GB2256197A - Process for the production of glycosyltransferases - Google Patents

Description

2 c 23 61 ? 7 Improved process for the production of glycosyltransferases

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 cylycosidic linka-e. Based on the type of sugar transferred, these enzymes are grouped into families, e.(Y. galactosyltransferases, sialyltransferases and fucosyltransferases. Being, resident membrane proteins primarily located in the Golaj 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 sicnal-anchor domain acts as both uncleavable signal peptide and as Z) 1) membrane spanning region and orients the catalytic -domain of the glycosyltransferase C; 1:1 0 within the lumen of the Gol 4 gi apparatus. The luminal stem or spacer region is supposed to serve as a flexible tether, allowing the catalytic domain to glycosylate carbohydrate groups of membrane-bound and soluble proteins of the secretory pathway enroute through I - 11.

the Golgi apparatus. Furthermore, the stem portion was discovered to function as retention Zn signal to keep the enzyme bound to the Golgi membrane (PCT 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 Z 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 reCgioselectivity, 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 occurriny in very low amounts, isolation from natural sources and subsequent purification are difficult. Therefore, production using r recombinant DNA technology has been worked on. For example, calactosyltransferases have been expressed in E. coli (PCT 90/07000) and Chinese hamster ovary (CHO) cells (Smith, D.F. et al. (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 cells (Potvin, B. (1990) J. Biol. Chem- 265, 1615-1622). Considering the facts that heterolo-ous expression in prokaryotes has the disadvantage of providing unglycosylated products, glycosyl- C 1) IM transferases, however, being glycoproteins, and that olycosyltransferase production by use 1 Z.D Zn 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 I 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 a glycosyltransferases by a recombinant DNA technology using a yeast vector expression system.

More specifically, the present invention provides 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, said process comprising culturing a yeast strain which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for said alycosyltransferase or variant which DNA is controlled by said promoter, and recovering the enzymatic activity.

In a first embodiment, the invention relates to a process for the production ofa membrane-bound glycosyltransferase selected from the group consisting of a galactosyl- 1 1 1 C> 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 l> - 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 0 C recovering the enzymatic activity.

I 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, 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.

I The term "olycosyltransferase" whenever used hereinbefore or hereinafter is intended to embrace the family of z-,,alactosyltransferases, the family of sialyltransferases and the family of fucosyltransferases. Said glycosyltransferases are naturally occurring enzymes I 1 of mammalian, e.g. bovine, murine, rat and human origin. Preferred are naturally occurring human full-length glycosyltransferases including those enzymes identified I I'D 0 hereinafter by their EC-numbers.

The membrane-bound galactosyltransferases and their variants obtainable according to the b 1 1 inventive process catalyse the transfer of a galactose residue from an activated donor, 1 usually a nucleotide activated donor such as uridine diphosphate galactose (UDP-Gal), to a carbohydrate group.

0 Examples of membrane-bound galactosyltransferases are UDP-Galactose: Pgalactoside (x(I-3)-galactosyltransferase (EC 2.4.1.151) which uses galactose as acceptor substrate forming an a(1-3)-linkage and UDPGalactose: P-N-acetylglucosamine P(1-4)-galactosyltransferase (EC 2.4.1. 22) which transfers galactose to N-acetylglucosamine (GIcNAc) forming a P(1-4)-linkage, including variants thereof, respectively. In the presence of cc-lactalbumin, said P(14)-galactosyltransferase also accepts glucose as an acceptor substrate, thus catalysing the synthesis of lactose.

1 0 The most preferred membrane-bound galactosyltransferase is the enzyme having the 1 C1 amino acid sequence depicted in the sequence listing with the SEQ ID NO. 1.

The membrane-bound sialyltransferases and their variants obtainable according to the 1 process of the invention catalyse the transfer of sialic acids (for example N-acetyl neuraminic 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 si"-"yltransferase obtainable according to the inventive method is the CMP-NeuAc: 0-alactoside (x(2-6)-sialyltransferase (EC 2.4.99.1) which forms the D NeuAc-(x(2-6)Gal-0(1-4)GlcNAc-sequence common to many Winked carbohydrate groups.

1 The most preferred membrane-bound sialfitransferase 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 process of the invention catalyse the transfer of a fucose residue from an activated donor, usually a nuelcotide-activated donor, such as guanosine diphosphate fucose (GDP-Fuc), to a carbohydrate group. Examples of such fucosyltransferases are Z-GDP-Fucose:p-galactoside cc(1-2)-fucosyltransferase (EC 2.4.1.69) and GDPFucose:N-acetyle,lucosamine (x(1-314)-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 membranebound 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 I I 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 I ID embraces glycosyltransferases produced from a DNA which has been subjected to in vitro Z__ - - mutagencsis, with the provision that the protein encoded by said DNA has the enzymatic activity of the native ()-lycosyltransferase. Such modifications may consist in an addition, it, exchange and/or deletion of amino acids, the latter resulting in shortened variants.

I 1 Preferred 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 galactosyltransferase, 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 membranebound form naturally located in the endoplasmic reticulum or the Golgi complex, by lack of the cytoplasmic tail, the sienal-anchor and, optionally, part of the stem region. The term "part of the stem" region as used herein is defined to be a minor part of the N-terininal 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 0 Z) full-length forms by the absence of an NH2-terminal pe' tide consisting of 26 to 67, p 0 particularly 26 to 61, amino acid residues, with the provision that those forms lacking part 1b of the stem region only lack the above-defined minor part therof. Preferred are soluble variants obtainable from the membrane-bound glycosyltransferases identified hereinbefore 41:1 by their EC-numbers and additionally soluble variants obtainable from the membrane-bound fucosyltransferase with SEQ. ID NO. 5.

Preferred soluble variants of -alactosyltransferases are distinct from the corresponding tp 0 full-lenath forms in that they lack an NH2-terminal peptide consisting of 37 to 55, 1 I= particularly 41 to 44, amino acids. The most preferred soluble variant prepared according to the inventive proc.-ss is the enzyme having the amino acid sequence depicted in the sequence listing with the SEQ ID NO. 2.

0 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 1 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 NH27terminal peptide consisting of 56 to 67, particularly 56 1 to 61, amino acids. Especially preferred is the soluble variant designated FT(Arg62-Ar.(-3,'405) 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 cast strains are cultured using methods known in the art.

y Z-5 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 sourcds include, for example, amino acids, such as casamino acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore yeast extract, malt extract, corn 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, In the nutrient medium may also contain growth promoting substances. Substances which promote growth include, for example, trace elements, such as iron, zinc, manganese and the like, or individual amino acids.

Due to the incompatbility between the endogenous two-micron DNA and hybrid vectors 1 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 Z) require the expression of a plasmid-encoded gene for growth. Most selective markers currentl in use and present in the hybrid vectors according to the invention (infra) are genes y 0 coding for enzymes of amino acid or purine biosynthesis. This makes it necessary to use 1 synthetic minimal media deficient in the corresponding amino acid or purine base. How- Z> ever, genes conferring resistance to an appropriate biocide may be used as well [e.g. a c t) gene conferring resistance to the amino-glycoside G4181. Yeast cells transformed with 1 W vectors containing antibiotic resistance genes are grown in complex media containing the Ily 1t5 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 Saccharomyces cerevisia-e which are devoid of endogenous two-micron plasmids (so-called cir' strains) so that the cultivation can be carried out under non-selective crowth conditions, i.e. in a complex medium.

0 Yeast cells containing hybrid plasmids with a constitutive promoter express the DNA encoding a glycosyltransferase, or a variant thereof, controlled by said promoter without 1 1 induction. However, if said DNA is under the control of a regulated promoter the C composition of the growth medium has to be adapted in order to obtain maximum levels 1 of mRNA transcripts, e.g. when using the PH05 promoter the growth medium must 1 1 1 contain a low concentration of inorganic phosphate for derepression of this promoter.

The cultivation is carried out by employing conventional techniques. The culturing 0 t> 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 Ion. 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 For example, the first step usually consists in separating the cells from the culture fluid by lt centrifugation. In cas- the a, ycosyltransferase, 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 Z ID such as shaking with glass beads, by ultrasonic vibration, osmotic shock and/or by enzymatic digestion of the cell wall. In case the (3lycosyltransferase, 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. Methods suitable for the purification of the crude olycosyltransferase, or the variant thereof include standard chromatographic procedures such as affinity chromatography, for example with a Z C:

suitable substrate, antibodies or Concanavalin A, ion exchange chromatography, gel filtra- tion, 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 ()lycosyltransferase 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.

Z:

In order to detect ()-lycosyltransferase activity assays known from the literature can be used. For example, galactosyltransferase activity can be measured by determing the amount of radioactively labelled galactose incorporated into a suitable acceptor molecule I such as aglycoprotein or a free sugar residue. Analogously, sialyltransferase activity may be assayed e.g. by the incorporation of sialic acid into suitable substrates, and fucosyltransferase 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 I recombinant DNA techniques comprisin(),, the steps of: preparing a hybrid vector comprising a yeast promoter and a DNA sequence coding for a membrane-bound alycosyltransferase, or a variant thereof, which DNA sequence is controlled by said promoter, transforming a yeast host strain with said hybrid vector, 1 and selectin a tran eformed yeast cells from untransformed yeast cells.

0 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 C1 W glycosyltransferase, or a variant thereof, which DNA sequence is controlled by said 1:1 promoter.

In a first embodiment, the yeast hybrid vector according to the invention comprises an 1 expression cassette comprising a yeast promoter, a DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, which DNA sequence is Z5 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 I 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 containino, , yeast transcription termination signals.

The yeast promoter is a regulated or a constitutive promoter preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the 1P promoter of the TRP1 acne, the ADHI or ADHII acne, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or cc-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, (vlyceraldehyde-3- phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (LGK), 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 h brid promoters comprising upstream activation sequences (UAS) of one yeast gene y 0 I'D and downstream promoter elements including a functional TATA box of another yeast acne, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and C5 - ID 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, especially functional frajogments thereof starting, at nueleotides between positions -550 and -180, in particular at nucleotide -540, -263 or -198, and ending at nucleotide -5 of the.GAP,c,ene. Another prefered promoter of the regulated type is the PH05 promoter. As a n c 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 C C_ nucleotide -173 and ending at nucleotide -9 of the PH05ggene.

The DNA sequence encoding a signal peptide ("siggnal 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, (x-factor, pheromone peptidase (KEXI), "killer toxin" and repressible acid phosphatase (PH05) genes and the glucoamylase signal sequence from Aspergillus awamori. Alternatively, fused signal sequences may be constructed by ligating part of the siinal 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 cleavaoge between the signal sequence and the glycosyltransferase amino acid sequence. Additional sequences, such as pro- or spacersequences which may or may not carry specific processing signals can also be included in I C the constructions to facilitate accurate processing of precursor molecules. Alternatively, I 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-Argg, which CP is recognized by a yeast endopeptidase located in the Goloi 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.

ZP 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 sequence containing yeast transcription termination signals.

0 b DNA encoding a mem- branc-bound glycosyltransferase, or a variant thereof, can be 1 1 prepared by methods known in the art and comprises genomic DNA, c. a. isolated from a 1 W mammalian genomic DNA library, e... 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 eDNA which can be isolated from a mammalian cDNA library or produced from the corresponding mRNA. The eDNA 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)IRNA 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 1 In 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 complementarity 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.

Inp W C Furthermore, DNA encoding a membrane-bound -lycosyltransferase, 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 more amino acids are deleted (DNA fragments) and/or exchanaed with one or more other 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). Stich a mutant sequence is also a degenerated DNA sequence. Degenerated DNA sequences are ID degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotideswithout resulting in a change of the amino acid sequence originally encoded. Such degenerated DNA sequences may be useful due to I 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 alycosyltransferase by using restriction enzymes. The availability of an appropriate restriction site is advantalyeous therefor.

A DNA sequence containing yeast transcription termination signals is preferably the 3' 0 0 flanking sequence of a yeast gene, which contains proper signals for transcription Z-- 0 11 termination and polyadenylation. Suitable Tflanking 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 11 1 DNA sequence containing yeast transcription termination signals are operably linked in a t:

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 a cosyltransferase, or a variant thereof, 1 Ily (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 or synthetic oli-odeoxynucleotide 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 C5 In cell after transformation and are replicated autonomously during mitosis. Also, hybrid vectors that contain sequences homologous to the yeast 2g plasmid DNA can be used. Such hybrid vectors are integrated by recombination in 2g plasmids already present within 0 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 I -- the selection for transformants due to the phenotypic expression of the marker gene.

C Suitable markers for yeast are, for example, those expressing antibiotic resistance or, in the case of auxotrophic yeast mutants, genes which com lement host lesions. p Corresponding genes confer, for example, resistance to the antibiotics G418, hygromycin Z: ID or bleomycin or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2 or TRP I aene.

C As the amplification of the hybrid vectors is conveniently done in E. coli, an E. coli (Yenetic marker and an E. coli replication origin are included advantageously. These can be -- C" 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 fraaments containino, selective crenetic 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 described below.

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

Z:' 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 1 0 sequence coding for a membrane-bound glycosyltransferase, or a variant therof, which Z-- 0 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 In 113 DNA sequence coding for a membrane-bound glycosyltransferase, or a variant thereof, C) which DNA sequence is controlled by said promoter, and a DNA sequence containing yeast transcription termination signals.

1 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.

It) 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 exam le according to the methods P 1.1 described by Hinnen et al. (Proc. NaLl. 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 0 process according to the invention can be used in a manner known per se, e.g. for the n C" synthesis and/or modification of glycoproteins, oligosaccharides and glycolipids 4; C 1) (US Patent 4,925,796; EP Application 414 17 1).

The invention concerns especially the method for the production of membrane-bound,o,lycosyltransferases, 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 are used: GT,=^ galactosyltransferase (EC 2.4.1.22), PCR 12, polymerase chain reaction; ST,-' sialyltransferasc (EC 2.4.99.1); FT= fucosyltransferase.

Example 1: Cloning of the!ZalactOSY1transferase (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)IRNA 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-HCI 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)IRNA is achieved by affinity chromatography on oligo(dT)-cellulose according to the method described in the Maniatis manual (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Habor, USA), applying 4 mg of total RNA on a 400 gl column. 3 % of the loaded RNA are recovered as enriched poly(A)IRNA which is stored in aliquots precipitated with 3 volumes of ethanol at -701C until it is used.

1.2 First strand cDNA synthesis for PCR Poly(A)IRNA (mRNA) is reversetranscribed into DNA by Moloney Murine Leukemia Virus RNase H- Reverse Transcriptase (M-MLV H- RT) (BRL). In setting up the 20 gl reaction mix, the protocol provided by BRL is followed with minor variations: I gg of HeLa cell poly(A)'RNA and 500 ng, Oli!zo(dT)12-18 (Pharmacia) in 11.5 gl sterile H20 are heated to 70'C for 10 min and then quickly chilled on ice. Then 4 gl reaction buffer provided by BRI, (250 mM Tris-11C1 pH 8-3, 375 mM KCl, 15 MM MS.P2), 2 gI 0. 1 M dithiothreitol, 1 ptl mixed dNTP (10 mM each dATP, dCTP, dGTP, TTP, Pharmacia), 0.5 gI (17.5 U) RNAguard (RNase Inhibitor of Pharmacia) and 1 gI (200 U)M-MIVH- RT are added. The reaction is carried out at 42'C and stopped after 1 h by heating the tube to 951C for 10 min.

In order to check the efficiency of the reaction an aliquot of the mixture (5 gl) is incubated 32P dCTP. By measuring the incorporated dCTP, the amount of in the presence of 2 gCi (x- Z 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., Verlao, Chemie, Weinheim, FRG) on an Applied Biosystems Model 380B synthesizer. They are listed in Table 1.

Table 1: PCR-primers corresponding to primer sequence (Y to Y)1) bp in CT cDNA2) Plup (KpnI) capgel,2y,,,tAAC-CLIC'ITCTTAAAGCGGCGGCGGGAAGATG (-26)P1 (EcoRI) úrcco,,aattcATGAGGCTTCGGGAGCCGCTCCTGAGCG Z - (SacI) CTGGAGCTCGTGGCAAAGCAGAACCC 3 1 - 28 448- 473 P2d (EcoRI) gccgaa'ITCAGTCTCTTATCCGTGTACCAAAACGCCTA 1222-1192 1 P4 (HindIll) cccaa2ctTGGAATGATGATGGCCACCTTGTGAGG 546- 520 Capital letters represent sequences from GT, small letters are additional sequences, sites for restriction enzymes are underlined. Codons for 'starC and 'stop' of RNA translation are highlighted in boldface.

cl GT cDNA sequence from human placenta as published in GenBank (Accession Nr. M22921).

Standard PCR-conditions for a 30 gl incubation mixture are: 1 gl of the Reverse Transcriptase reaction (see Example 1.2), containing about 5 ng first strand cDNA, 15 pmol each of the relevant primers, 200 gmol each of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and TTP) in PCRbuffor (10 mM Tris-HCl pH 8.3 (at 23'C), 50 mM KCI, 1.5 mM MgC12, 0'001 % gelatine) and 0.5 U AmpliTaq Polymerase (Perkin Elmer).

I The amplification is performed in the Thermocycler 60 (Biomed) using the following conditions: 0.5 min do,-naturim, at 95'C, 1 min annealing at 56T, and 1 min 15 sec exten- C 41D sion at 72T, for a total of 20 cycles. In the last cycle, primer extension at 72T is carried out for 5 min.

For sequencing and subcloning, the HeLa GT cDNA is amplified in two overlapping W 1 pieces, usine different primer combinations:

c (1)Fragment P1 -P4: Primers P1 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 c 0 covering nucleotide positions 457 - 1232 (SEQ ID NO. 1).

In order to avoid errors during amplification four independent PCRs are carried out for z: 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, frazment PI - P4 is digested with the restriction enzymes EcoRl and Hind111, analysed on a 1.2 % kgarose 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 SacJ and EcoRI, analysed on a 1.2 % gel, eluted and subcloned into pUC18, digested with SacI and EcoRL The resulting subclones are pUC18/Pl P4 and pUC18/P3 - P2d, respectively. For subcloning, ligation and I C) transformation of E. coli strain DH5cc, standard protocols are followed as described in Example 2. Minipreparations of Plasmids pUC 1 8/P 1 - P4 and pUC 18/P3 - P2d are used for dideoxy-sequencing of denatured doublestranded DNA with the T7 polymerase Sequencing kit (Pharmacia). M13/pUC sequencing primers and reverse sequencing 0 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 C, 1!D ID fragments of both strands. The sequence of fragments amplified by independent PCRs shows that the error of amplification is less than I in 3000 nucleotides. The complete nucleotide sequence of the HeLa cell GT cDNA which is presented in SEQ ID NO. I is 99.2 % homologous to that of human placenta (Genbank Accession No. M22921). Three differences are found:

(a) Three extra base pairs at nucleofide positions 37-39 (SEQ ID NO. 1) resulting in one 0 extra amino acid (Ser) in the N-terminal region of the protein; (b) bp 98 to 101 are 'CTCT' instead of 'T(-'TG' 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 1 position 1047 is changed from W to 'G' without ensuing a change in amino acid 1 b 1= sequence.

The two overlapping DNA-fragnlents P1 - P4 and P3 - P2d encoding the HeLa GT cDNA are joined via the NotI restriction site at nucleotide position 498 which is present in both frao,ments.

g The complete HeLa cell GT cDNA (SEQ ID No. 1) is cloned as a 1.2 kb EcoRl- EcoRI restriction fragment in plasmid pIC-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, 1 48 1-485), resulting, in vector p4AD 113. For the purpose of creating the GT expression cassette the EcoRI restriction site (bp 1227) at the Tend of the cDNA sequence is deleted as follows: vector p4AD 113 is first linearized by digestion with EcoRV and then treated with alkaline phosphatase. Furthermore, 1 gg 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 ael by GENECLEAN (Bio 101). The protrudino, EcoRI end is filled in with Klenow polymerase C as described in the Maniatis manual (supra). After phenolisation and ethanol precipitation the plasmid is religated and used to transform E. coli DH5(x (Gibco/BRL). Minipreparation of plasmids are prepared from six transformants. The plasmids obtained are checked by restriction analysis for the absence of the EcoRI and EcoRV restriction sites at the 3' end of HeLa GT cDNA. The plasmid designated p4AE1 13 is chosen for the following I I experiments, its DNA sequence being identical to that of plasmid p4AD 113, with the exception -that bp 1232-1238 with the EcoRI-EcoRV restriction sites are deleted.

Example 2: Construction of expression cassettes for full length QT For heterologous expression in Saccharomyces cerevisiae the full len,-,th 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 titne (EH05 C> - W 1 fl5) (EP 100561). The full-length PH05 promoter is regulated by the supply of inorganic phosphate in the culture medium. High Pi C) 0 concentrations 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 p4AE1 13 with the full length GT cDNA sequence is dicrested with the restriction 0 It> enzymes EcoRI and B oIII. The DNA fragments are clectrophoretically separated on a 1 % agarose gel. A 1.2 kb DNA fragment containing the complete cDNA sequence for HeLa 1 It: -- GT is isolated from the -cl by adsorption to glasmilk, using the GENECLEAN kit t 0 (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 Yuntranslated region of HeLa GT and the multiple cloning c Itp site of the vector with the BgIII 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 SaII. The restriction fragments are 1 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 lar.e Sall - HindIII vector fra-ment of 1 0 CP the pBR322 derivative as well as a 337 bp PH05 transcription terminator sequence in place of the HindIII 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 p3 1 R (cf. EP 10056 1) by digestion with the restriction enzymes SaII and EcoRI. The 0.8 kb Sall - EcoRI DNA fragment comprises the 276bp Sall - BamHI pBR322 sequence and the 534 bp BamHlEcoRl PH05 promoter fragment 1.

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 pi lination mixture: 100 na of DNA W c, fragment (a) and 30 no, each of fragments (b) and (c) are li-ated using 0. 3 U T4 In Z W -- DNA ligase (Boehringer) in the supplied ligase buffer (66 mM Tris-HCI pH 7.5, 1 mM Z> C1 dithioerythritol, 5 MM M-01C12, 1 mM ATP) at 15'C for 18 hours. Half of the ligation mix is used to transform competent cells of E. coli strain DH5(x (Gibco/BRL). For preparing competent cells and for transformation, the standard protocol 0 as given in the Maniatis manual (supra) is followed. The cells are plated on selective LB-medium, supplemented with 75 go,/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 Birnboim, 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, PstI, HindIII, SalI, 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 SalI - HindIII fragm nt c referred to as DNA fragment (IA).

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 p31LPH05(173)RIT.

(a) Construction of plasmid p31/PHOSG173)RIT Plasmid p31 RITI2 (EP 288435) comprises the full length, regulated PH05 promoter (with an EcoRI site introduced at nueleotide position -8 on a 534bp BamHI - EcoRI fragm nt, c followed by the coding sequence for the yeast invertase signal sequence (72bp EcoRI - 1 1 XhoI) and the PH05 transcription termination signal (135bp XhoI - HindIII) cloned in a tandem array between BamHI and HindHI of the pBR322 derived vector.

The constitutive PH05(A73) promoter element from plasmid pJDB207LPH05(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 PH05(A73) promoter, therefore, behaves like a constitutive promoter. This example describes the replacement of the regulated PH05 promoter in plasmid p31RIT12 by the short, constitutive PH05 (A73) promoter element in order to obtain plasmid p3 1/PH05 (-173) RIT.

Plasmids p3 1RIT12 'EP 288435) and pJDB207/PH05(-173)-YHIR (EP 340170) are digested with restriction endonucleases SaII and EcoRl. The respective 3.6 kb and 0.4 kb SaII - EcoRI fragments are isolated on a 0.8 % ag 0 In garose gel, eluted from the gel, ethanol precipitated and resuspended in H20 at a concentration of 0. 1 pmoles/gI. Both DNA fragments are ligated and 1 gI aliquots of the ligation mix are used to transform E. coli I 1 -1 - HB101 (ATCC) competent cells. Ampicillin resistant colonies are grown individually in LB medium supplemented with ampicillin (100 gg/ml). Plasmid DNA is isolated according to the method of Holmes, D.S. et al. (Anal. Biochem. (1981) 144, 193) and analysed Z:' by restriction digests with SaII and EcoRL The plasmid of one clone with the correct I restriction fragments is referred to as p3l/PH05(-173)RIT.

(b) Construction of plasmid pGTB 1135 Plasmid p3 1/PH05(-173)RIT is digested with the restriction enzymes EcoRI and Sail. After separation on a 1 % agarose -cl, a 0.45 kb Sail - EcoRI fragment is isolated from the C 1:1 -- gel by GENECLEAN (BIO 101). This fragment contains tile 276 bp SaII- BarnIII sequence of pBR322 and the 173bp BamM(BstEfl)-EcoRI constitutive PH05 promoter fragment. The 0.45 kb SaII-EcoRI fragment is ligated to the 1.2 kb EcoRI - Bg1II GT cDNA (fragment (Q) 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 1 0 E. coli strain DH5oc 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 frairment. This expression cassette can be excised from the vector pGTB 1135 as a 2 kb Sail - HindIH fragment, referred to as DNA fracyment (1B).

0 0 Example 3: Construction of the expression vectors pDPGTA8 and PDPGTB5 The yeast vector used for heterolog, ous 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 URA3 and dLEU2 yeast selective markers. Vector pDP34 (cf. EP 340170) is diaested with the restriction enzyme BamHl. 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 SaH and subjected to gel electrophoresis on a 0. 8 % a arose gel.

Z) g 0 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 I digested with HindlH. The protruding ends of the linearized plasmids are filled in by C C.

Klenow polymerase treatment and subsequently subjected to SaII digestion, resulting in (2A) a 2.35 kb (HindII1)blunt end - SalI fragment with the phosphate regulated expression cassette, or (2B) a 2.0 kb (HindIH)blunt end - SalI fragment with the constitutive expression cassette.

Ligation of the blunt end-SalI pDP34 vector part with fragment 2A or fragment 2B and transformation of competent cells of E. coli strain DH5ot is carried out as described in Example 2 using 80 ng of the vector part and 40 no, of fragment 2A or 2B, respectively. 58 C5 0 Z= 15 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 (MATot, his4, leu2, ura3, pra 1, prb 1, prc 1, cps 1) and H 449 (MATa, prb I, cps I, ura3A5, leu 2-3, 2112, cir') are each transformed with 5 go, each of plasmids pDPGTA8, pDPGTB5 and pDP34 (control without expression cassette) according to the lithium-acetate transformation method (Ito, H. et al., supra). Uraltransformants are isolated and screened for GT activity (infra). Single transformed yeast cells are selected and referred to as Saccharomyces cerevisiae BT 150lpDPGTA8 Saccharomyces cerevisiae BT 150/pDPGTB5 Saccharomyces cerevisiae BT 150ffiDP34 Saccharomyces cerevisiae H 449/pDPWA8 Saccharomyces cere-isiae H 449/pDPGTB5 Saccharomyces cerevisiae H 449/pl)P34 Example 5: Enzyme activity of full-length GT expressed in yeast 5.1 Preparation of cell extracts Cells of transformed Saccharomyces cerevisiae strains BT 150 and H 449 are each arown Z:

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 OD578 of 0.5) or stationary cells are collected by centrifugation, washed once with 50 mM Tris-HCl buffer pH 7.4 (buffer 1)

I and resuspended in buffer I at a concentration corresponding to 0. 1 0. 2 OD578Mechanical breakatle of the cells is effected b viaorous shaking on a vortex mixer with lt y 1 alass beads (0.45 - 0.5 mm diameter) for 4 min with intermittent coolina. 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 for 5 min; secondly, the supernatant obtained is centrifued 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.

5.2 Protein assay The protein concentration is determined by use of the BCA-Protein Assay Kit (Pierce).

5.3 Assay for GT activity GT activity can be measured with radiochemicalmethods usina either ovalbumin, a,glycoprotein which solely exposes GIcNAc as acceptor site, or free GIcNAc as acceptor substrates. Cell extracts (of I - 2 ODS 578 of cells) are assayed for 45 or 60 min at 37'C in a 100 gI incubation mixture containing 100 mM Tris-HCI pH 7.4,50 nCi UDP- 14C-Gal (325 mCi/mmol), 80 nmol UDP-Gal, 1 gmol MnC'2, 1 % Triton X- 100 and 1 mg ovalbumin or 2 [Lmol 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 H20 and the unused UDP-14C_ galactose is separated from 14C products on an anion exchanged column (AG X1-8, 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.

GT specific activity (mU/ml, protein) plasmid PH05 promoter high Pi low Pi 1 pDP34 -- <0.01 n.d. 1) pDPWA8 phosphate regulated 0.1 0.6-1 pl)PGTB5 constitutive 0.6 n.d. 1) 1) not determined 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 I I increased by the addition of 1 - 2 % Triton X-100 to the enzyme assay. Both findings 1 suggest that recombinant GT is membrane bound in yeast cells, as it is in HeLa cells.

tn.) Table 3: Distribution of GT activity during fractionation of BT 150/pDPGT5 cells GT activity cpin UDP-14C-Gal incorporated Fraction in GlcNAc crude extract 19400 400 g pellet 1000 20000 a pellet 15800 Z:, 20000 supernatant 5000 total: 21800 4.6% 72.5% 22.9% 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 HC1 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 gra filtered and passed over a GIcNAc-p-aminophenyi- 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 mi/min at 4'C. The enzyme is eluted with 50 mM Tris-HC1 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: 1 ml). These fractions are pooled and dialyzed against 2x 11 of 50 mM Tris-HC1 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 p4AD 113 (Example 1) by digestion with EcoRI. The 1 1.2 kb fragment containing the complete GT cDNA is isolated and partially digested with MvnI ffloehringer) cutting the GT sequence at positions 43, 55, 140 and 288 of the C C nucleotide sequence depicted in SEQ ID NO. 1. When digesting 0.2 pin of the EcoRl 1 -- -- EcoRI 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 MviiI-EcoRI fragment.

(b) Vector for amplification in E. coli Plasmid pUC18 (Ph.--.macia) is digested with BamHI and EcoRI in the multiple cloning 0 Z-2 site. Then the plasmid is treated with alkaline phosphatase as described in the Maniatis manual (supra), subjected to agarose acl clectrophoresis and isolated from the eel as a 2.7 kb DNA fragment.

(c) Constitutive PH05 (-173) promoter and SUC2 signal sequence The vector p3 1/PH05 (A73) RIT (Example 2) is digested with the restriction enzymes 0 BamHI and Xhol. A 0.25 kb, BamH1-XhoI fragment with the constitutive PH05 (A73) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is 1 - isolated. Then the fragment is recut with Hgral (BioLabs). The HgaI 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 BarnHI and HgaI t> W 0 cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the 1'n 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) YCT GCA CTG GCT GGC CG Yand 5W GCC AGC CAC Tfor the complementary strand. The oligo- 1 nucleotides are annealed to each other by first heating to 95T and then slowly cooling to 200C. The annealed adaptor is stored frozen.

(e) Construction of plasmid psGT For liyation, 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 Z 1 molar ratio of 1: 2: 2: 30-100. Lication is carried out in 12 gl of ligase buffer (66 mM Tris-HCI pH 7.5, 1 mM dithioerythritol, 5 mM MLYC12, 1 mM ATP) at 16'C f6r 18 hours. The ligation mix is used to transform competent cells of E. coli strain DH5oc 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, PstI, EcoRI, Xhol, also in combination). A single clone with the expected restriction pattern is referred to as psGT.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide with the cDNA coding for soluble GT is confirmed for plasmid psGT by using the 1 1 T7-Sequencing kit (Pharmacia) and primer 5'AGTCGAGGTTAGTATGGC Tstarting at position -77 in the constitutive PH05 (-173) promoter.

Sequence for MvnI DNAl): 5' AAA ATA Tct gca ctg gct g9c cgC GAC CTG AGC 31 31 TTT TAT AGA CGT gac cga ccg gcG CAG GAC TCG 51 Protein: Lys Ile Ser Ala I Leu Ala Gly Arg Asp Leu Ser 16 19 42 inv ss GT sequence cleavage site for signal endopeptidase 1) 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 Sall (BamHI) - EcoRI fragment. The expression cassette is still lacking the PH05 terminator sequences to be added in the following cloning step.

C Examffle 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 blunt end - SalI vector fragment.

(b) Expression cassette Plasmid psGT is first linearized by digestion with SaH (in the multiple cloning site) and 1 then partially digested with EcoRl. A 1.35 kb DNA fragment is isolated containing the constitutive PH05 (-173) promotor, a DNA sequence encoding the yeast invertasesignal peptide and the partial GT cDNA.

(c) PH05 terminator sequence The PH05 terminator sequence is isolated from plasmid p31 which is constructed starting 1 from plasmid p30 as described in EP 100561. After digestion with the restriction enzyme HindIII the protruding ends are filled up by Klenow polymerase treatment as described 1 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. col! strain DH5cc 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 1 transform S. cerevisiae strains BT 150 and H 449. Ural-transformants are isolated and screened for GT activity. In each case one transformant is selected and referred to as Saccharomyces cerevisiae BT 1501pl)PM and Saccharomyces cerevisiae H 449lpDPGTS, respectively. Employing the assay described in Example 5 GT-activity is 1 found in the culture broths of both transformants.

Example 10: Cloning of the sialyltransferase (ST) cDNA from human Hej)G2 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.

Table 5: PCR-primers corresponding to bp primer sequence (Y to Y)1) in ST cDNA2) PstI/EcoRI SIA1 ccrctgcagaattcaaaATGATTCACACCAACCTGAAGAAAAAGT 1 28 BamHI SIA3 ú_e-atCCTGTGCTTAGCAGTGAATGGTCCGGAAGCC 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 NnXl7247).

HepG2 ST cDNA can be amplified as one DNA fragment of 1.2 kb using the primers c SIA l and SIA3. PCR is performed as described for GT cDNA under slightly modified cycling conditions: 0.5 min denaturing at 95T, 1 min. 15 sec annealing at 56T, and 1 min 30 sec extension at 72'C, for a total of 25-35 cycles. In the last cycle, primer extension at 72T is carried out for 5 min.

After PCR amplification, the 1.2 kb fragment is digested with the restriction enzymes BamHI and PstI, analysed on a 1.2 % agarose -cl, eluted from the gel and subcloned into 1 1 1 the vector pUC18. The resulting subclone is designated pSIA2.

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

Plasmid pSIA2 is first linearized by di,( gestion with the restriction enzyme BamHI and subsequently partially digested with EcoRL 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 EcoRl using 1 gg of DNA and 1 0 0.25 U EcoRI (I h, 37'Q. 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 EcoRl restriction site. Three adenosine phosphates (see PCR primer SIA 1) provide an 'A' 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 TAX and 5 bp of the 3' untranslated region of the gene are followed by the BamHI site.

The 1.2 kb EcoRI-BainHI ST cDNA fragment is ligated to the 0.45 kb SaflEcoRI fragment containing the constitutive PH05 (A73) promoter (Example 2. 2(b)) and a 3.5 kb BamHI-SalI 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 DH5oc 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 P1105 (A73) promotor as a 2.0 kb SalMindIII fragment, referred to as DNA fragment (IC).

Example 12: Expression of ST in yeast Vector pDP34 (cf. EP 340 170) is digested with the restriction enzyme BamHl. 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.

C.

After ethanol precipitation the plasmid is digested with Sall and subjected to gel D electrophoresis on a 0.8 % agarose eel. The (BamHI) blunt end-SalI cut vector pDP34 is isolated as an 11.8 kb DNA fragment with the GENECLEAN kit.

Plasmid pST2 is digested with the restriction enzyme HindIII and in analogy to the C c preparation of the vector part filled in at the HindIII site by Klenow polymerase treatment. The product is subjected to SalI digestion, resulting in a 2.0 kb (HindIII) blunt end - SalI 1 1 fragment comprising the constitutive ST expression cassette (2Q.

c -- Lisration of 80 no, of the pDP34 vector with 40 ng of fragment 2C and transformation of 0 W -- competent cells of E. coli strain DH5oc is performed as described in Example 2. One clone showing the expected restriction pattern is chosen and referred to as pDPST5.

C1 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 - of plasmid DNA All according to the lithium-acetate transformation method (Ito, H. et al, supra).

Z Ural-transformants are selected and screened for ST activity. In each case, one positive transformant is selected and referred to as Saccharomyces 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 Saccharomyces cerevisiae BT 150/pDPST5 are grown under uracil selection in CP yeast minimal media supplemented with histidine and leucine. Exponentionally growing cells (at OD578 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 OD578- Mechanical breakage of the cells is effected by vigorous 4:1 Z) Z:' shaking on a vortex mixer with -lass 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. After termination of the reaction by acid precipitation the precipitate is filtered using glass fiber filters (Whatman GFA), 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 gI cell extract corresponding to approximately 0.5 ing protein, 3 gl imidazol buffer 50 mMol/l, pH 7.0; 50 nMol CMP-N-acetylneuraminic acid (Sigma) to which CMp-3H-Nacetylneuraminic acid (Amersham) is added to give a final specific activity of 7.3 Ci/mol, and 75 tg asialo-fetuin (prepared by acid hydrolysis using 0. 1 M H2SO4 at 80'C for 60 min, followed by neutralization, dialysis and Iyophilization).

ST-activity is found in the crude extracts prepared from S. cerevisiae BT 150/pDPST5 and H 449/pIDPST5 cells.

Example 14: Construction of an expression cassette for soluble ST-(Lys39Cys4061 The soluble ST designated ST(LYS39-CYS406) is an N-terminally truncated variant and g consists of 368 amino acids (SEQ ID NO. 4).

- 322 - (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 dioCsted with BamHI and EcoRI in the multiple cloning In t> 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) RIT (Example 2) is digested with the restriction enzymes BarnHI and XhoL A 0.25 kb, BamHI-Xhol fragment with the constitutive PHOS (- 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 IlgaI recognition sequence is on the antisense DNA strand. The restriction enzyme cuts upstream of the recognition sequence in such a way that the 5 stagg red end of the antisense strand coincides with the gge end of the coding sequence of the invertase signal sequence. The 0.24 kb BarnHI and Hgal cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence.

t (d) Adaptor Fragment (a) is linked to fragment (c) by means of an adaptor sequence which is prepared 0 - from equimolar amounts of the synthetic oligonucleotides 0 5'CTGCAAAA'fTGCAAACCAAGG Yand 5'AA'ITCCTrGGMGCAAM Yfor the complementary strand. The oligonucleotides are annealed to each other by first heating to 950C and then slowly cooling to WC, The annealed adaptor is stored frozen.

lb (e) Construction of plasmid psST For ligati6n, 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 1 0 molar ratio of 1: 2: 2: 30-100. Ligation is carried out in 12 gI of ligase buffer (66 mM Tris-HCI pH 7.5, 1 mM dithioerythritol, 5 mM Moú12, 1 mM ATP) at WC for 18 hours. The ligation mix is used to transform competent cells of E. coli strain DH5(x as described above. Minipreparations of plasmid are performed from 24 independent transformants. A single clone showing the expected restriction pattern after characterisation by restriction W t analysis using four different enzymes (BamHl, PstI, EcoRI, XhoI, also in combination) is - 3 3 - referred to as psST.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide In Z with the cDNA coding for soluble ST(LyS39-CYS406) is confirmed for plasmid psST by 0 using, the'17-Sequencino, kit (Pharmacia) and primer 5'ACGAGGTTAATGGC 3'startino, D 'D 1 1 1 at position -77 in the constitutive PHOS (A73) promoter.

Sequenc,a for DNAU: 51 AAA ATA Tct gca aaa ttg caa acc aag gAA 31 31 TTT TAT AGA GTG ttt aac gtt tgg ttc ctt 5' Protein: Lys Ile Ser Ala I Lys Leu Gln Thr Lys Glu 16 19 39 inv ss ST sequence cleavage site for signal endopeptidase 1) Small letters represent the adaptor sequence.

The expression cassette for secreted ST containing the constitutive PH05 (-173) promoter, 1 - the DNA sequence encoding invertase signal peptide and the partial ST cDNA from 0 -1 plasmid psST can be excised as a 1.35 kb SalI (BamHI) - EcoRI fragment. The expression cassette is still lacking the PH05 terminator sequences to be added in the following cloning stop.

Example 15: Construction of the expression vector pDPSTS For construction of the expression vector for soluble ST(LYS39-CYS406) the following tp fragments are combined:

(a) Vector part Plasmid pDP34 is digested and pretreated as described in Example 3 resulting in a 11.8 kb l> C5 blunt end - SalI vector fragment.

(b) Expression cassette Plasmid psST is first linearized by digestion with Sall (in the multiple cloning site) and 11 then partially digested with EcoRl. A 1.35 kb DNA fragment is isolated containing the I Z' Z5 constitutive 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 HindIll the protruding ends are filled up by Klenow polymerase treatment as described p above. Then the plasmid is digested with EcoRI and a 0.39 kb blunt end EcoRI fragment 1 1 with the PH05 terminator sequences is isolated.

Ligation of fra gments (a), (b) and (c) and transformation of competent cells of E. coli strain DH5ec 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 yeas 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. Ural-transformants are isolated and screened for ST activity. In each case one transformant is selected and referred to as Saccharomyces cerevisiae BT 150/pDPSTS and Saccharomyces cerevisiae H 449/pDPSTS. Using the assay described in Example 13 STactivity is found in the 1 culture broths of both transformants.

Example 17: Construction of an expression cassette for soluble ST(LyS27:S 61 The soluble ST designated ST(LYS27-CYS406) is an N-terminally truncated variant containin-the 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 di gested with EcoRI and a 1. 1 kb EcoRI-EcoRI fra-ment is isolated.

(b) Vector for amplification in E. col! Plasmid pUC18 (Pharmacia) is digested with BamHI and EcoRI in the multiple cloning 0 J:P 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 1 -1 2.7 kb DNA fragment.

(c) Constitutive PH05 (A73) promoter and SUC2 signal sequence 0 The vector p31/PH05 (-173) RIT (Example 2) is digested with the restriction enzymes 1 BamHI and XhoL A 0.25 kb BamHI-XlioI fragment with the constitutive PH05 (-173) promoter and the adjacent coding sequence for the invertase signal sequence (inv ss) is 0 1 isolated. Then the fragment is recut with Heal (BioLabs). The HgaI recognition sequence ..5 0 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 1 40 end of the coding sequence of the invertase signal sequence. The 0.24 kb BamHI and Heal 0 0 0 cut DNA fragment contains the constitutive PH05 (-173) promoter sequence linked to the yeast invertase signal sequence.

1 (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 5'CTGCAAAGGAAAAGAAGAAAGGGAGTrACTATGAITCCMAAATTGCAAA CCAAGG T, and 5'AATFCC"fTG7TGCAAMAAAGGAATCATAGTAACTCCCMCTTCTI-IT CC7T 3' for the complementary strand. The oligonucleotides are annealed to each other by first heating to WC and then slowly cooling to 20'C. The annealed adaptor is stored frozen.

(c) Construction of plasmid psSTI, For ligation, linearized vector (b), the STeDNA 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. Liaation is carried out in 12 gl of ligase buffer (66 mM b W Tris-HCI pH 7.5, 1 mM dithioerythritol, 5 mM MgC12, 1 mM ATP) at 16T for 18 hours.

The ligatiOn mix is used to transform competent cells of E. coli strain DH5cx as described - above. Minipreparations of plasmid are performed from 24 independent transformants. A single clone showing the expected restriction pattern after characterisation by restriction 0 c analysis using four different enzymes (BamHI, PstI, EcoRI, XhoI, also in combination) is referred to as psSTI.

The correct sequence at the fusion site of the sequence encoding invertase signal peptide with the cDNA coding for soluble ST(LYS27-CYS406) is confirmed for plasmid psST1 by 1 using the T7-Sequencino, kit (Pharmacia) and primer YAGWGAGTAGTATGGC 3' Z starting at position -7 7 in the constitutive PH05 (-173) promoter.

0 sequence for DNAO: 51 AAA ATA Tct gca aag gaa aag aag aaa ggg 3' 3' TTT TAT AGA GTG ttc ctt ttc ttc ttt ccc 51 Protein: Lys Ile Ser Ala I Lys Glu Lys Lys Lys Gly 16 19 27 inv ss ST sequenc cleavage site for signal endopeptidase 1) Small letters represent the adaptor sequence.

The expression cassette for secreted ST(Lys27-CYS406) containing the constitutive PH05 (-173) promoter, the DNA sequence encoding invertase signal peptide and the partial 1 -- ST cDNA from Plasmid psST1 can be excised as a 1.35 kb SalI (BamHl) - EcoRI fragment. The expression cassette is still lacking the PH05 terminator sequences to be 1 1 added in the following cloning step.

Example 18: Construction of the expression vector pDPSTS 1 For construction of the expression vector for soluble ST(LYS27-CYS406) the following fragments are combined:

1 (a) Vector part Plasmid pDP34 is digested and pretreated as described in Example 3 resulting in a 11.8 kb it) - blunt end'- SalI vector fragment.

(b) Expression cassette Plasmid psST1 is first linearized by dig stion with Sall (in the multiple cloning site) and Ole 11.1 then partially digested with EcoRl. 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.

(c) PH05 terminator sequence The PH05 terminator sequence is isolated from plasmid p31 which is constructed starting Z:1 from plasmid p30 as -described in EP 100561. After digestion with the restriction enzyme HindIll the protruding ends are filled up by Klenow polymerase treatment as described 1 above. Then the plasmid is digested with EcoRI and a 0.39 kb blunt end - EcoRI fragment t> C with the PH05 terminator sequences is isolated.

Ligation of fragments (a), (b) and (c) and transformation of competent cells of E. coli strain DH5(x 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 1.

Example 19: Expression of soluble ST(LVS27!CY-S406)-i-n-Y-CaSt In analogy to Example 4, CsCl-purified DNA of the expression vector pDPSTS l is used to transform S. cerevisiae strains BT 150 and H 449. Ural-transform ants are isolated and screened for ST activity. In each case one transfonnant is selected and referred to as Saccharomyces cerevisiae BT 150/pl)PSTS l and Saccharomyces cerevisiae H 449/pl)PSTS1. Using the assay described in Example 13, ST-activity is found in the It) 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 (X(1-3)1 1 fucosyltransferase the following oligonucleotide primers are designed to amplify a DNA I I I fragment encompassing the open reading frame of the ELFI'cDNA by PCR technology:

Z) 5'-CAGCGCTGCCTGTrCGCGCCAT-3'(ELFr-lB) and 5'-GGAGATGCACAGTAGAGGATCA3'(ELFr-2B). ELFT- I B 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% with the following cycle:

DMSO and 2 MM M,,,C'2 951C 5 min, 25x (I min 951C, I min 600C, 1.5 min721C), lOx (I min 951C, I min 601C, 1.5 min + 15 sec/cycle 720C). A(Yarose 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 I purified using, a Gene-Clean kit (Bio 10 1) and is subcloned into the pCR 1000 vector (Invitrogen). A single clone with the correct 1.3 kb insert is selected and referred to as Z) 11;1 BRB.ELFT/pCR100()-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 FT(Ar 627Are 051 in yeas j Soluble FT(Arto,'62-Ar. 05) is expressed from the FT cDNA starting at nucleotide position 64 241 (NruI restriction site) omitting the N-terminal region coding- for the cytoplasmic tail W CY and the membrane spanning domain (see sequence ID NO. 5).

Plasmid BRB.ELFT/pCRIOOO-13 is digested with HindIII, which cuts in the multicloning region 3' of the FT cDNA insert. The sticky ends are converted I.P to blunt ends in a reaction with Klenow DNA polymerase. XhoI linker (5' CCTCGAGG 3', Biolabs) are kinased, annealed and lioated to the blunt ends of the plasmid, using a 100-fold molar excess of In 11) linkers. Unreacted linkers are removed by isopropanol precipitation of the DNA, which is further di(rested with XhoI and Nrul (cleavagge at nucleotide position 240 of the FT cDNA I=> according to Sequence ID NO. 5). The 1.1 kb NruI-Xhol fragment (a) contains the FT I'D ZD cDNA sequence lacking the region which codes for the cytoplasmic tail and the membrane-spannina, domain up to amino acid 61.

Plasmids p31 RITI2 and p3 1/PH05(A73)RIT (see Example 2) are each digested with SalI and XhoL The 0.9 kb and 0.5 kb fra,(,,.ments, respectively, are isolated and cut with HgaL Z The resulting sticky ends are filled by Klenow DNA polymerase. The created blunt ends 0 coincide with the 3' end of the coding sequence for the yeast invertase signal sequence.

b Cb Subsequent BamHI cleavagge releases a 596 bp Baniffi-blunt end fragement (b) which comprises the inducible PH05 promoter and the invertase signal sequence with its own 1 ATG or a 234 bp BamHl-blunt end fracyment (c) comprising the short, constitutive 0 0 PH05(A73) promoter and the invertase signal sequence.

1 Plasmid p31RIT12 is linearized with restriction endonuclease SaIL Partial HindlIl digestion in the presence of ethidiumbromide results in a I kb Sall-Hindlll fragment comprising the 276 bp Sall-BamHI pBR322 sequence, the 534 bp promoter of the yeast I acid phosphatase PH05, the yeast invertase signal sequence (coding for 19 amino acids) I I=) and the PH05 transcriptional terminator. The 1 kb SalPHindIII fragment of p31RIT12 is Cp cloned into the yeast-Ecoli shuttle vector pW207 (Beggs, J.D. in: Molecular Genetics in yeast, Alfred Ben-on Symposium 16, Copenhagen, 1981, pp. 383-389), which has been cut with SalI and HindHI. The resulting plasmid containing the 1 kb insert is referred to as pJDB207/PH05-RIT12.

Plasmid pJDB207/PH05-RIT12 is digested with BamW and XhoI and the large, 6.8 kb c) 1 BamHI-Xh-)I fragment (d) is isolated. This fragment contains all the pJ1)B207 vector sequences and the PH05 transcriptional terminator.

The 596 bp B amHl-blunt end fragment (b), the 1. 1 kb Nrul-XhoI fagment (a) and the 6.8 I kb XhoI-BamHI vector fragment (d) are ligated using standard conditions for blunt end 1).D 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/PH05-1-FT. Ligation of DNA fragments (c), (a) and (d) leads to expression plasmid pJDB207/PH05(-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 (x(1-3)fucosyltransferase which s expressed under the control of the inducible PH05 or the constitutive PH05(-173) promoter, respectively. The expression cassettes are cloned into the yeast-E. coli shuttle vector pJDB207 between the BamHI and HindIII restriction sites. The nucleotide sequence at the site of the fusion between the invertase signal sequence and cDNA codino, for soluble FT is confirmed by DNA sequencing on double stranded 4D 11 plasmid DNA using the primer 5' AGTCGAGGTTAGTATGGC 3' representing the I nucleotide sequence at position -77 to -60 of the PH05, as well as the PH05(-173) promoter. The correctjunction is:

5' AAA ATA TCT GCA CGA CCG GTG 3' 3' TTT'TAT AGA CGT GCT GGC CAC 5' Lys Ile Ser Ala Arg Pro Val 19 62 Inv. ss FT cleavage site for signal peptidase Example 22: Construction of plasmids for inducible and constitutive expression of membrane-bound FT in yeast These constructs use the coding sequence of the FT cDNA with its own ATG. The 0 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 0 0 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 bp in FT cDNA primer sequence (5' to Y)1) FT1 cgagaattcataATGGGGGCACCGTGGGGC 58 to 75 FT2 ccggaGAGCGCGGCTTCACCGCTCG 1285 to 1266 1) Capital letters represent nucIeotides from FT, small letters are additional new sequences, restriction sites are underlined, "starC and "stop" codons are highlighted.

0 Standard PCR conditions are used to amplify the FT cDNA with primers FIl and FT2 (see Table 6) in 30 cycles of DNA synthesis (Taq DNA polymerase, Perkin- Elmer, 5 U/gl, I min at 721Q, denaturation (10 sec at 930C) and annealing (40 sec at 601Q. The resultinli, 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 reparative 4% Nusieve 3:1 agarose (FMC BioProducts, Rockland, ME, USA) oel in P CI) tris-borate buffer PH 8.3, ael-eluted and ethanol precipitated. Frag!zment (e) comprises the 5' part of the FT gene codin for amino acids 1 to 6 1. The DNA has a 5' extension with I the EcoRI site as in PCR primer FTL Plasmids p3 IRITI2 and p3 11PH05(-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, cluted and purified. The 4.1 kb XhoI- EcoRI fragment (f) comprises the Z7 0 pBR322-derived vector, the 534 bp PH05 promoter (Y EcoRI site) and the 131 bp PH05 transcriptional terminator (Y XhoI site). The 3.7 kb XhoPEcoRI fragment (g) only differs t) by the short, constitutive, 172 bp PH05(-17-3) promoter (Y EcoRI site) instead of the full length PH05 promoter.

The 191 bp EcoRI-NruI fragment (e), the 1.1 kb NruI-XhoI fragment (a) and the 4.1 kb iz 11:1 XhoI-EcoRI fragment (f) are ligated. A I [tl aliquot of the ligation mix is used to transform c,)mpetent E. coli HB 10 1 cells. Plasmid DNA from ampicillin resistant colonies is analysed. PlasMid DNA from a single clone is referred to as p31R/PH05-ssFT.

I Ligation of DNA fragments (c), (a) and (g) leads to plasmid p31R/PH05(A73)-ssFT.

1:1 1 These plasmids comprise the coding sequence of the membrane-bound FT under the 0 control of the inducible PH05 or the constitutive PH05(-173) promoter, respectively.

Example 23: Cloning of the FT expression cassettes into pDP34:

Plasmids p31R/PH05-ssFT and p31R/PH05(-173)-ssFT are digested with HindIII, which 1 cuts 3' of the PH05 transcriptional terminator. After a reaction with Klenow DNA polymerase, the DNA is digested with SalL The 2.3.kb and 1.9 kb Sallblunt end fragments, respectively, are isolated.

Plasmids pJDB207/PH05-1-FT and pJDB207/PH05(-173)-I-FT are partially digested with HindIII in the presence of 0. 1 m,-/ml of ethidium bromide (to avoid cleavage at an 1 additional HindIII site in the invertase signal sequence) and then treated with Klenow 1 DNA polymcrase and SalI as above. The 2.1 kb and 1.8 kb fragments, respectively, are isolated.

The four DNA fragments are each lia gated to the 11.8 kb SaII-blunt end vector fragment of pDP34 (sde 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/PH05-I-FT; pDP34/PH05(-173)-I-FT; pW34RIPH05- ssFT and pDP34R/PH05(-173)-ssFT.

S. cerevisiae strains BT150 and H449 are transformed with 5 g. each of the four expression plasmids (above) according to Example 4. Single transformed yeast colonies are selected and referred to as Saccharomyces cerevisiae 11 BT1501pDP34/PH05-PFT; BT150lpDP34/PH05(-173)-I-FT; BT150/pDP34RIPH05-ssFT; BT1501pDH4RIPH05(A73)-ssFT; H4491pDP34/PH05-I-FT; H4491pDP34/PH05(473)-I- FT; H4491pl)P34R/PH05-ssFT; H4491pDH4RIPH05(A73)-ssFT 1, 29 11 12 21 I@ 11 11 11 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 BT1501pDP34RIPH05-ssFT, BT150/pDP34RIPH05(-173)-ssFT, H449/pDP34R/PHOSssFT and H4491pl)P34R/PH05(-173)-ssFT, and in the culture broth of strains H4491pM4/PH05-I-FT, H449/pDP34/PH05(A73)-I-FT, BT1501pl)P34/PH05- 1-FT and BT150lpDP34/PH05(-173)I-Fr.

Deposition of microorganisms The following microorganism strains were deposited with the Deutsche SammlunI-g von Mikroorganismen (DSM), Mascheroder Wet, 16, D-3300 Braunschweig (deposition dates I C, and accession numbers given): Escherichia coli JM109/pDP34: March 14,1988; DSM 4473 Escherichia coli HB 10 I/p3O: October 23, 1987; DSM 4297 Escherichia coli HB 10 I/p3 IR: December 19, 1988; DSM 5116 Saccharomyces cerevisiae H 449: February 18, 1988; DSM 4413 Saccharomyces cerevisiae BT 150: May 23, 1991; DSM 6530 Sequence listing SEQ ID NO. 1 SEQUENCE TYPE: Nucleotide with corresponding protein SEQUENCE LENGTH: 1265 bp STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: recombinant IMMEDIATE EXPERIMENTAL SOURCE: Plasmid p4AD 113 from E. coli DH5cc/p4AD 113 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 NotI site EcoRI site EcoRV site B (Y1II site c PROPERTIES: EcoRI-HindIll fragment from plasmid p4AD1 13 comprising HeLa cell cDNA coding for full-length galactosyltransferase (EC 2.4.1.22) GAATTC ATG AGG C= CGG GAG CW CTC CTG AGC GGC AGC Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser 10 39 GCC GCG ATG CCA GGC GCG TCC CTA CAG CGG GCC TGC CGC 78 Ala Ala Met Pro Gly Ala Ser Leu Gln Arg Ala Cys Arg 20 CTG CTC GTG GCC GTC TGC GCT CTG CAC CTT GGC GTC ACC Leu Leu Val Ala Val Cys Ala Leu His Leu Gly Val Thr 30 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 CTG GTC GGA GTC TCC ACA CCG CTG CAG GGC GGC 195 Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly 60 TCG AAC AGT GCC GCC GCC ATC GGG CAG TCC TCC GGG GAG 234 Ser Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu 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 85 GCC TCC TCC CAG CCG CGC CW GGT GGC GAC TCC AGC CCA 312 Ala Ser Ser G1n Pro Arg Pro Gly Gly Asp Ser Ser Pro 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 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 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 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 150 GCA AAG CAG AAC CCA AAT GTG AAG ATG GGC GGC CGC TAT 507 Ala Lys Gln Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 160 165 GCC CCC AGG GAC TGC GTC TCT CCT CAC AAG GTG GCC ATC 546 Ala Pro Arg As) Cys Val Ser Pro His Lys Val Ala Ile 175 180 ATC ATT CCA TTC CGC AAC CGG CAG GAG CAC CTC AAG TAC 585 Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys Tyr 190 TGG CTA TAT TAT TTG CAC CCA GTC CTG CAG CGC CAG CAG 624 Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln 200 205 CTG GAC TAT GGC ATC TAT GTT ATC AAC CAG GCG GGA GAC 663 Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp 210 215 ACT ATA TTC AAT CGT GCT AAG CTC CTC AAT GTT GGC TTT 702 Thr Ile Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 220 225 230 CAA GAA GCC TTG AAG GAC TAT GAC TAC ACC TGC TTT GTG 741 Gln Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys Phe Val 235 240 245 TTT AGT GAC GTG GAC CTC ATT CCA ATG AAT GAC CAT AAT 780 Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn 250 255 GCG TAC AGG TGT TTT TCA CAG CCA CGG CAC ATT TCC GTT 819 Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val 260 265 270 GCA ATG GAT AAG TTT GGA TTC AGC CTA CCT TAT GTT CAG 858 Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln 275 280 TAT TTT GGA GGT GTC TCT GCT CTA AGT AAA CAA CAG TTT 897 Tyr Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe 285 290 295 CTA ACC ATC AAT GGA TTT CCT AAT AAT TAT TGG GGC TGG 936 Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp 300 305 310 GGA GGA GAA GAT GAT GAC ATT TTT AAC AGA TTA GTT TTT Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe 315 320 AGA GGC ATG TCT ATA TCT CGC CCA AAT GCT GTG GTC GGG 1014 Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 325 330 335 AGG TGT CGC ATG ATC CGC CAC TCA AGA GAC AAG AAA AAT 1053 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn 340 345 GAA CCC AAT CCT CAG AGG TTT GAC CGA ATT GCA CAC ACA 1092 Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr 350 355 360 AAG GAG ACA ATG CTC TCT GAT GGT TTG AAC TCA CTC ACC 1131 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 Gln Val Leu Asp Val Gln Arg Tyr Pro Leu Tyr Thr 380 385 CAA ATC ACA GTG GAC ATC GGG ACA CCG AGC TAGGACTTTT 1210 Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 390 395 GGTACAGGTA AAGACTGAAT TCATWATAT CTAGAT=G AG=GWAA AG= 1250 1265 SEO ID NO. 2 SEQUENCE TYPE: Protein SEQUENCE LENK 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 Leu Ser Arg Leu 5 Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly 15 20 Ser Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly GI 30 Leu Arg Thr Gly Gly Ala Arg Pro Pro Pro Pro Leu Gly 45 Ala Ser Ser Gln Pro Arg Pro Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr 70 Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 80 Ala Cy Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met 95 100 Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val 110 Ala Lys Gln Asn Pro Asn Val Lys Met Gly Gly Arg Tyr 120 125 Ala Pro Arg Asp Cys Val Ser Pro His Lys Val Ala I 135 le Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys Tyr 145 150 Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln 160 165 Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp 175 Thr Ile Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 185 190 Gln Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys Phe Val 200 Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn 205 210 215 Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val 220 225 230 Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln 235 240 Tyr Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe 245 250 255 Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp 260 265 Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe 270 275 280 Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 285 290 295 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn 300 305 Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr 310 315 320 Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr 325 330 Tyr Gln Val Leu Aso Val Gln Arg Tyr Pro Leu Tyr Thr 335 340 345 Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 350 355 SEQ ID NO. 3 SEQUENCE TYPE: Nucleotide with corresponding protein C7 SEQUENCE LENH: 1246 bp STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: recombinant IMMEDIATE EXPERIMENTAL SOURCE: Plasmid pSIA2 from E. coli DH5otIpSIA2 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 eDNA sequence coding for IlepG2 cell sialyltransferase PstI site EcoRI site EcoRI site BamHI site PROPERTIES: PstI-Barnffi fragment from plasmid pSIA2 comprising HepG2 cDNA In coding for full-length sialyltransferase (EC 2.4.99. 1) 1 CTGCAGAATT CAAA ATG ATT CAC ACC AAC CTG AAG AAA Met Ile His Thr Asn Leu Lys Lys 5 38 AAG TTC AGC TGC TGC GTC CTG GTC TTT CTT CTG TTT GCA 77 Lys Phe Ser Cys Cys Val Leu Val Phe Leu Leu Phe Ala 15 20 GTC ATC TGT GTG TGG AAG GAA AAG AAG AAA GGG AGT TAC 116 Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly Ser Tyr 30, TAT GAT TCC TTT AAA TTG CAA ACC AAG GAA TTC CAG GTG Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val 40 45 522 - TTA AAG AGT CTG GGG AAA TTG GCC ATG GGG TCT GAT TCC 194 Leu Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser 55 GO CAG TCT GTA TCC TCA AGC AGC ACC CAG GAC CCC CAC AGG 233 Gln Ser Val Ser Ser Ser Ser Thr Gln Asp Pro His Arg 70 GGC CGC CAG ACC CTC GGC AGT CTC AGA GGC CTA GCC AAG 272 Gly Arg Gln Thr Leu Gly Ser Leu Arg Gly Leu Ala Lys 80 85 GCC AAA CCA GAG GCC TCC TTC CAG GTG TGG AAC AAG GAC 311 Ala Lys Pro Glu Ala Ser Phe Gln Val Trp Asn Lys Asp 95 AGC TCT TCC AAA AAC CTT ATC CCT AGG CTG CAA AAG ATC 350 Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile 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 120 125 TAC AAG GGG CCA GGA CCA GGC ATC AAG TTC AGT GCA GAG 428 Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu 135 GCC CTC 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 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 160 TGG GAG GGT TAT CTG CCC AAG GAG AGC ATT AGG ACC AAG 545 Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr Lys 170 175 GCT GGG CCT TGG GGC AGG TGT GCT GTT GTG TCG TCA GCG 584 Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala 185 190 GGA TCT CTG AAG TCC TCC CAA CTA GGC AGA GAA ATC GAT 623 Gly Ser Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp 200 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 Gln Gln Asp Val Gly Thr Lys Thr Thr Ile 220 225 CGC CTG ATG AAC TCT CAG TTG GTT ACC ACA GAG AAG CGC 740 Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu Lys Arg 230 235 240 TTC CTC AAA GAC AGT TTG TAC AAT GAA GGA ATC CTA ATT 779 Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile 245 250 255 GTA TGG GAC CCA TCT GTA TAC CAC TCA dAT ATC CCA AAG 818 Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys 260 265 TGG TAC CAG AAT CCG GAT TAT AAT TTC TTT AAC AAC TAC 857 Trp Tyr Gln 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 Lys Leu His Pro Asn Gln Pro Phe Tyr 283 290 ATC CTC AAG CCC CAG ATG CCT TGG GAG CTA TGG GAC ATT 935 Ile Leu Lys Pro Gln Met Pro Trp Glu Leu Trp Asp Ile 295 300 305 CTT CAA GAA ATC TCC CCA GAA GAG ATT CAG CCA AAC CCC 974 Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln 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 Ile Ile Ile Met Met Thr 325 330 CTG TGT GAC CAG GTG GAT ATT TAT GAG TTC CTC CCA TCC 1052 Leu Cys Asp Gln Val Asp Ile 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 Gln 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 TArt GAG AAG AAT TTG GTG AAG CAT CTC AAC CAG GGC 11G9 Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln Gly 375 380 385 ACA GAT GAG GAC ATC TAC CTG CTT GGA AAA GCC ACA CTG 1208 Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu 390 395 CCT GGC TTC CGG ACC ATT CAC TGC TAAGCACAGG ATCC Pro Gly Phe Arg Thr Ile His Cys 400 405 1246 SEO ID NO. 4 SEQUENCE TYPE: Protein SEQUENCE LENII: 368 amino acids MOLECULE TYPE: C-terminal fragment of full-length sialfitransferase PROPERTIES: soluble sialyltransferase (EC 2.4.99. 1) from human HepG2 cells Lys Leu r--',ln Thr Lys Glu Phe Gln Val 5 Leu Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser 15 20 Gln Ser Val Ser Ser Ser Ser Thr Gln Asp Pro His Arg 30 35 Gly Arg Gln Thr Leu Gly Ser Leu Arg Gly Leu Ala Lys 45 Ala Lys Pro Glu Ala Ser Phe Gln Val Trp Asn Lys Asp 55 60 Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile 70 Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser 80 85 Tyr Lys Gly Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu 95 100 Ala Leu Arg Cys His Leu Arg Asp His Val Asn Val Ser 110 Met Val Glu Val Thr Asp Phe Pro Phe Asn Thr Ser Glu 120 125 Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr Lys 135 Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala 145 150 Gly Ser Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp 160 165 Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala Pro Thr 175 Ala Asn Phe Gln Gln Asp Val Gly Thr Lys Thr Thr Ile 185 190 Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu Lys Arg 200 Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile 205 210 215 Val Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys 220 225 230 Trp Tyr Gln Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr 235 240 Lys Thr Tyr Arg Lys Leu His Pro Asn Gln Pro Phe Tyr 245 250 255 Ile Leu Lys Pro Gln Met Pro Trp Glu Leu Trp Asp Ile 260 265 Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn Pro 270 275 280 Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met Thr 285 290 295 Leu Cys Asp Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser 300 305 Lys Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gln Lys Phe 310 315 320 Phe Asp Ser Ala Cys Thr Met Gly Ala Tyr His Pro Leu 325 330 Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln Gly 335 340 345 Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu 350 355 360 Pro Gly Phe Arg Thr Ile His Cys 365 SEQ ID NO.5 SEQUENCE TYPE: Nucleotide sequence with corresponding protein SEQUENCE LENGTH: 1400 bp STRANDEDNESS: double TOPOLOGY: linear IMMEDIATE EXPERIMENTAL SOURCE: BRB.ELFr/pCR1000-13 FEATURES:

* from 58 to 1272 bp cDNA sequence coding forhuman(x(1-3) fucosyltransferase from 238 to 243 bp NruI site PROPERTIES: HL60 cDNA coding for full-length (x(1-3) fucosyl transferase CG==CA WCCTGWGA CG=GWGA GCGGAGGCAG CGCTGCCTGT 50 TCGCGCC ATG GGG GCA CCG TGG GGC TCG CCG ACG GCG GCG Met Gly Ala Pro Trp Gly Ser Pro Thr Ala Ala 10 GCG GGC GGG CGG CGC GGG TGG CGC CGA GGC CGG GGG CTG Ala Gly Gly Arg Arg Gly Trp Arg Arg Gly Arg Gly Leu 20 CCA TGG ACC GTC TGT GTG CTG GCG GCC GCC GGC TTG ACG Pro Trp Thr Val Cys Val Leu Ala Ala Ala Gly Leu Thr 30 35 TGT ACG GCG CTG ATC ACC TAC GCT TGC TGG GGG CAG CTG Cys Thr Ala Leu Ile Thr Tyr Ala Cys Trp Gly Gln Leu 45 50 CCG CCG CTG CCC TGG GCG TCG CCA ACC CCG TCG CGA CCG Pro Pro Leu Pro Trp Ala Ser Pro Thr Pro Ser Arg Pro 60 129 168 207 246 GTG GGC GTG CTG CTG TGG TGG GAG CCC TTC GdG GGG CGC Val Gly Val Leu Leu Trp Trp Gilu Pro Phe Gly Gly Arg 70 75 GAT AGC GCC CCG AGG CCG CCC CCT GAC TGC CGG CTG CGC Asp Ser Ala Pro Arg Pro Pro Pro Asp Cys Arg Leu Arg 85 TTC AAC ATC AGC GGC TGC CGC CTG CTC ACC GAC CGC GCG Phe Asn Ile Ser Gly Cys Arg Leu Leu Thr Asp Arg Ala 95 100 TCC TAC GGA GAG GCT CAG GCC GTG CTT TTC CAC CAC CGC Ser Tyr Gly Glu Ala Gln Ala Val Leu Phe His His Arg 110 115 GAC CTC GTG AAG GGG CCC CCC GAC TGG CCC CCG CCC TGG Asp Leu Val Lys Gly Pro Pro Asp Trp Pro Pro Pro Trp 125 GGC ATC CAG GCG CAC ACT GCC GAG GAG GTG GAT CTG CGC Gly Ile Gln Ala His Thr Ala Glu Glu Val Asp Leu Arg 135 140 GTG TTG GAC TAC GAG GAG GCA GCG GCG GCG GCA GAA GCC Val Leu Asp Tyr Glu Glu Ala Ala Ala Ala Ala Glu Ala 150 CTG GCG ACC TCC AGC CCC AGG CCC CCG GGC CAG CGC TGG Leu Ala Thr Ser Ser Pro Arg Pro Pro Gly Gln Arg Trp 160 165 GTT TGG ATG AAC TTC GAG TCG CCC TCG CAC TCC CCG GGG Val Trp Met Asn Phe Glu Ser Pro Ser His Ser Pro Gly 175 180 285 324 363 402 480 519 558 597 1 CTG CGA AGC CTG GCA AGT AAC CTC TTC AAC TGG ACG CTC Leu Arg Ser Le-a Ala Ser Asn Leu Phe Asn Trp Thr Leu 190 TCC TAC CGG GCG GAC TCG GAC GTC TTT GTG CCT TAT GGC Ser Tyr Arg Ala Asp Ser Asp Val Phe Val Pro Tyr Gly 200 205 TAC CTC TAC CCC AGA AGC CAC CCC GGC GAC CCG CCC TCA 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 Gly Leu Ala Pro Pro Leu Ser Arg Lys Gln Gly Leu Val 220 225 230 GCA TGG GTG GTG AGC CAC TGG GAC GAG CGC CAG GCC CGG Ala Trp Val Val Ser His Trp Asp Glu Arg Gln Ala Arg 235 240 245 GTC CGC TAC TAC CAC CAA CTG AGC CAA CAT GTG ACC GTG Val Arg Tyr Tyr His Gln Leu Ser Gln His Val Thr Val 250 - 255 GAC GTG TTC GGC CGG GGC GGG CCG GGG CAG CCG GTG CCC Asp Val Phe Gly Arg Gly Gly Pro Gly Gln Pro Val Pro 260 265 270 GAA ATT GGG CTC CTG CAC ACA GTG GCC CGC TAC AAG TTC Glu Ile Gly Leu Leu His Thr Val Ala Arg Tyr Lys Phe 275 280 TAC CTG GCT TTC GAG AAC TCG CAG CAC CTG GAT TAT ATC Tyr Leu Ala Phe Glu Asn Ser Gln His Leu Asp Tyr Ile 285 290 295 636 675 71 792 831 870 909 948 ACC GAG AAG CTC TGG CGC AAC GCG TTG CTC GCT GGG GCG Thr Glu Lys Lea Trp Arg Asn Ala Leu Leu Ala Gly Ala 300 305 310 GTG CCG GTG GTG CTG GGC CCA GAC CGT GCC AAC TAC GAG Val Pro Val Val Leu Gly Pro Asp Arg Ala Asn Tyr Glu 315 320 987 1026 CGC TTT GTG CCC CGC GGC GCC TTC ATC CAC GTG GAC GAC 1065 Arg Phe Val Pro Arg Gly Ala Phe Ile His Val Asp Asp 325 330 335 TTC CCA AGT GCC TCC TCC CTG GCC TCG TAC CTG CTT TTC 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 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 Trp Arg Arg Ser Tyr Ala Val His Ile Thr Ser Phe Trp 365 370 375 1104 1182 GAC GAG CCT TGG TGC CGG GTG TGC CAG GCT GTA CAG AGG 1221 Asp Glu Pro Trp Cys Arg Val Cys Gln Ala Val Gln Arg 380 385 GCT GGG GAC CGG CCC AAG AGC ATA CGG AAC TTG GCC AGC 1260 Ala Gly Asp Arg Pro Lys Ser Ile Arg Asn Leu Ala Ser 390 395 400 TGG TTC GAG CGG TGAAGCCGCG CTCCCCTGGA AGWACCCAG Trp Phe Glu Arg 405 1302 WGAGGCCAA GTTGTCAWT =TGATCCT CTACT=CA TC=TTGAC 1352 TWWCATCA MGGAGTAAG =TTCAAAC ACCCATT= GCTCTATG 1400

Claims (19)

Claims:.

1. Process for the production of a membrane-bound mammalian glycosyltransferase selected from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a variant thereof, respectively, comprising culturing a yeast strain which has been 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.

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

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 IM DNA sequence coding for said variant which DNA sequence is controlled by said promoter, and recovering the enzymatic activity.

5. Process according to claim 1, wherein the glycosyltransferase is a galactosyltransferase.

6. Process according to claim 5, wherein the galactosyltransferase is selected from the group consisting of UDP-Galactose: 0-galactoside (x(13)-galactosyltransferase (EC 2.4.1.151) and UDP-Galactose: P-Nacetylglucosamine P(1-4)-galactosyltransferase (EC 2.4.1.22).

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 sequence depicted in SEQ ID NO. 2.

9. Process according to claim 1, wherein the glycosyltransferase is a sialyltransferase.

C

10. Process according to claim 9, wherein the sialyltransferase is CMPNeuAc 0-galactoside (x(2-6)-sialyltransferase (EC 2.4.99.1).

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

12. Process according to claim 9, wherein the sialyltransferase is designated ST(LyS27-CYS406) 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. Process according to claim 14, wherein the fucosyltransferase is selected from the group consisting of G1)P-Fucose:p-galactoside a(1-2)fucosyltransferase (EC 2.4.1.69) and GDP-Fucose:N-acetylglucosainine (x(13/4)-fucosyltransferase (EC 2.4.1.65).

16. Process according to claim 14, wherein the fucosyltransferase has the amino acid sequence depleted in the sequence listing with SEQ ID NO. 5.

17. Process according to claim 14, wherein the fucosyltransferase is designated FT(Arg62-Arg40.5) 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 DNA ' sequence coding for a membrane-bound mammalian glycosyltransferase selected from the group consisting of a galactosyltransferase, a sialyltransferase and a fucosyltransferase, or a variant thereof, respectively, which DNA sequence is controlled by said promoter.

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

FD 4.411)VC