessed for radioactivity by liquid scintillation counting (Hesford et al. (1984), Glycoconjugate J. 1, 141-153). Cell extracts are assayed for 45 min in an incubation mixture containing 37 ul cell extract corresponding to approximately 0.5mg protein, 3 ul lmldazol buffer 50 mMol/1 pH 7.0; 50 nMol CMP-N-acetylneuramlnic acid (Sigma) to which CMP-3H-Nacetylneuramlnlc acid (Amersham) is added to give a final specific activity of 7.3 Cl/mol, and 75 ug asialo-fetuin (prepared by acid hydrolysis using 0.1 M H2SO4 at 80 C for 60 min, followed by neutralization, dialysis and lyophilization).

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

Example 14: Construction of an expression cassette lor soluble ST (Lysw-Cvs^) The soluble ST designated ST(Lys39-Cys4o6) is an N-lcrminally truncated variant and consists of 368 amino acids (SEQ ID NO. 4). -32(a) Partial HepG2 ST cDNA sequence Plasmid pSIA2 is digested with EcoRI and a 1.1 kb EcoRI-EcoRI fragment is isolated. (b) Vector for amplification in E. coli Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRI in the multiple cloning site. Then the plasmid is treated with alkaline phosphatase as described in the Maniatis manual (supra), subjected to agarose gel electrophoresis and isolated from the gel as a 2.7 kb DNA fragment (c) Constitutive PH05 (-173) promoter and SUC2 signal sequence The vector p31/PH05 (-173) R1T (Example 2) is digested with the restriction enzymes BamHI and Xhol. 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. Then the fragment is recut with Hgal (BioLabs). The Hgal recognition sequence is on the antisense DNA strand. The restriction enzyme cuts upstream of the recognition sequence in such a way that the 5 staggered end of the antisense strand coincides with the end of the coding sequence of the invertase signal sequence. The 0.24 kb,BamHI and Hgal cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence. (d) Adaptor Fragment (a) is linked to fragment (c) by means of an adaptor sequence which is prepared from equimolar amounts of the synthetic oligonucleotides 'CTGCAAAATTGCAAACCAAGG 3’ and 5'AATTCCTTGGTTTGCAATTT 3’ for the complementary strand. The oligonucleotides are annealed to each other by first heating to 95°C and then slowly cooling to 20°C. The annealed adaptor is stored frozen. (e) Construction of plasmid psST For ligation, linearized vector (b), the STcDNA fragment (a), fragment (c) containing the promoter and the sequence encoding the signal peptide and the adaptor (d) are used in a molar ratio of 1:2:2: 30-100. Ligation is carried out in 12 gl of ligase buffer (66 mM Tris-HCl pH 7.5,1 mM dithioerythritol, 5 mM MgCl2,1 mM ATP) at 16°C for 18 hours. The ligation mix is used to transform competent cells of E. coli strain DH5a as described above. Minipreparations of plasmid are performed from 24 independent transformants. A single clone showing the expected restriction pattern after characterisation by restriction analysis using four different enzymes (BamHI, Pstl, EcoRI, Xhol, also in combination) is -33referred to as psST.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide with the cDNA coding for soluble ST(Lys39-Cys406) is confirmed for plasmid psST by using the T7-Sequencing kit (Pharmacia) and primer 5’ACGAGGTTAATGGC 3' starting at position -77 in the constitutive PH05 (-173) promoter.

Sequence for DNA1): 5' AAA ATA Tct gca aaa ttg caa acc aag gAA 3' 3' TTT TAT AGA GTG ttt aac gtt tgg ttc ctt 5' Protein: Lys lie Ser Ala I Lys Leu Gin Thr Lys Glu 16 19 39 inv ss ST sequence cleavage site for signal endopeptidase 11 Small letters represent the adaptor sequence.

The expression cassette for secreted ST containing the constitutive PH05 (-173) promoter, the DNA sequence encoding invertase signal peptide and the partial ST cDNA from plasmid psST can be excised as a 1.35 kb Sail (BamHI) - EcoRl fragment The expression cassette is still lacking the PH05 terminator sequences to be added in the following cloning step.

Example 15: Construction of the expression vector pDPSTS For construction of the expression vector for soluble ST(Lys39-Cys406) the following fragments are combined: (a) Vector part Plasmid pDP34 is digested and pretreated as described in Example 3 resulting in a 11.8 kb blunt end - Sail vector fragment. (b) Expression cassette Plasmid psST is first linearized by digestion with Sail (in the multiple cloning site) and then partially digested with EcoRl. A 1.35 kb DNA fragment is isolated containing the -34constitutive PH05 (-173) promotor, a DNA sequence encoding the yeast invertase signal peptide and the partial ST cDNA. (c) PH05 terminator sequence The PH05 terminator sequence is isolated from plasmid p31 which is constructed starting from plasmid p30 as described in EP 100561. After digestion with the restriction enzyme Hindin the protruding ends are filled up by Klenow polymerase treatment as described above. Then the plasmid is digested with EcoRI and a 0.39 kb blunt end - EcoRI fragment with the PH05 terminator sequences is isolated.

Ligation of fragments (a), (b) and (c) and transformation of competent cells of E. coli strain DH5a is carried out as described in Example 2. From the obtained transformants plasmids are isolated and characterized by restriction analysis. The plasmid of one clone with the correct restriction fragments is referred to as pDPSTS.

Example 16: Expression of soluble ST in yeast In analogy to Example 4, CsCl-purified DNA of the expression vector pDPSTS is used to transform S. cerevisiae strains BT 150 and H 449. Ura+-transformants are isolated and screened for ST activity. In each case one transformant is selected and referred to as Saccharomvces cerevisiae BT 150/pDPSTS and Saccharomvces cerevisiae H 449/pDPSTS. Using the assay described in Example 13 ST-activity is found in the culture broths of both transformants.

Example 17: Construction of an expression cassette for soluble STflA^-Cvs^nJ The soluble ST designated ST(Lys27-Cys406) is an N-terminally truncated variant containingthe catalytic domain and the entire stem region and consisting of 380 amino acids, i.e. amino acids 27 to 406 of the amino acid sequence listed in SEQ ID NO. 3. (a) Partial HepG2 ST cDNA sequence Plasmid pSIA2 is digested with EcoRI and a 1.1 kb EcoRI-EcoRI fragment is isolated. (b) Vector for amplification in E. coli Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRI in the multiple cloning site. Then the plasmid is treated with alkaline phosphatase as described in the Maniatis manual (supra), subjected to agarose gel electrophoresis and isolated from the gel as a -352.7 kb DNA fragment. (c) Constitutive PH05 (-173) promoter and SUC2 signal sequence The vector p31/PH05 (-173) RTT (Example 2) is digested with the restriction enzymes BamHl and Xhol. 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. Then the fragment is recut with Hgal (BioLabs). The Hgal recognition sequence is on the antisense DNA strand. The restriction enzyme cuts upstream of the recognition sequence in such a way that the 5 staggered end of the antisense strand coincides with the end of the coding sequence of the invertase signal sequence. The 0.24 kb BamHl and Hgal cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence. (d) Adaptor Fragment (a) is linked to fragment (c) by means of an adaptor sequence which is prepared from equimolar amounts of the synthetic oligonucleotides ’CTGCAAAGGAAAAGAAGAAAGGGAGTTACTATGATTCCTTTAAATTGCAAA CCAAGG 3*, and ’ AATTCCTTGTTGCAATTTAAAGGAATCATAGTAACTCCCTTTCTTCTTTT CCTT3* for the complementary strand. The oligonucleotides are annealed to each other, by first, heating to 95°C and then slowly cooling to 20°C. The annealed adaptor is stored frozen. (e) Construction of plasmid psSTl For ligation, linearized vector (b), the STcDNA fragment (a), fragment (c) containing the promoter and the sequence encoding the signal peptide and the adaptor (d) are used in a molar ratio of 1:2:2:30-100. Ligation is carried out in 12 gl of ligase buffer (66 mM Tris-HCl pH 7.5,1 mM dithioeiythritol, 5 mM MgCl2,1 mM ATP) at 16°C for 18 hours.

The ligation mix is used to transform competent cells of E. coli strain DH5a as described above. Minipreparations of plasmid are performed from 24 independent transformants. A single clone showing the expected restriction pattern after characterisation by restriction analysis using four different enzymes (BamHl, Pstl, EcoRI, Xhol, also in combination) is referred to as psSTl. 3Q The correct sequence at the fusion site of the sequence encoding invertase signal peptide -36with the cDNA coding for soluble ST(Lys27-Cys406) is confirmed for plasmid psSTl by using the T7-Sequencing kit (Pharmacia) and primer 5'AGTCGAGTAGTATGGC 3' starting at position -77 in the constitutive PH05 (-173) promoter.

Sequence for DNAn: 5' AAA ATA Tct gca aag gaa aag aag aaa ggg 3 3' TTT TAT AGA GTG ttc ctt ttc ttc ttt CCC 5 Protein: Lys Ile Ser Alai Lys Glu Lys Lys Lys Gly 16 19 27 inv ss ST sequence cleavage site for signal endopeptidase l) Small letters represent the adaptor sequence.

The expression cassette for secreted STO-ys^Cys^) containing the constitutive PH05 15 (-173) promoter, the DNA sequence encoding invertase signal peptide and the partial ST cDNA from plasmid psSTl can be excised as a 1.35 kb Sail (BamHI) - EcoRI fragment The expression cassette is still lacking the PH05 terminator sequences to be added in the following cloning step.

Example 18: Construction of the expression vector pDPSTSl 20 For construction of the expression vector for soluble ST(Lys27-Cys406) the following fragments are combined: (a) Vector part Plasmid pDP34 is digested and pretreated as described in Example 3 resulting in a 11.8 kb blunt end - Sail vector fragment. (b) Expression cassette Plasmid psSTl is first linearized by digestion with Sail (in the multiple cloning site) and then partially digested with EcoRI. A 1.35 kb DNA fragment is isolated containing the constitutive PH05 (-173) promotor, a DNA sequence encoding the yeast invertase signal peptide and the partial ST cDNA. -37(c) PH05 terminator sequence The PH05 terminator sequence is isolated from plasmid p31 which is constructed starting from plasmid p30 as described in EP 100561. After digestion with the restriction enzyme Hindm the protruding ends are filled up by Klenow polymerase treatment as described above. Then the plasmid is digested with EcoRI and a 0.39 kb blunt end - EcoRI fragment with the PH05 terminator sequences is isolated.

Ligation of fragments (a), (b) and (c) and transformation of competent cells of E. coli strain DH5a is carried out as described in Example 2. From the obtained transformants plasmids are isolated and characterized by restriction analysis. The plasmid of one clone with the correct restriction fragments is referred to as pDPSTSl.

Example 19: Expression of soluble ST(Lvsr Cvs^a) in yeast In analogy to Example 4, CsCl-purified DNA of the expression vector pDPSTSl is used to transform S. cerevisiae strains BT 150 and H 449. Ura+-transformants are isolated and screened for ST activity. In each case one transformant is selected and referred to as Saccharomyces cerevisiae BT 150/pDPSTSl and Saccharomyces cerevisiae H 449/pDPSTSl. Using the assay described in Example 13, ST-activity is found in the culture broths of both transformants.

Example 20: Cloning of the FTcDNA from the human HL60 cell line On the basis of the published cDNA sequence (Goelz, S.E. et al. (1990) Cell 63, 1349-1356) for ELFT (ELAM-1 ligand fucosyltransferase) coding for a( 1-3)fucosyltransferase the following oligonucleotide primers are designed to amplify a DNA fragment encompassing the open reading frame of the ELFT cDNA by PCR technology: 5’-CAGCGCTGCCTGTTCGCGCCAT-3’ (ELFT-1B) and ’-GGAGATGCACAGTAGAGGATCA-3’(ELFT-2B). ELFT-IB primes at base pairs 38-59 of the published sequence, and ELFT-2B primes at bp 1347-1326 in the antisense. The primers are used to amplify a 1.3 kb fragment using the Perkin-Elmer Cetus Taq polymerase kit. The fragment is amplified from fresh HL60 cDNA in the presence of 5% DMSO and 2 mM MgCl2 with the following cycle: 95°C 5 min, 25x (1 min 95°C, 1 min 60°C, 1.5 min 72°C), lOx (1 min 95°C, 1 min 60°C, 1.5 min + 15 sec/cycle 72°C).

Agarose gel electrophoresis reveals a prominent 1.3 kb band which when digested with Smal or Apal gives the pattern predicted by the published sequence. The 1.3 kb band is -38purified using a Gene-Clean kit (Bio 101) and is subcloned into the pCRIOOO vector (Invitrogen). A single clone with the correct 1.3 kb insert is selected and referred to as BRB.ELET/pCR1000-13. The FTcDNA is inserted in the vector in a reverse orientation with respect to the T7 promoter. The open reading frame of the FTcDNA has been fully sequenced and is identical to the published sequence (SEQ.ID NO.5).

Example 21: Construction of plasmids for inducible and constitutive expression of soluble FTfArg^-Argzny) in yeast Soluble FT(Arg62-Arg405) is expressed from the FT cDNA starting at nucleotide position 241 (Nrul restriction site) omitting the N-terminal region coding for the cytoplasmic tail and the membrane spanning domain (see sequence ID NO. 5).

Plasmid BRB.ELFT/pCR1000-13 is digested with Hindin, which cuts in the multicloning region 3’ of the FT cDNA insert The sticky ends are convened to blunt ends in a reaction with Klenow DNA polymerase. Xhol linker (5’ CCTCGAGG 3’, Biolabs) are kinased, annealed and ligated to the blunt ends of the plasmid, using a 100-fold molar excess of linkers. Unreacted linkers are removed by isopropanol precipitation of the DNA, which is further digested with Xhol and Nrul (cleavage at nucleotide position 240 of the FT cDNA according to Sequence ID NO. 5). The 1.1 kb Nrul-Xhol fragment (a) contains the FT cDNA sequence lacking the region which codes for the cytoplasmic tail and the membrane-spanning domain up to amino acid 61.

Plasmids p31 RIT12 and p31/PHO5(-173)RlT (see Example 2) are each digested with Sail and Xhol. The 0.9 kb and 0.5 kb fragments, respectively, are isolated and cut with Hgal. The resulting sticky ends are filled by Klenow DNA polymerase. The created blunt ends coincide with the 3' end of the coding sequence for the yeast invertase signal sequence. Subsequent BamHI cleavage releases a 596 bp BamHI-blunt end fragment (b) which comprises the inducible PHO5 promoter and the invertase signal sequence with its own ATG or a 234 bp BamHI-blunt end fragment (c) comprising the short, constitutive PHO5(-173) promoter and the invertase signal sequence.

Plasmid p31RIT12 is linearized with restriction endonuclease Sail. Partial HindIU digestion in the presence of ethidiumbromide results in a 1 kb Sall-HindlU fragment comprising the 276 bp Sall-BamHI pBR322 sequence, the 534 bp promoter of the yeast acid phosphatase PHO5. the yeast invertase signal sequence (coding for 19 amino acids) -39and the PHO5 transcriptional terminator. The 1 kb Sall-Hindin fragment of p31RIT12 is cloned into the veast-E.coli shuttle vector pJDB207 (Beggs, J.D. in: Molecular Genetics in yeast, Alfred Benzon Symposium 16, Copenhagen, 1981, pp. 383-389), which has been cut with Sail and Hindm. The resulting plasmid containing the 1 kb insert is referred to as pJDB207/PHO5-RIT12.

Plasmid pJDB207/PHO5-RIT12 is digested with BamHI and Xhol and the large, 6.8 kb BamHI-XhoI fragment (d) is isolated. This fragment contains all the pJDB207 vector sequences and the PHO5 transcriptional terminator.

The 596 bp BamHI-blunt end fragment (b), the 1.1 kb Nrul-Xhol fagment (a) and the 6.8 kb XhoI-BamHI vector fragment (d) are ligated using standard conditions for blunt end ligation. Aliquots of the ligation mix are used to transform competent E. coli HB101 cells. Plasmid DNA from ampicillin-resistant colonies is analysed by restriction digests. A single clone with the correct expression plasmid is referred to as pJDB207/PHO5-I-FT. Ligation of DNA fragments (c), (a) and (d) leads to expression plasmid pJDB207/PHO5(-173)-I-FT. The expression cassettes of these plasmids comprise the coding sequence of the invertase signal sequence fused in frame to that of the soluble a(l-3)fucosyltransferase which is expressed under the control of the inducible PHO5 or the constitutive PHO5(-173) promoter, respectively. The expression cassettes are cloned into the yeast-E. coli shuttle vector pJDB207 between the BamHI and HindHI restriction sites.

The nucleotide sequence at the site of the fusion between the invertase signal sequence and cDNA coding for soluble FT is confirmed by DNA sequencing on double stranded plasmid DNA using the primer 5’ AGTCGAGGTTAGTATGGC 3’ representing the nucleotide sequence at position -77 to -60 of the PHO5. as well as the PHO5(-173) promoter. The correct junction is: AAA ATA TCT GCA CGA CCG GTG TTT TAT AGA CGT GCT GGC CAC Lys lie Ser Ala Arg Pro Val 19 62 Inv.ss FT cleavage site for signal peptidase -40Example 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 its own ATG. The nucleotide sequence immediately upstream of the ATG, however, is rather unfavourable for expression in yeast due to its high G-C content. This region has been replaced by an A-T rich sequence using PCR methods. At the same time an EcoRI restriction site is introduced at the new nucleotide positions -4 to -9.

Table 6: PCR primers corresponding to primer sequence (5’ to 3’)!) bp in FT cDNA FT1 cqaqaat1cataATGGGGGCACCGTGGGGC 58 to 75 FT2 ccqctcqaqGAGCGCGGCTTCACCGCTCG 1285 to 1266 Capital letters represent nucleotides from FT, small letters are additional new sequences, restriction sites are underlined, start and stop* codons are highlighted.

Standard PCR conditions are used to amplify the FT cDNA with primers FT1 and FT2 (see Table 6) in 30 cycles of DNA synthesis (Taq DNA polymerase, Perkin-Elmer, 5 U/μΙ, 1 min at 72°C), denaturation (10 sec at 93°C) and annealing (40 sec at 60°C). The resulting 1.25 kb DNA fragment is purified by phenol extraction and ethanol precipitation, then digested with EcoRI, Xhol and Nrul. The 191 bp EcoRI-Nrul fragment (e) is isolated on a preparative 4% Nusieve 3:1 agarose (FMC BioProducts, Rockland, ME, USA) gel in tris-borate buffer pH 8.3, gel-eluted and ethanol precipitated. Fragment (e) comprises the 5’ part of the FT gene coding for amino acids 1 to 61. The DNA has a 5’ extension with the EcoRI site as in PCR primer FT1.

Plasmids p31RIT12 and p31/PHO5(-173)RIT are each digested with EcoRI and Xhol. The large vector fragments (f and g, respectively) are isolated on a preparative 0.8% agarose gel, eluted and purified. The 4.1 kb XhoI-EcoRI fragment (f) comprises the -41 pBR322-derived vector, the 534 bp PHO5 promoter (3’ EcoRl site) and the 131 bp PHO5 transcriptional terminator (5’ Xhol site). The 3.7 kb XhoI-EcoRI fragment (g) only differs by the short, constitutive, 172 bp PHO5(-173) promoter (3’ EcoRl site) instead of the full length PHO5 promoter.

The 191 bp EcoRI-NruI fragment (e), the 1.1 kb Nrul-Xhol fragment (a) and the 4.1 kb XhoI-EcoRI fragment (f) are ligated. A 1 μΐ aliquot of the ligation mix is used to transform competent E. coli HB101 cells. Plasmid DNA from ampicillin resistant colonies is analysed. Plasmid DNA from a single clone is referred to as p31R/PHO5-ssFT.

Ligation of DNA fragments (e), (a) and (g) leads to plasmid p31R/PHO5(-173)-ssFT. These plasmids comprise the coding sequence of the membrane-bound FT under the control of the inducible PHO5 or the constitutive PHO5(-173) promoter, respectively.

Example 23: Cloning of the FT expression cassettes into pDP34: Plasmids p31R/PHO5-ssFT and p31R/PHO5(-173)-ssFT are digested with Hindlll, which cuts 3’ of the PHO5 transcriptional terminator. After a reaction with Klenow DNA polymerase, the DNA is digested with Sail. The 2.3 kb and 1.9 kb Sail-blunt end fragments, respectively, are isolated.

Plasmids pJDB207/PHO5-I-FT and pJDB207/PHO5(-173)-I-FT are partially digested with Hindlll in the presence of 0.1 mg/ml of ethidium bromide (to avoid cleavage at an additional Hindlll site in the invertase signal sequence) and then treated with Klenow DNA polymerase and Sail as above. The 2.1 kb and 1.8 kb fragments, respectively, are isolated.

The four DNA fragments are each ligated to the 11.8 kb Sail-blunt end vector fragment of pDP34 (see Example 3). Upon transformation of competent E. coli HB101 cells and analysis of plasmid DNA of individual transformants, four correct expression plasmids are referred to as pDP34/PHO5-I-FT; pDP34/PHO5(-173)-I-FT; pDP34R/PHO5-ssFT and pDP34R/PHO5(- 173)-ssFT.

S. cerevisiae strains BT150 and H449 are transformed with 5 pg each of the four expression plasmids (above) according to Example 4. Single transformed yeast colonies are selected and referred to as -42Saccharomvces cerevisiae / It «I n ti n ft BT150/pDP34/PHO5-I-FT; BT150/pDP34/PHO5(- 173)-I-FT; BT150/pDP34R/PHO5-ssFT; BT150/pDP34R/PHO5(-173)-ssFT; H449/pDP34/PHO5-I-FT; H449/pDP34/PHO5(-173)-I-FT; H449/pDP34R/PHO5-ssFT; H449/pDP34R/PHO5(-173)-ssFT Fermentation and preparation of the cell extracts is performed according to Example 5.

Using an assay analogous to that described by Goelz et al. (supra) FT-activity is found in the crude extracts prepared from strains BT150/pDP34R/PHO5-ssFT, BT150/pDP34R/PHO5(-173)-ssFT, H449/pDP34R/PHO5-ssFT and H449/pDP34R/PHO5(-173)-ssFT, and in the culture broth of strains H449/pDP34/PHO5-I-FT, H449/pDP34/PHO5(-173)-I-FT, BT150/pDP34/PHO5-I-FT and BT150/pDP34/PHO5(-173)-I-FT.

Deposition of microorganisms The following microorganism strains were deposited with the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg 16, D-3300 Braunschweig (deposition dates and accession numbers given): Escherichia coli JM109/pDP34: March 14,1988; DSM 4473 Escherichia coli HB101/p30: October 23,1987; DSM 4297 Escherichia coli HB101/p31R: December 19,1988; DSM 5116 Saccharomyces cerevisiae H 449: February 18,1988; DSM 4413 Saccharomyces cerevisiae BT 150: May 23,1991; DSM 6530 -43Sequence listing SEQ ID NO. 1 SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENGTH: 1265 bp STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: recombinant IMMEDIATE EXPERIMENTAL SOURCE: Plasmid p4ADl 13 from E. coli DH5a/p4AD113 FEATURES: from 6 to 1200 bp from 1 to 6 bp from 497 to 504 bp from 1227 to 1232 bp from 1236 to 1241 bp from 1243 to 1248 bp cDNA sequence coding for HeLa cell galactosyltransferase EcoRI site Notl site EcoRI site EcoRV site Bglll site PROPERTIES: EcoRI-HindHI fragment from plasmid p4ADl 13 comprising HeLa cell cDNA coding for full-length galactosyltransferase (EC 2.4.1.22) GAATTC ATG AGG CTT CGG GAG CCG CTC CTG AGC GGC AGC 39 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser GCC Ala GCG ATG CCA GGC GCG TCC CTA CAG CGG GCC TGC CGC 78 Ala Met Pro Gly 15 Ala Ser Leu Gin 20 Arg Ala Cys Arg CTG CTC GTG GCC GTC TGC GCT CTG CAC CTT GGC GTC ACC 117 Leu Leu Val Ala Val Cys Ala Leu His Leu Gly Val Thr 25 - 30 35 CTC GTT TAC TAC CTG GCT GGC CGC GAC CTG AGC CGC CTG 156 Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser Arg Leu 45 50 CCC CAA Pro Gin CTG GTC GGA GTC TCC ACA CCG CTG CAG GGC GGC 195 Leu Val Gly Val 55 Ser Thr Pro Leu 60 Gin Gly Gly TCG AAC AGT GCC GCC GCC ATC GGG CAG TCC TCC GGG GAG 234 Ser Asn Ser Ala Ala Ala Ile Gly Gin Ser Ser Gly Glu 65 70 75 CTC CGG ACC GGA GGG GCC CGG CCG CCG CCT CCT CTA GGC 273 Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly 80 85 GCC TCC TCC CAG CCG CGC CCG GGT GGC GAC TCC AGC CCA 312 Ala Ser Ser Gin Pro Arg Pro Gly Gly Asp Ser Ser Pro 90 95 100 GTC GTG GAT TCT GGC CCT GGC CCC GCT AGC AAC TTG ACC 351 Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr 105 110 115 TCG GTC CCA GTG CCC CAC ACC ACC GCA CTG TCG CTG CCC 390 Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 120 125 GCC TGC CCT GAG GAG TCC CCG CTG CTT GTG GGC CCC ATG 429 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 120 - . 135 140 CTG ATT GAG TTT AAC ATG CCT GTG GAC CTG GAG CTC GTG 468 Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val 145 150 GCA AAG CAG AAC CCA AAT GTG AAG ATG GGC GGC CGC TAT Ala Lys Gin Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 155 160 165 GCC Ala CCC AGG GAC Asp TGC GTC TCT CCT CAC AAG GTG GCC ATC 546 Pro Arg 170 Cys Val Ser Pro 175 His Lys Val Ala He 180 ATC ATT CCA TTC CGC AAC CGG CAG GAG CAC CTC AAG TAC 585 5 He lie Pro Phe Arg Asn Arg Gin Glu His Leu Lys Tyr 185 190 TGG CTA TAT TAT TTG CAC CCA GTC CTG CAG CGC CAG CAG 624 Trp Leu Tyr Tyr Leu His Pro Val Leu Gin Arg Gin Gin 195 200 205 10 CTG GAC TAT GGC ATC TAT GTT ATC AAC CAG GCG GGA GAC 663 Leu Asp Tyr Gly He Tyr Val lie Asn Gin Ala Gly Asp 210 215 ACT ATA TTC AAT CGT GCT AAG CTC CTC AAT GTT GGC TTT 702 Thr He Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 15 220 225 230 CAA GAA GCC TTG AAG GAC TAT GAC TAC ACC TGC TTT GTG 741 Gin Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys Phe Val 235 240 245 TTT AGT GAC GTG GAC CTC ATT CCA ATG AAT GAC CAT AAT 780 Phe Ser Asp Val Asp Leu He Pro Met Asn Asp His Asn 20 - 250 255 • GCG TAC AGG TGT TTT TCA CAG CCA CGG CAC ATT TCC GTT 819 Ala Tyr Arg Cys Phe Ser Gin Pro Arg His lie Ser Val 260 - . 265 270 GCA ATG GAT AAG TTT GGA TTC AGC CTA CCT TAT GTT CAG 858 25 Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gin 275 280 -4610 TAT TTT GGA GGT GTC Val TCT GCT CTA AGT AAA CAA CAG TTT Ser Ala Leu Ser Lys Gin Gin Phe 897 Tyr 285 Phe Gly Gly 290 295 CTA ACC ATC AAT GGA TTT CCT AAT AAT TAT TGG GGC TGG 936 Leu Thr He Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp 300 305 310 GGA GGA GAA GAT GAT GAC ATT TTT AAC AGA TTA GTT TTT 975 Gly Gly Glu Asp Asp Asp He Phe Asn Arg Leu Val Phe 315 320 AGA GGC ATG TCT ATA TCT CGC CCA AAT GCT GTG GTC GGG 1014 Arg Gly Met Ser He Ser Arg Pro Asn Ala Val Val Gly 325 330 335 AGG TGT CGC ATG ATC CGC CAC TCA AGA GAC AAG AAA AAT 1053 Arg Cys Arg Met He Arg His Ser Arg Asp Lys Lys Asn 340 345 GAA CCC AAT CCT CAG AGG TTT GAC CGA ATT GCA CAC ACA 1092 Glu Pro Asn Pro Gin Arg Phe Asp Arg He Ala His Thr 350 355 360 AAG GAG ACA ATG CTC TCT GAT GGT TTG AAC TCA CTC ACC 1131 Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr 365 - - 370 375 TAC CAG GTG CTG GAT GTA CAG AGA TAC CCA TTG TAT ACC 1170 Tyr Gin Val Leu Asp Val Gin Arg Tyr Pro Leu Tyr Thr - 380 385 CAA ATC ACA GTG GAC ATC GGG ACA CCG AGC TAGGACTTTT 1210 Gin lie Thr Val Asp He Gly Thr Pro Ser 390 395 -47GGTACAGGTA AAGACTGAAT TCATCGATAT CTAGATCTCG AGCTCGCGAA AGCTT 1250 1265 -48SEO ID NO. 2 SEQUENCE TYPE: Protein SEQUENCE LENH: 357 amino acids MOLECULE TYPE: C-terminal fragment of full-length HeLa cell galactosyl-transferase PROPERTIES: * soluble galactosyltransferase (EC 2.4.1.22) from HeLa cells Leu Ala Gly Arg Asp 5 Leu Ser Arg Leu Pro Gin Leu Val Gly Val Ser Thr Pro Leu Gin Gly Gly 10 15 20 Ser Asn Ser Ala Ala Ala He Gly Gin Ser Ser Gly Glu 25 30 35 Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly 40 45 Ala Ser Ser Gin Pro Arg Pro Gly Gly Asp Ser Ser Pro 50 55 60 Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr 65 70 Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 75 80 85 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 90 95 100 Leu lie Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val - 105 110 Ala Lys Gin Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 115 120 125 Ala Pro Arg Asp 130 Cys Val Ser Pro His 135 Lys Val Ala lie lie 140 He Pro Phe Arg Asn 145 Arg Gin Glu His Leu 150 Lys iyr Trp Leu Tyr 155 Tyr Leu His Pro Val 160 Leu Gin Arg Gin Gin 165 Leu Asp Tyr Gly lie 170 Tyr Val lie Asn Gin 175 Ala Gly Asp Thr He 180 Phe Asn Arg Ala Lys 185 Leu Leu Asn Val Gly 190 Phe Gin Glu Ala Leu 195 Lys Asp Tyr Asp Tyr 200 Thr Cys Phe Val Phe 205 Ser Asp Val Asp Leu 210 lie Pro Met Asn Asp 215 His Asn Ala Tyr Arg 220 Cys Phe Ser Gin Pro 225 Arg His lie Ser Val 230 Ala Met Asp Lys Phe 235 Gly Phe Ser Leu Pro 240 Tyr Val Gin Tyr Phe 245 Gly Gly Val Ser Ala 250 Leu Ser Lys Gin Gin 255 Phe Leu Thr He Asn 260 Gly Phe Pro Asn Asn 265 Tyr Trp Gly Trp Gly 270 Gly Glu Asp Asp Asp 275 He Phe Asn Arg Leu 280 Val Phe -50Arg Gly Met Ser lie Ser Arg Pro Asn Ala Val Val Gly 285 290 295 Arg Cys Arg Met He Arg His Ser Arg Asp Lys Lys Asn 300 305 Glu Pro Asn Pro Gin Arg Phe Asp Arg lie Ala His Thr 310 315 320 Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr 325 330 Tyr Gin Val Leu Asp Val Gin Arg Tyr Pro Leu Tyr Thr 335 340 345 Gin He Thr Val Asp He Gly Thr Pro Ser 350 355 -51 SEQ ID NO. 3 SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENH: 1246 bp STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: recombinant IMMEDIATE EXPERIMENTAL SOURCE: Plasmid pSIA2 from E. coli DH5o/pSIA2 FEATURES: from 15 to 1232 bp from 1 to 6 bp from 6 to 11 bp from 144 to 149 bp from 1241 to 1246 bp sialyltransferase Pstl site EcoRI site EcoRI site BamHI site PROPERTIES: Pstl-BamHI fragment from plasmid pSIA2 comprising HepG2 cDNA coding for full-length sialyltransferase (EC 2.4.99.1) CTGCAGAATT CAAA ATG ATT CAC ACC AAC CTG AAG AAA lie His Thr Asn Leu Lys Lys 5 38 77 AAG Lys TTC Phe 10 Met AGC TGC TGC Ser Cys Cys GTC CTG GTC TTT CTT CTG TTT GCA Val Leu 15 Val Phe Leu Leu Phe 20 Ala GTC ATC TGT GTG TGG AAG GAA AAG AAG AAA GGG AGT TAC 116 Val lie Cys Val Trp Lys Glu Lys Lys Lys Gly Ser Tyr 25 30 TAT GAT TCC TTT AAA TTG CAA ACC AAG GAA TTC CAG GTG 155 Tyr Asp Ser Phe Lys Leu Gin Thr Lys Glu Phe Gin Val 35 40 45 TTA AAG AGT CTG GGG AAA TTG GCC ATG GGG TCT GAT TCC 194 Leu Lys Ser 50 Leu Gly Lys Leu Ala Met 55 Gly Ser Asp Ser 60 CAG TCT GTA TCC TCA AGC AGC ACC CAG GAC CCC CAC AGG 233 Gin Ser Val Ser Ser Ser Ser Thr Gin Asp Pro His Arg 65 70 GGC CGC CAG ACC CTC GGC AGT CTC AGA GGC CTA GCC AAG 272 Gly Arg Gin Thr Leu Gly Ser Leu Arg Gly Leu Ala Lys 75 80 85 GCC AAA CCA GAG GCC TCC TTC CAG GTG TGG AAC AAG GAC 311 Ala Lys Pro Glu Ala Ser Phe Gin Val Trp Asn Lys Asp 90 95 AGC TCT TCC AAA AAC CTT ATC CCT AGG CTG CAA AAG ATC 350 Ser Ser Ser Lys Asn Leu lie Pro Arg Leu Gin Lys He 100 105 110 TGG AAG AAT TAC CTA AGC ATG AAC AAG TAC AAA GTG TCC 389 Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser 115 120 125 TAC AAG GGG CCA GGA CCA GGC ATC AAG TTC AGT GCA GAG 428 Tyr Lys Gly Pro Gly Pro Gly He Lys Phe Ser Ala Glu 130 135 GCC CTG CGC TGC CAC CTC CGG GAC CAT GTG AAT GTA TCC 467 Ala Leu Arg Cys His Leu Arg Asp His Val Asn Val Ser 140 145 150 ATG GTA GAG GTC ACA GAT TTT CCC TTC AAT ACC TCT GAA 506 Met Val Glu Val Thr Asp Phe Pro Phe Asn Thr Ser Glu 155 160 TGG Trp 165 GAG GGT TAT CTG CCC AAG GAG AGC Ser ATT AGG ACC AAG 545 Glu Gly Tyr Leu Pro 170 Lys Glu lie Arg 175 Thr Lys GCT GGG CCT TGG GGC AGG TGT GCT GTT GTG TCG TCA GCG 584 5 Ala Gly Pro- Trp Gly Arg Cys Ala Val Val Ser Ser Ala 180 185 190 GGA TCT CTG AAG TCC TCC CAA CTA GGC AGA GAA ATC GAT 623 Gly Ser Leu Lys Ser Ser Gin Leu Gly Arg Glu lie Asp 195 200 10 GAT CAT GAC GCA GTC CTG AGG TTT AAT GGG GCA CCC ACA 662 Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala Pro Thr 205 210 215 GCC AAC TTC CAA CAA GAT GTG GGC ACA AAA ACT ACC ATT 701 Ala Asn Phe Gin Gin Asp Val Gly Thr Lys Thr Thr lie 15 220 225 CGC CTG ATG AAC TCT CAG TTG GTT ACC ACA GAG AAG CGC 740 Arg Leu Met Asn Ser Gin Leu Val Thr Thr Glu Lys Arg 230 235 240 TTC CTC AAA GAC AGT TTG TAC AAT GAA GGA ATC CTA ATT 779 20 Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly lie Leu lie 245 250 255 GTA TGG GAC CCA TCT GTA TAC CAC TCA GAT ATC CCA AAG 818 Val Trp Asp Pro Ser Val Tyr His Ser Asp lie Pro Lys 260 265 25 TGG TAC CAG AAT CCG GAT TAT AAT TTC TTT AAC AAC TAC 857 Trp Tyr Gin Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr 270 275 280 AAG ACT TAT CGT AAG CTG CAC CCC AAT CAG CCC TTT TAC 896 Lys Thr Tyr Arg 285 Lys Leu His Pro Asn 290 Gin Pro Phe Tyr ATC CTC AAG CCC CAG ATG CCT TGG GAG CTA TGG GAC ATT 935 lie Leu Lys Pro Gin Met Pro Trp Glu Leu Trp Asp He 295 300 305 CTT CAA GAA ATC TCC CCA GAA GAG ATT CAG CCA AAC CCC 974 Leu Gin Glu He Ser Pro Glu Glu He Gin Pro Asn Pro 310 315 320 CCA TCC TCT GGG ATG CTT GGT ATC ATC ATC ATG ATG ACG 1013 Pro Ser Ser Gly Met Leu Gly He He He Met Met Thr 325 330 CTG TGT GAC CAG GTG GAT ATT TAT GAG TTC CTC CCA TCC 1052 Leu Cys Asp Gin Val Asp He Tyr Glu Phe Leu Pro Ser 335 340 345 AAG CGC AAG ACT GAC GTG TGC TAC TAC TAC CAG AAG TTC 1091 Lys Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gin Lys Phe 350 355 TTC GAT AGT GCC TGC ACG ATG GGT GCC TAC CAC CCG CTG 1130 Phe Asp Ser Ala Cys Thr Met Gly Ala Tyr His Pro Leu 360 365 370 CTC TAT GAG AAG AAT TTG GTG AAG CAT CTC AAC CAG GGC 1169 Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gin Gly 375 380 385 ACA GAT GAG GAC ATC TAC CTG CTT GGA AAA GCC ACA CTG 1208 Thr Asp Glu Asp lie Tyr Leu Leu Gly Lys Ala Thr Leu 390 395 -55CCT GGC TTC CGG ACC ATT CAC TGC TAAGCACAGG ATCC Pro Gly Phe Arg Thr lie His Cys 400 405 1246 -56SEO ID NO. 4 SEQUENCE TYPE: Protein SEQUENCE LENH: 368 amino acids MOLECULE TYPE: C-terminal fragment of full-length sialyltransferase PROPERTIES: - soluble sialyltransferase (EC 2.4.99.1) from human HepG2 cells Lys Leu Gin Thr Lys 5 Glu Phe Gin Val Leu 10 Lys Ser Leu Gly Lys 15 Leu Ala Met Gly Ser 20 Asp Ser Gin Ser Val 25 Ser Ser Ser Ser Thr 30 Gin Asp Pro His Arg 35 Gly Arg Gin Thr Leu 40 Gly Ser Leu Arg Gly 45 Leu Ala Lys Ala Lys 50 Pro Glu Ala Ser Phe 55 Gin Val Trp Asn Lys 60 Asp Ser Ser Ser Lys 65 Asn Leu lie Pro Arg 70 Leu Gin Lys He Trp 75 Lys Asn Tyr Leu Ser 80 Met Asn Lys Tyr Lys 85 Val Ser Tyr Lys Gly 90 Pro Gly Pro Gly He 95 Lys Phe Ser Ala Glu 100 Ala Leu Arg Cys His 105 Leu Arg Asp His Val 110 Asn Val Ser Met Val 115 Glu Val Thr Asp Phe 120 Pro Phe Asn Thr Ser 125 Glu -5715 Trp Glu Gly Tyr 130 Leu Pro Lys Glu Ser 135 Ile Arg Thr Lys Ala 140 Gly Pro Trp Gly Arg 145 Cys Ala Val Val Ser 150 Ser Ala Gly Ser Leu 155 Lys Ser Ser Gin Leu 160 Gly Arg Glu Ile Asp 165 Asp His Asp Ala Val 170 Leu Arg Phe Asn Gly 175 Ala Pro Thr Ala Asn 180 Phe Gin Gin Asp Val 185 Gly. Thr Lys Thr Thr 190 Ile Arg Leu Met Asn 195 Ser Gin Leu Val Thr 200 Thr Glu Lys Arg Phe 205 Leu Lys Asp Ser Leu 210 Tyr Asn Glu Gly Ile 215 Leu Ile Val Trp Asp 220 Pro Ser Val Tyr His 225 Ser Asp Ile Pro Lys 230 Trp Tyr Gin Asn Pro 235 Asp Tyr Asn Phe Phe 240 Asn Asn Tyr Lys Thr 245 Tyr Arg Lys Leu His 250 Pro Asn Gin Pro Phe 255 Tyr Ile Leu Lys Pro 260 Gin Met Pro Trp Glu 265 Leu Trp Asp Ile Leu Gin Glu Ile Ser Pro Glu Glu Ile Gin Pro Asn Pro 270 275 280 -58Pro Ser Ser Gly Met Leu Gly lie lie lie Met Met Thr . 285 290 295 Leu Cys Asp Gin Val Asp He Tyr Glu Phe Leu Pro Ser 300 305 Lys Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gin Lys Phe 310 315 320 Phe Asp Ser Ala Cys Thr Met Gly Ala Tyr His Pro Leu 325 t 330 Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gin Gly 10 335 340 345 Thr Asp Glu Asp He Tyr Leu Leu Gly Lys Ala Thr Leu 350 355 360 Pro Gly Phe Arg Thr He His Cys 365 -59SEO ID NO.5 SEQUENCE TYPE: Nucleotide sequence with corresponding protein SEQUENCE LENGTH: 1400 bp STRANDEDNESS: double TOPOLOGY:linear IMMEDIATE EXPERIMENTAL SOURCE: BRB.ELFT/pCR1000-13 FEATURES : from 58 to 1272 bp cDNA sequence coding for human a (1-3) fucosyltransferase from 238 to 243 bp Nrul site PROPERTIES: HL 60 cDNA coding for full-length a (1-3) fucosyltransferase CGCTCCTCCA CGCCTGCGGA CGCGTGGCGA GCGGAGGCAG CGCTGCCTGT 50 TCGCGCC ATG GGG GCA CCG Ala Pro TGG GGC TCG CCG ACG GCG GCG Trp Gly Ser Pro Thr Ala Ala 90 Met Gly 5 10 GCG GGC GGG CGG CGC GGG TGG CGC CGA GGC CGG GGG CTG 129 Ala Gly Gly Arg Arg Gly Trp Arg Arg Gly Arg Gly Leu 15 20 CCA TGG ACC GTC TGT GTG CTG GCG GCC GCC GGC TTG ACG 168 20 Pro Trp Thr Val Cys Val Leu Ala Ala Ala Gly Leu Thr 25 30 35 TGT ACG GCG CTG ATC ACC TAC GCT TGC TGG GGG CAG CTG 207 Cys Thr Ala Leu lie Thr Tyr Ala Cys Trp Gly Gin Leu 40 45 50 CCG CCG CTG CCC TGG GCG TCG CCA ACC CCG TCG CGA CCG 246 25 Pro Pro Leu Pro Trp Ala Ser Pro Thr Pro Ser Arg Pro 55 60 GTG GGC GTG CTG CTG TGG TGG GAG CCC TTC GGG GGG CGC 285 Val Gly Val Leu Leu Trp Trp 70 Glu Pro Phe Gly Gly 75 Arg 65 GAT AGC GCC- CCG AGG CCG CCC CCT GAC TGC CGG CTG CGC 324 Asp Ser Ala Pro Arg Pro Pro Pro Asp Cys Arg Leu Arg 80 85 TTC AAC ATC AGC GGC TGC CGC CTG CTC ACC GAC CGC GCG 363 Phe Asn lie Ser Gly Cys Arg Leu Leu Thr Asp Arg Ala 90 95 100 TCC TAC GGA GAG GCT CAG GCC GTG CTT TTC CAC CAC CGC 402 Ser Tyr Gly Glu Ala Gin Ala Val Leu Phe His His Arg 105 110 115 GAC CTC GTG AAG GGG CCC CCC GAC TGG CCC CCG CCC TGG 441 Asp Leu Val Lys Gly Pro Pro Asp Trp Pro Pro Pro Trp 120 125 GGC ATC CAG GCG CAC ACT GCC GAG GAG GTG GAT CTG CGC 480 Gly lie Gin Ala His Thr Ala Glu Glu Val Asp Leu Arg 130 135 140 GTG TTG GAC TAC GAG GAG GCA GCG GCG GCG GCA GAA GCC 519 Val Leu Asp iyr Glu Glu Ala Ala Ala Ala Ala Glu Ala 145 150 CTG GCG ACC TCC AGC CCC AGG CCC CCG GGC CAG CGC TGG 558 Leu Ala Thr Ser Ser Pro Arg Pro Pro Gly Gin Arg Trp 155 - 160 165 GTT TGG ATG AAC TTC GAG TCG CCC TCG CAC TCC CCG GGG 597 Val Trp Met Asn Phe Glu Ser Pro Ser His Ser Pro Gly 170 175 180 -61 25 CTG CGA AGC CTG GCA AGT AAC CTC TTC AAC Phe Asn 190 TGG ACG CTC 636 Leu Arg Ser Leu Ala 185 Ser Asn Leu Trp Thr Leu TCC TAC CGG GCG GAC TCG GAC GTC TTT GTG CCT TAT GGC 675 Ser Tyr Arg Ala Asp Ser Asp Val Phe Val Pro Tyr Gly 195 200 205 TAC CTC TAC CCC AGA AGC CAC CCC GGC GAC CCG CCC TCA 714 Tyr Leu Tyr Pro Arg Ser His Pro Gly Asp Pro Pro Ser 210 215 GGC CTG GCC CCG CCA CTG TCC AGG AAA CAG GGG CTG GTG 753 Gly Leu Ala Pro Pro Leu Ser Arg Lys Gin Gly Leu Val 220 225 230 GCA TGG GTG GTG AGC CAC TGG GAC GAG CGC CAG GCC CGG 792 Ala Trp Val Val Ser His Trp Asp Glu Arg Gin Ala Arg 235 240 245 GTC CGC TAC TAC CAC CAA CTG AGC CAA CAT GTG ACC GTG 831 Val Arg Tyr Tyr His Gin Leu Ser Gin His Val Thr Val 250 255 GAC GTG TTC GGC CGG GGC GGG CCG GGG CAG CCG GTG CCC 870 Asp Val Phe Gly Arg Gly Gly Pro Gly Gin Pro Val Pro 260 265 270 GAA ATT GGG CTC CTG CAC ACA GTG GCC CGC TAC AAG TTC 909 Glu lie Gly Leu Leu His Thr Val Ala Arg Tyr Lys Phe 275 280 TAC CTG GCT TTC GAG AAC TCG CAG CAC CTG GAT TAT ATC 948 Tyr Leu Ala Phe Glu Asn Ser Gin His Leu Asp Tyr He 285 290 295 ACC Thr GAG AAG CTC TGG CGC AAC GCG TTG CTC GCT GGG GCG Ala Leu Leu Ala Gly Ala 987 Glu Lys Leu 300 Trp Arg Asn 305 310 GTG CCG GTG- GTG CTG GGC CCA GAC CGT GCC AAC TAC GAG 1026 Val Pro Val Val Leu Gly Pro Asp Arg Ala Asn Tyr Glu 315 320 CGC TTT GTG CCC CGC GGC GCC TTC ATC CAC GTG GAC GAC 1065 Arg Phe Val Pro Arg Gly Ala Phe lie His Val Asp Asp 325 330 335 TTC CCA AGT GCC TCC TCC CTG GCC TCG TAC CTG CTT TTC 1104 Phe Pro Ser Ala Ser Ser Leu Ala Ser Tyr Leu Leu Phe 340 345 CTC GAC CGC AAC CCC GCG GTC TAT CGC CGC TAC TTC CAC 1143 Leu Asp Arg Asn Pro Ala Val Tyr Arg Arg Tyr Phe His 350 355 360 TGG CGC CGG AGC TAC GCT GTC CAC ATC ACC TCC TTC TGG 1182 Trp Arg Arg Ser Tyr Ala Val His lie Thr Ser Phe Trp 365 370 375 GAC GAG CCT TGG TGC CGG GTG TGC CAG GCT GTA CAG AGG 1221 Asp Glu Pro Trp Cys Arg Val Cys Gin Ala Val Gin Arg 380 385 GCT GGG GAC CGG CCC AAG AGC ATA CGG AAC TTG GCC AGC 1260 Ala Gly Asp Arg Pro Lys Ser lie Arg Asn Leu Ala Ser 390 395 400 1302 TGG TTC GAG CGG TGAAGCCGCG CTCCCCTGGA AGCGACCCAG Trp Phe Glu Arg 405 · -63GGGAGGCCAA GTTGTCAGCT TTTTGATCCT CTACTGTGCA TCTCCTTGAC TGCCGCATCA TGGGAGTAAG TTCTTCAAAC ACCCATTTTT GCTCTATG

Claims (23)

Claims: 1. o

1. Process for the production of a membrane-bound mammalain glycosyltransferase selected from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a membrane-bound or a soluble variant thereof, respectively, consisting of essentially the whole stem region and the catalytic domain, said process comprising culturing a yeast strain which has been

2. Process according to claim 1, wherein the glycosyltransferase is of human origin.

3. Process for the production of a variant according to claim 1. wherein the variant differs

4. Process for the production of a variant according to claim 3 comprising culturing a yeast strain comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second

5. Process according to claim 1, wherein the glycosyltransferase is a galactosyltransferase. * 5 transformed with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for said glycosyltransferase or variant which DNA is controlled by said promoter, and recovering the enzymatic activity.

6. Process according to claim 5, wherein the galactosyltransferase is selected from the group consisting of UDP-Galactose: β-galactoside α( 1 -3 )-galac tosyl transferase

7. Process according to claim 5, wherein the galactosyltransferase has the amino acid sequence depicted in SEQ ID NO. 1.

8. Process according to claim 5, wherein the galactosyltransferase has the amino acid 25 sequence depicted in SEQ ID NO. 2.

9. Process according to claim 1, wherein the glycosyltransferase is a sialyltransferase. -6510. Process according to claim 9, wherein the sialyltransferase is CMP-NeuAc β-galactoside a(2-6)-sialyltransferase (EC 2.4.99.1).

10. From the corresponding full-length glycosyltransferase by lack of the cytoplasmic tail, the signal anchor and, optionally, a minor part of the stem region.

11. Process according to claim 9, wherein the sialyltransferase has the amino acid depicted in SEQ ID NO. 3. 5

12. Process according to claim 9, wherein the sialyltransferase is designated ST(Lys27-CyS4oe) and consists of amino acids 27 to 406 of the amino acid sequence listed in SEQ. ID NO. 3.

13. Process according to claim 9, wherein the sialyltransferase has the amino acid depicted in SEQ ID NO. 4.

14. Process according to claim 1, wherein the glycosyltransferase is a fucosyltransferase. 15. Sequence depicted in the sequence listing with SEQ ID NO. 5.

15. Process according to claim 14, wherein the fucosyltransferase is selected from the group consisting of GDP-Fucose^-galactoside a( 1-2)-fucosyltransferase (EC 2.4.1.69) and GDP-Fucose:N-acetylglucosamine α( 1 -3/4)-fucosyltransferase (EC 2.4.1.65). 15 DNA sequence coding for said variant which DNA sequence is controlled by said promoter, and recovering the enzymatic activity.

16. Process according to claim 14, wherein the fucosyltransferase has the amino acid

17. Process according to claim 14, wherein the fucosyltransferase is designated FT(Arg 62 -Arg^) and consists of amino acids 62 to 405 of the amino acid sequence depicted in the sequence listing with SEQ ID. NO. 5.

18. A yeast hybrid vector comprising an expression cassette comprising a yeast promoter and a 20 DNA sequence coding for a membrane-bound mammalian glycosyltransferase selected from the * group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a membrane-bound or a soluble variant thereof, respectively, consisting of essentially the whole stem region and the catalytic domain which DNA sequence is controlled by said promoter. 25

19. A yeast strain which has been transformed with a hybrid vector according to claim 18. 66

20. A process according to claln 1 for the production of a Ϊ membrane-bound mammalian glycosyltransferase, substantially as , j hereinbefore described and exemplified. 20 (EC 2.4.1.151) and UDP-Galactose: β-Ν-acetylglucosamine P(l-4)-galac tosyl transferase (EC 2.4.1.22).

21. A membrane-bound mammalian glycosyltransferase, whenever produced by a process claimed in a preceding claim.

22. A yeast hybrid vector according to claim 18, substantially as hereinbefore described and exemplified.

23. A yeast strain according to claim 19, substantially as hereinbefore described.