AP411A - DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas. - Google Patents

Abstract

The present invention relates to a DNA molecule containing

Description

The present invention relates to a DNA molecule containing intron sequences and encoding a human protein which is, depending on the site of action, called Bile Salt-Stimulated Lipase (BSSL) or Carboxyl Ester Lipase (CEL). The DNA molecule is advantageously used in the production of recombinant human BSSL/CEL, preferably by means of production in transgenic nonhuman mammals. The recombinant human BSSL/CEL can be used as a constituent of infant formulas used for feeding infants as a substitute for human milk, or in the manufacture of medicaments against e.g. fat malabsorption, cystic fibrosis and chronic pancreatitis .

(56) Documents cited: WO 9115234 bad original $

PRIORITY DATA CONTINUED

2.	SE	9201826-6	12.06.92
3.	SE	9202088-2	03.07.92
4.	SE	9300902-5	19.03.93
INVENTORS	CONTINUED

2. PETER NILS IVAR CARLSSON Kommendorsgatan 30A

S-414 59 Goteborg SWEDEN

3. CURT SVEN MAGNUS ENERBACK Slatthultsliden 13A S-431 69 Mondal SWEDEN

4. STIG LENNART HANSSON Bjorkvagen 50 S-902 40 Umea SWEDEN

5. ULF FREDRIK PONTUS LIDBERG Teknologgatan 4

S-411 32 Goteborg SWEDEN

6. JEANETTE ANNIKA NILSSON Seagatan 16

S-413 14 Goteborg SWEDEN

7. JAN BIRGER FREDRIK TORNELL Klaveskarsgatan 56 S-421 59 Bastra Frolunda SWEDEN

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TECHNICAL FIELD

The present invention relates tc- a DNA molecule containing intron sequences and encoding a human protein which is, depending on the site of action, called Bile Salt-Stimulated Lipase (BSSL) or Carboxyl Ester Lipase (CEL). The DNA molecule is advantageously used in the production of recombinant human BSSL/CEL, preferably by means of production in transgenic nonhuman mammals. The recombinant human BSSL/CEL can be used as a constituent of infant formulas used for feeding infants as a substitute for human milk, or in the manufacture of medicaments against e.g. fat malabsorption, cystic fibrosis and chronic pancreatitis .

BACKGROUND OF THE INVENTION

Hydrolysis of dietary lipids

Dietary lipids are an important source of energy. The energy-rich triacylglycerols constitute more than 95% of these lipids. Some of the lipids, e.g. certain fatty acids and the fat-soluble vitamins, are essential dietary constituents. Before gastro-intestinal absorption the triacylglycerols as well as the minor components, i.e. esterified fat-soluble vitamins and cholesterol, and diacylphosphatidylglycerols, require hydrolysis of the ester bonds to give rise to less hydrophobic, absorbable products. These reactions are catalyzed by a specific group of enzymes called lipases .

In the human adult the essential lipases involved are considered to be Gastric Lipase, Pancreatic ColipaseDependent Lipase (hydrolysis of tri- and bad original $

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HX 1147 —2— diacylglycerols), Pancreatic Phospholipase A2 (hydrolysis of diacylphosphatidylglycerols) and Carboxylic Ester Lipase (CEL) (hydrolysis of cholesteryl- and fat soluble vitamin esters). In the breast-fed newborn, Bile Salt-Stimulated Lipas- (BSSL) plays an essential part in the hydrolysis of several of the above mentioned lipids. Together with bile salts the products of lipid digestion form mixed micelles from which absorption occurs.

Bile Salt-Stimulated Lipase

The human lactating mammary gland synthesizes and secretes with the milk a Bile Salt-Stimulated Lipase (BSSL) (Blackberg et al., 1987) that, after specific activation by primary bile salts, contributes to the breast-fed infant's endogenous capacity of intestinal fat digestion. This enzyme, which accounts for approximately 1% of total milk protein (Blackberg & Hernell, 1981), is not degraded during passage with the milk through the stomach, and in duodenal contents it is protected by bile salts from inactivation by pancreatic proteases such as trypsin and chymotrypsin. It is, however, inactivated when the milk is pasteurized, e.g. heated to 62.5°C, 30 min (Bjorksten et al., 1980) .

Model experiments in vitro suggest that the end products of triacylglycerol digestion are different in the presence of BSSL (Bernback et al., 1990; Hernell &

Blackberg, 1982). Due to lower intraluminal bile salt concentrations during the neonatal period this may be beneficial to product absorption.

Carboxylic Ester Lipase

The Carboxylic Ester Lipase (CEL) of human pancreatic juice (Lombardo et al., 1978) seems functionally to be identical, or at least very similar, to BSSL (Blackberg et al, 1981) . They also share common epitopes, have

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HX 1147 —3— identical N-terminal amino acid sequences (Abouakil et al., 1988) and are inhibited by inhibitors of serine esterases, e.g. eserine and diisopropylfluorophopsphate. In recent studies from several laboratories the cDNA structures from both the milk lipase and the pancreas lipase have been characterized (Baba et al., 1991; Hui et al., 1991; Nilsson et al., 1990; Reue et al., 1991) and the conclusion is that the milk enzyme and the pancreas enzyme are products of the same gene (in this application referred to as the CEL gene, EC 3.1.1.1). The cDNA sequence and deduced amino acid sequence of the CEL gene are described in WO 91/15234 (Oklahoma Medical Research Foundation) and in WO 91/18923 (Aktiebolaget Astra).

CEL is thus assumed to be identical to BSSL, and the polypeptide encoded by the CEL gene is in the present context called BSSL/CEL.

Lipid malabsorption

Common causes of lipid malabsorption, and hence malnutrition, are reduced intraluminal levels of Pancreatic Colipase-Dependent Lipase and/or bile salts. Typical examples of such lipase deficiency are patients suffering from cystic fibrosis, a common genetic disorder resulting in a life-long deficiency in 80% of the patients, and chronic pancreatitis, often due to chronic alcoholism.

The present treatment of patients suffering from a deficiency of pancreatic lipase is the oral administration of very large doses of a crude preparation of porcine pancreatic enzymes. However, Colipase-Dependent Pancreatic Lipase is inactivated by the low pH prevalent in the stomach. This effect cannot be completely overcome by the use of large doses of enzyme. Thus the large doses administered are

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HX 1147 —4 — inadequate for most patients, and moreover the preparations are impure and unpalatable.

Certain tablets have been formulated which pass through 5 the acid regions of the stomach and discharge the enzyme only in the relatively alkaline environment of the jejunum. However, many patients suffering from pancreatic disorders have an abnormally acid jejunum and in those cases the tablets may fail to discharge the enzyme.

Moreover, since the preparations presently on the market are of a non-human source there is a risk of immunoreactions that may cause harmful effects to the patients or result in reduced therapy efficiency. A further drawback with the present preparations is that their content of other lipolytic activities than Colipase-Dependent Lipase are not stated. In fact, most of them contain very low levels of BSSL/CEL-activity.

This may be one reason why many patients, suffering from cystic fibrosis in spite of supplementation therapy, suffer from deficiencies of fat soluble vitamins and essential fatty acids.

Thus, there is a great need for products with ( properties and structure derived from human lipases and with a broad substrate specificity, which products may be orally administered to patients suffering from deficiency of one or several of the pancreatic lipolytic enzymes. Products that can be derived from the use of the present invention fulfil this need by themselves, or in combination with preparations containing other lipases.

Infant formulas

It is well known that human milk-feeding is considered superior to formula-feeding for infants. Not only does human milk provide a well-balanced supply of nutrients, bad original J)

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-5but it is also easily digested by the infant. Thus, several biologically active components which are known tc have physiological functions in the infant are either a constituent of human milk or produced during the digestion thereof, including components involved in the defense against infection and components facilitating the uptake of nutrients from human milk.

In spite of the great efforts which have been invested 10 in preparing infant formulas, it has not been possible to produce a formula which to any substantial extent has the advantageous properties of human milk. Thus, infant formulas, often prepared on the basis of cow milk, is generally incompletely digested by the infant and is lacking substances known to have effect on the physiological functions of the infant. In order to obtain an infant formula with a nutritional value similar to human milk, a number of additives including protein fragments, vitamins, minerals etc., which are normally formed or taken up during the infant's digestion of human milk, are included in the formula with the consequent risk of posing an increased strain on and possible long-term damage of important organs such as liver and kidney. Another disadvantage associated with the use of cow milk-based formulas is the increased risk for inducing allergy in the infant against bovine proteins.

As an alternative to cow milk-based infant formulas, human milk obtainable from so-called milk banks has been used. However, feeding newborn infants with human milk from milk banks has in the recent years to an increasing extent been avoided, because of the fear for the presence of infective agents such as HIV and CMV in human milk. In order to destroy the infective agents in human milk it has become necessary to pasteurize the milk before use. However, by pasteurization the nutritional value and the biological effects of the

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-6— milk components are decreased, for example is BSSL inactivated, as mentioned above.

Addition of lipases to infant formulas

The pancreatic and liver functions are not fully developed at birth, most notably in infants born before term. Fat malabsorption, for physiological reasons, is a common finding and thought to result from low intraluminal Pancreatic Colipase-Dependent Lipase and bile salt concentrations. However, because of BSSL, such malabsorption is much less frequent in breast-fed infants than in infants fed pasteurized human milk or infant formulas (Bernback et al., 1990).

To avoid the above disadvantages associated with pasteurized milk and bovine milk-based infant formulas, it would thus be desirable to prepare an infant formula with a composition closer to that of human milk, i.e. a formula comprising human milk proteins.

BSSL/CEL has several unique properties that makes it ideally suited for supplementation of infant formulas:

• It has been designed by nature for oral administration. Thus, it resists passage through the stomach and is activated in contents of the small intestine.

• Its specific activation mechanism should prevent hazardous lipolysis of food or tissue lipids during storage and passage to its site of action.

• Due to its broad substrate specificity it has the potential to, on its own, mediate complete digestion of most dietary lipids, including the fat soluble vitamin esters.

• BSSL/CEL may be superior to Pancreatic ColipaseDependent Lipase to hydrolyze ester bonds containing long-chain polyunsaturated fatty acids.

• In the presence of Gastric Lipase and in the absence of, or at low levels of Colipase-Dependent

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Lipase, BSSL/CEL can ascertain a complete triacylglycerol digestion in vitro even if the bile salt levels are low such as in newborn infants. In the presence of BSSL/CEL the end products of triacylglycerol digestion become free fatty acids and free glycerol rather than free fatty acids and monoacylglycerol generated by the other two lipases (Bernback et al. , 1990). This may favour product absorption particularly when the intraluminal bile salt levels are low.

The utilization of BSSL/CEL for supplementation of infant formulas requires however access to large quantities of the product. Although human milk proteins may be purified directly from human milk, this is not a realistic and sufficiently economical way to obtain the large quantities needed for large scale formula production, and other methods must consequently be developed before an infant formula comprising human milk proteins may be prepared. The present invention provides such methods for preparation of BSSL/CEL in large quantities.

Production of proteins in milk of transgenic animals

The isolation of genes encoding pharmacologically active proteins has permitted cheaper production of such proteins in heterologous systems. An appealing expression system for milk proteins is the transgenic animal (For a review see Hennighausen et al., 19 90) .

Dietary compositions comprising bile salt-activated lipase derived from e.g. transgenic animal technology, is described in EP 317,355 (Oklahoma Medical Research Foundation).

In the transgenic animal, the protein coding sequence can be introduced as cDNA or as a genomic sequence. Since introns may be necessary for regulated gene expression in transgenic animals (Brinster et al.,

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1988; Whitelaw et al., 1991) it is in many cases preferable to use the genomic form rather than the cDNA form of the structural gene. WO 90/05188 (Pharmaceutical Proteins Limited) describes the use in 5 transgenic animals of protein-coding DNA comprising at least one, but not all, of the introns naturally occurring in a gene coding for the protein.

PURPOSE OF THE INVENTION

It is an object of the present invention to provide a means for producing recombinant human BSSL/CEL, in a high yield and at a realistic price, for use in infant formulas in order to avoid the disadvantages with pasteurized milk and formulas based on bovine proteins.

BRIEF DESCRIPTION OF THE INVENTION

The purpose of the invention has been achieved by cloning and sequencing the human CEL gene. In order to improve the yield of BSSL/CEL, the obtained DNA molecule containing intron sequences, instead of the known cDNA sequence, of the human CEL gene has been used for production of human BSSL/CEL in a transgenic non-human mammal.

Accordingly, in one aspect the present invention relates to a DNA molecule shown in the Sequence Listing as SEQ ID NO: 1, or an analogue of the said DNA molecule which hybridizes with the DNA molecule shown in the Sequence Listing as SEQ ID NO: 1, or a specific part thereof, under stringent hybridization conditions.

The procedure used for isolating the human BSSL/CEL DNA molecule is outlined in the Examples below.

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-9The stringent hybridization conditions referred to above are to be understood in their conventional meaning, i.e. that hybridization is carried out according to an ordinary laboratory manual such as

Sambrook et al. (1989).

In another aspect the present invention provides a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a non-human mammal so as to form a hybrid gene which is expressible in the mammary gland of an adult female of a mammal harbouring said hybrid gene so that human BSSL/CEL is produced when the hybrid gene is expressed.

In yet a further aspect, the present invention relates to a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL, comprising injecting a mammalian expression system as defined above into a fertilized egg or a cell of an embryo of a mammal so as to incorporate the expression system into the germline of the mammal and developing the resulting injected fertilized egg or embryo into an adult female mammal.

DETAILED DESCRIPTION OF THE INVENTION

The DNA molecule shown in the Sequence Listing as SEQ

ID NO: 1, which has an overall length of 11531 bp, has the following features:

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Feature	-10- from base	to b<
	5'-Flanking	region	1	1640
	TATA box		1611	1617
	Exon 1		1641	1727
5	Translation	start	1653	1653
	Exon 2		4071	4221
	Exon 3		4307	4429
	Exon 4		4707	4904
	Exon 5		6193	6323
10	Exon 6		6501	6608
	Exon 7		6751	6868
	Exon 8		8335	8521
	Exon 9		8719	8922
	Exon 10		10124	10321
15	Exon 11		10650	11490
	3'-Flanking	region	11491	11531

In the present context, the term gene is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5'-upstream or 3'-downstream region. The 5'upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter. The 3'-downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.

The DNA molecules of the invention explained herein may comprise natural as well as synthetic DNA sequences, the natural sequence typically being derived directly from genomic DNA, normally of mammalian origin, e.g. as described below. A synthetic sequence may be prepared

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-11by conventional methods for synthetically preparing DNA molecules. The DNA sequence may further be of mixed genomic and synthetic origin.

In a further aspect, the present invention relates to a replicable expression vector which carries and is capable of mediating the expression of a DNA sequence encoding human BSSL/CEL.

In the present context, the term “replicable means that the vector is able to replicate in a given type of host cell into which it has been introduced. Immediately upstream of the human BSSL/CEL DNA sequence there may be provided a sequence coding for a signal peptide, the presence of which ensures secretion of the human

BSSL/CEL expressed by host cells harbouring the vector. The signal sequence may be the one naturally associated with the human BSSL/CEL DNA sequence or of another origin.

The vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which ( exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromosome or virus. Alternatively, the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome (s) into which it has been integrated. Examples of suitable vectors are a bacterial expression vector and a yeast expression vector. The vector of the invention may carry any of the DNA molecules of the invention as defined above.

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-12The present invention further relates to a cell harbouring a replicable expression vector as defined above. In principle, this cell may be of any type of cell, i.e. a prokaryotic cell, a unicellular eukaryotic organism or a cell derived from a multicellular organism, e.g. a mammal. The mammalian cells are especially suitable for the purpose and are further discussed below.

In another important aspect, the invention relates to a method of producing recombinant human BSSL/CEL, in which a DNA sequence encoding human BSSL/CEL is inserted in a vector which is able to replicate in a specific host cell, the resulting recombinant vector is introduced into a host cell which is grown in or on an appropriate culture medium under appropriate conditions for expression of human BSSL/CEL and the human BSSL/CEL is recovered.

The medium used to grow the cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. The recombinant human BSSL/CEL expressed by the cells may be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.

If the human BSSL/CEL is produced intracellularly by the recombinant host, that is, is not secreted by the cell, it may be recovered by standard procedures comprising cell disrupture by mechanical means, e.g.

sonication or homogenization, or by enzymatic or chemical means followed by purification.

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In order to be secreted, the DNA sequence encoding human BSSL/CEL should be preceded by a sequence coding for a signal peptide, the presence of which ensures secretion of human BSSL/CEL from the cells so that at least a significant proportion cf the human BSSL/CEL expressed is secreted into the culture medium and recovered.

The presently preferred method of producing recombinant 10 human BSSL/CEL of the invention is by use of transgenic non-human mammals capable of excreting the human BSSL/CEL into their milk. The use of transgenic nonhuman mammals has the advantage that large yields of recombinant human BSSL/CEL are obtainable at reasonable costs and, especially when the non-human mammal is a cow, that the recombinant human BSSL/CEL is produced in milk which is the normal constituent of, e.g., infant formulae so that no extensive purification is needed when the recombinant human BSSL/CEL is to be used as a nutrient supplement in milk-based products.

Furthermore, production in a higher organism such as a non-human mammal normally leads to the correct processing of the mammalian protein, e.g. with respect to post-translational processing as discussed above and proper folding. Also large quantities of substantially f pure human BSSL/CEL may be obtained.

Accordingly, in a further important aspect, the present invention relates to a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a nonhuman mammal so as to form a hybrid gene which is expressible in the mammary gland of an adult female of a mammal harbouring said hybrid gene.

The DNA sequence encoding human BSSL/CEL is preferably a DNA sequence as shown in the Sequence Listing as SEQ

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-14ID NO: 1 or a genomic human BSSL/CEL gene or an analogue thereof .

The mammary gland as a tissue of expression and genes 5 encoding milk proteins are generally considered to be particularly suitable for use in the production of heterologous proteins in transgenic non-human mammals as milk proteins are naturally produced at high expression levels in the mammary gland. Also, milk is readily collected and available in large quantities. In the present connection the use of milk protein genes in the production of recombinant human BSSL/CEL has the further advantage that it is produced under conditions similar to the its natural production conditions in terms of regulation of expression and production location (the mammary gland).

In the present context the term hybrid gene denotes a DNA sequence comprising on the one hand a DNA sequence encoding human BSSL/CEL as defined above and on the other hand a DNA sequence of the milk protein gene which is capable of mediating the expression of the hybrid gene product. The term “gene encoding a milk protein denotes an entire gene as well as a subsequence thereof capable of mediating and targeting the expression of the hybrid gene to the tissue of interest, i.e. the mammary gland. Normally, said subsequence is one which at least harbours one or more of a promoter region, a transcriptional start site, 3' and 5' non-coding regions and structural sequences. The DNA sequence encoding human BSSL/CEL is preferably substantially free from prokaryotic sequences, such as vector sequences, which may be associated with the DNA sequence after, e.g., cloning thereof.

The hybrid gene is preferably formed by inserting in vitro the DNA sequence encoding human BSSL/CEL into the milk protein gene by use of techniques known in the bad original £

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HX 1147 —15— art. Alternatively, the DNA sequence encoding human BSSL/CEL can be inserted in vivo by homologous recombinant ion .

Normally, the DNA sequence encoding human BSSL/CEL will be inserted in one of the first exons of the milk protein gene of choice or an effective subsequence thereof comprising the first exons and preferably a substantial part of the 5' flanking sequence which is believed to be of regulatory importance.

The hybrid gene preferably comprises a sequence encoding a signal peptide so as to enable the hybrid gene product to be secreted correctly into the mammary gland. The signal peptide will typically be the one normally found in the milk protein gene in question or one associated with the DNA sequence encoding human BSSL/CEL. However, also other signal sequences capable of mediating the secretion of the hybrid gene product to the mammary gland are relevant. Of course, the various elements of the hybrid gene should be fused in such a manner as to allow for correct expression and processing of the gene product. Thus, normally the DNA sequence encoding the signal peptide of choice should be precisely fused to the N-terminal part of the DNA sequence encoding human BSSL/CEL. In the hybrid gene, the DNA sequence encoding human BSSL/CEL will normally comprise its stop codon, but not its own message cleavance and polyadenylation site. Downstream of the

DNA sequence encoding human BSSL/CEL, the mRNA processing sequences of the milk protein gene will normally be retained.

A number of factors are contemplated to be responsible for the actual expression level of a particular hybrid gene. The capability of the promoter as well of other regulatory sequences as mentioned above, the integration site of the expression system in the genome

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-16of the mammal, the integration site of the DNA sequence encoding human BSSL/CEL in the milk protein encoding gene, elements conferring post-transcriptional regulation and other similar factors may be of vital importance for the expression level obtained. On the basis of the knowledge of the various factors influencing the expression level of the hybrid gene, the person skilled in the art would know how to design an expression system useful for the present purpose.

A variety of different milk proteins are secreted by the mammary gland. Two main groups of milk proteins exist, namely the caseins and the whey proteins. The composition of milk from different species varies qualitatively as well as quantitatively with respect to these proteins. Most non-human mammals produces 3 different types of casein, namely a-casein, β-casein and κ-casein. The most common bovine whey proteins are α-lactalbumin and β-lactalbumin. The composition of milk of various origins are further disclosed in Clark et al. (1987) .

The milk protein gene to be used may be derived from the same species as the one in which the expression system is to be inserted, or it may be derived from another species. In this connection it has been shown that the regulatory elements that target gene expression to the mammary gland are functional across species boundaries, which may be due to a possible common ancestor (Hennighausen et al., 1990).

Examples of suitable genes encoding a milk protein or effective subsequences thereof to be used in the construction of an expression system of the invention are normally found among whey proteins of various mammalian origins, e.g. a whey acidic protein (WAP) gene, preferably of murine origin, and a βlactoglobulin gene, preferably of ovine origin. Also

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HX 1147 —17— casein genes of various origins may be found to be suitable for the transgenic production of human BSSL/CEL, e.g. bovine aSl-casein and rabbit β-casein. The presently preferred gene is a murine WAP gene as this has been found to be capable of providing a high level of expression of a number of foreign human proteins in milk of different transgenic animals (Hennighausen et al, 1990) .

Another sequence preferably associated with the expression system of the invention is a so-called expression stabilizing sequence capable of mediating high-level expression. Strong indications exist that such stabilizing sequences are found in the vicinity of and upstreams of milk protein genes.

The DNA sequence encoding human BSSL/CEL to be inserted in the expression system of the invention may be of genomic or synthetic origin or any combination thereof.

Some expression systems have been found to require the presence of introns and other regulatory regions in order to obtain a satisfactory expression (Hennighausen et al., 1990) . In some cases it may be advantageous to introduce genomic structures, rather than cDNA elements, as polypeptide encoding element in vector constructs (Brinster et al.). The intron and exon structure may result in higher steady state mRNA levels that obtained when cDNA based vectors are used.

In a further aspect, the present invention relates to a hybrid gene comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a non-human mammal, the DNA sequence being inserted in the milk protein gene in such a manner that it is expressible in the mammary gland of an adult female of a mammal harbouring the hybrid gene. The hybrid gene and its constituents have been discussed in detail above. The hybrid gene constitutes an important

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-18intermediate in the construction of an expression system of the invention as disclosed above.

In another aspect, the present invention relates to a 5 non-human mammalian cell harbouring an expression system as defined above. The mammalian cell is preferably an embryo cell or a pro-nucleus. The expression system is suitably inserted in the mammalian cell using a method as explained in the following and specifically illustrated in the Example below.

In a further important aspect, the present invention relates to a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL, comprising injecting an expression system of the invention as defined above into a fertilized egg or a cell of an embryo of a mammal so as to incorporate the expression system into the germline of the mammal and developing the resulting injected fertilized egg or embryo into an adult female mammal.

The incorporation of the expression system into the germline of the mammal may be performed using any suitable technique, e.g. as described in Manipulating the Mouse Embryo; A Laboratory Manual, Cold Spring ( Harbor Laboratory Press, 1986. For instance, a few hundred molecules of the expression system may be directly injected into a fertilized egg, e.g. a fertilized one cell egg or a pro-nucleus thereof, or an embryo of the mammal of choice and the microinjected eggs may then subsequently be transferred into the oviducts of pseudopregnant foster mothers and allowed to develop. Normally, not all of the injected eggs will develop into adult females expressing human

BSSL/CEL. Thus, about half of the mammals will from a statistically point of view be males from which, however, females can be bred in the following generations .

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-19Once integrated in the germ line, the DNA sequence encoding human BSSL/CEL may be expressed at high levels to produce a correctly processed and functional human BSSL/CEL in stable lines of the mammal in question.

Of further interest is a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL and substantially incapable of expressing BSSL/CEL from the mammal itself, comprising (a) destroying the mammalian BSSL/CEL expressing capability of the mammal so that substantially no mammalian BSSL/CEL is expressed and inserting an expression system of the invention as defined above or a DNA sequence encoding human BSSL/CEL into the germline of the mammal in such a manner that human BSSL/CEL is expressed in the mammal; and/or (b) replacing the mammalian BSSL/CEL gene or part thereof with an expression system of the invention as defined above or a DNA sequence encoding human BSSL/CEL.

The mammalian BSSL/CEL expressing capability is conveniently destroyed by introduction of mutations in the DNA sequence responsible for the expression of the BSSL/CEL. Such mutations may comprise mutations which make the DNA sequence out of frame, or introduction of a stop codon or a deletion of one or more nucleotides of the DNA sequence.

The mammalian BSSL/CEL gene or a part thereof may be replaced with an expression system as defined above or a DNA sequence encoding human BSSL/CEL by use of the well known principles of homologous recombination.

In a further aspect, the present invention relates to a transgenic non-human mammal prepared by a method as described above.

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-2 0While the transgenic non-human mammal of the invention in its broadest aspect is not restricted to any particular type of mammal, the mammal will normally be selected from the group consisting of mice, rats, rabbits, sheep, pigs, goats and cattle. For large scale production of human BSSL/CEL the larger animals such as sheep, goats, pigs and especially cattle are normally preferred due to their high milk production. However, also mice, rabbits and rats may be interesting due to the fact that the manipulation of these animals is more simple and results in transgenic animals more quickly than when, e.g. cattle, are concerned.

Also progeny of a transgenic mammal as defined above, capable of producing human BSSL/CEL is within the scope of the present invention.

In a further aspect the present invention includes milk from a non-human mammal comprising recombinant human

BSSL/CEL.

In a still further aspect, the present invention relates to an infant formula comprising recombinant human BSSL/CEL, in particular a polypeptide of the invention as defined above. The infant formula may be ( prepared by adding the recombinant human BSSL/CEL or polypeptide in a purified or partly purified form to the normal constituents of the infant formula. However, normally it is preferred that the infant formula is prepared from milk of the invention as defined above, especially when it is of bovine origin. The infant formula may be prepared using conventional procedures and contain any necessary additives such as minerals, vitamins etc.

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-21EXAMPLES

EXAMPLE 1: GENOMIC ORGANIZATION, SEQUENCE ANALYSIS AND CHROMOSOMAL LOCALIZATION OF THE CEL GENE

Standard molecular biology techniques were used (Maniatis et al., 1982 ; Ausubel et al., 1987; Sarrbrook et al., 1989) if nothing else is mentioned.

Isolation of Genomic Recombinants

Two different human genomic phage libraries, XDASH (Clcnentech Laboratories Inc., Palo Alto, Ca, USA, and XEMBL-3 SP6/T7 (Stratagene, La Jolla, CA, USA), were screened by plaque hybridization using various subcloned cDNA restriction fragments (Nilsson et al., 1990) as probes, labeled with [a-²²P]dCTP by the oligolabeling technique (Feinberg et al., 1983).

Mapping, Subcloning and Sequencing of Genomic Clones

Positive clones were digested with various restriction enzymes, electrophoresed on 1% agarose gels and then vacuumtransfered (Pharmacia LKB BTG, Uppsala, Sweden) to a nylon membrane. The membrane was hybridized with various cDNA probes. Restriction fragments, hybridizing with the probes, were isolated using the ( isotachophoreses method (Ofverstedt et al., 1984).

Smaller fragments, <800 bp, were directly inserted into M13mpl8, M13mpl9, M13BM20 or M13BM21 vectors and sequenced, using E. coli TGI as host bacteria, whereas larger fragments were subcloned into pTZ18R or pTZ19R vectors, using E. coli DH5a as host bacteria, and further digested. (The plasmids pS309, pS310 and pS451 used in Example 2 below were produced accordingly.)

Some of the isolated fragments were also used as probes in hybridizations. All of the nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using Klenow enzyme and either the M13 universal sequencing primer of specific

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-22oligonucleotides. Sequence information was retrieved from autoradiograms by the use of the software MS-EdSeq as described by Sjoberg et al. (1989). The sequences were analyzed using the programs obtained from the

UWGCG software package (Devereux et al., 1984) .

Primer Extension

Total RNA was isolated from human pancreas, lactating mammary gland and adipose tissue by the guanidinium isothiocyanate-CsCl procedure (Chirgwin et al. , 1979).

Primer extension was performed according to (Ausubel et al., 1987) using total RNA and an antisense 26-mer oligonucleotide (5'-AGGTGAGGCCCAACACAACCAGTTGC-3'), nt position 33-58. Hybridization of the primer with 20 gg of the total RNA was performed in 30 μΐ of 0.9 M NaCl, 0.15 M Hepes pH 7.5 and 0.3 M EDTA at 30°C overnight. After the extension reaction with reverse transcriptase, the extension products were analyzed by electrophoresis through a 6% denaturing polyacrylamide gel.

Somatic Cell Hybrids

DNA from 16 human-rodent somatic cell hybrid lines, obtained from NIGMS Human Genetic Mutant Cell

Repository (Coriell Institute for Medical Research,

Camden, NJ) were used for the chromosomal assignment of the CEL gene. Human-mouse somatic cell hybrids GM09925 through GM09940 were derived from fusions of fetal human male fibroblasts (IMR-91), with the thymidine kinase deficient mouse cell line B-82 (Taggart et al. , 1985; Mohandas et al., 1986). Hybrids GM10324 and GM02860 with the HPRT and APRT deficient mouse cell line A9 (Callen et al., 1986), while hybrid GM10611 resulted from a microcell fusion of the retroviral vector SP-1 infected human lymphoblast cell line

GM07890 with the Chinese hamster ovary line UV-135 (Warburton et al., 1990). Hybrid GM10095 was derived from the fusion of lymphocytes from a female with a

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HX 1147 —23— balanced 46,X,t(X;9)(ql3;34) karyotype with the Chinese hamster cell line CHW1102 (Mohandas et al., 1979). The human chromosome content of the hybrid lines, which was determined by cytogenetic analysis as well as by

Southern blot analysis and in situ hybridization analysis, are shown in Table 1. High molecular weight DNAs isolated from mouse, Chinese hamster and human parental cell line and the 16 hybrid cell lines were digested with FcoRI, fractionated in 0.8% agarose gels, and transferred to nylon filters. A [a-²²P]dCTP-labeled CEL cDNA probe (a full-length cDNA) was prepared by oligolabeling (Feinberg and Vogelstein, 1983) and hybridized to the filters. The filters were washed for 60 min each at 65°C in 6xSSC/0.5%SDS and in

2xSSC/0.5%SDS.

Polymerase Chain Reaction

Total human genomic DNA isolated from leukocytes, DNA from somatic cell hybrids and from some of the positive genomic recombinants and total RNA from human lactating mammary gland and human pancreas were amplified for exon 10 and exon 11. Two gg of DNA were used. The primers used are listed in Table 2. Thirty cycles of PCR were performed in 100 gl volume [10 mM Tris-HCl, pH

8.3, 50 mM KC1, 1.5 mM MgCl₂, 200 gM of each dNTP, 100 gg/ml gelatin, 100 pmol of each primer, 1.5 U Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT, USA)] and the annealing temperature 55°C for all the primer pairs. The RNA sequence was amplified by the use of combined complementary DNA (cDNA) and PCR methodologies. cDNA was synthesized from 10gg total RNA in 40 Ml of a solution containing 50 mM Tris-HCl, pH 8.3, 50 mM KC1, 10 mM MgCl₂, 10 gg/ml BSA, 1 mM of each dNTP, 500 ng of oligo(dt)i₂_iq» 40 U ribonuclease inhibitor, and 200 U reverse transcriptase (MoMuLV), (BRL, Bethesda Research Laborataries, N.Y., USA) for 30 min at 42°C. The cDNA was precipitated and resuspended in 25 gl H₂O; 2 gl of this was amplified, as described

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-24above. The amplified fragments were analyzed on a 2% agarose gel. Some of the fragments were further subcloned and sequenced.

Gene Structure of the Human CEL Gene

In each genomic library, 10^ recombinants were screened and the screenings yielded several positive clones, which were all isolated and mapped. Two clones, designated XBSSLl and XBSSL5A, were further analyzed.

Restriction enzyme digestions with several enzymes,

Southern blotting followed by hybridization with cDNA probes, indicated that the XBSSLSA clone covers the whole CEL gene and that the XBSSLl clone covers the 5'half and about 10 kb of 5'-flanking region (Fig. 1).

Together these two clones cover about 25 kb of human genome.

After subcloning and restriction enzyme digestion, suitable fragments for sequencing were obtained and the entire sequence of the CEL gene could be determined, including 1640 bp of the 5'-flanking region and 41 bp of the 3'-flanking region. These data revealed that the human CEL gene (SEQ ID NO: 1) span a region of 9850 bp, containing 11 exons interrupted by 10 introns (Fig. 1).

This means that the exons and especially the introns are relatively small. In fact, exons 1-10 range in sizes from 87-204 bp respectively while exon 11 is 841 bp long. The introns range in sizes from 85-2343 bp respectively. As can be noted in Table 3, all exon/intron boundaries obey the AG/GT rule and conform well to the consensus sequence suggested by Mount et al. (1982). When the coding part of the CEL gene was compared with the cDNA (Nilsson et al·., 1990), only one difference in nucleotide sequence was found; the second nt in exon 1, a C, which in the cDNA sequence is a T. Since this position is located 10 nt upstream the translation start codon ATG, this difference does not influence the amino acid sequence.

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-25Seven members of the Alu class of repetitive DNA elements are present in the sequenced region, labeled Alul-Alu7(5'-3')(Fig. 1), one in the 5'-flanking region and the six others within the CEL gene.

Transcription Initiation Sites and 5'-Flanking region

To map the human CEL gene transcription initiation site(s), primer extension analysis was performed using total RNA from human pancreas, lactating mammary gland and adipose tissue. The results indicated a major transcription start site located 12 bp, and a minor start site located 8 bases, upstream of the initiator methionine. The transcription initiation sites are the same in both pancreas and lactating mammary gland whereas no signal could be detected in adipose tissue (Fig. 2). The sequenced region includes 1640 nt of 5'flanking DNA. Based on sequence similarities a TATAbox-like sequence, CATAAAT was found 30 nt upstream the transcription initiation site (Fig. 4). Neither a CAAT20 box structure nor GC boxes were evident in this region.

The 5'-flanking sequence was computer screened, in both strands, for nucleotide sequences known as transcription factor binding sequences in other mammary gland- and pancreatic-specific genes. Several putative recognition sequences were found, see Fig. 4.

Chromosomal Localization of the CEL Gene

In human control DNA the CEL cDNA probe detected four

EcoRI fragments of approximately 13 kb, 10 kb, 2.2 kb and 2.0 kb, while in the mouse and hamster control DNAs single fragments of about 25 kb and 8.6 kb, respectively, were detected. The presence of human CEL gene sequences in the hybrid clones correlated only with the presence of human chromosome 9 (Table 1). Only one of the 16 hybrids analyzed were positive for the human CEL gene; this hybrid contained chromosome 9 as the only human chromosome. No discordancies for

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HX 1147 —26— localization to this chromosome were found, whereas there were at least two discordancies for localization to any other chromosome (Table 1). To further sublocalize the CEL gene we utilized a human-Chinese 5 hamster hybrid (GM 10095) retaining a der(9) translocation chromosome (9pter —> 9q34:Xql3 —> Xqter) as the only human DNA. By Southern blot we failed to detect any CEL gene sequences in this hybrid, indicating that the CEL gene resides within the 9q3410 qter region.

EXAMPLE 2: CONSTRUCTION OF EXPRESSION VECTORS

To construct an expression vector for production of recombinant human CEL in milk from transgenic animals the following strategy was employed (Fig. 5).

Three pTZ based plasmids (Pharmacia, Uppsala, Sweden) containing different parts of the human CEL gene, pS309, pS310 and pS311 were obtained using the methods described above. The plasmid pS309 contains a Sphl fragment covering the the CEL gene from the 5' untranscribed region to part of the fourth intron. The plasmid pS310 contains a Sacl fragment covering the CEL gene sequence from part of the first intron to a part of the sixth intron. Third, the plasmid pS311 contains a BamHI fragment covering a variant of the CEL gene from a major part of the fifth intron and the rest of the intron/exon structure. In this plasmid, the repetitive sequence of exon 11 that normally encodes the 16 repeats was mutated to encode a truncated variant having 9 repeats.

Another plasmid, pS283, containing a part of the human CEL cDNA cloned into the plasmid pUC19 at the Bindlll and Sacl sites was used for fusion of the genomic sequences. pS283 was also used to get a convenient

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-27restriction enzyme site, Kpnl, located in the 5' untranslated leader sequence of CEL. Plasmid pS283 was then digested with Ncol and Sacl and a fragment of about 2.7 kb was isolated. Plasmid pS309 was digested with Ncol and BspEI and a fragment of about 2.3 kb containing the 5'-part of the CEL gene was isolated. Plasmid pS310 was digested with BspEI and Sacl and a fragment of about 2.7 kb containing a part of the middle region of the CEL gene was isolated. These three fragments were ligated and transformed into competent

E. coli, strain TG2 , and transformants were isolated by ampicillin selection. Plasmids were prepared from a number of transformants, and one plasmid called pS312 (Fig. 6), containing the desired construct was used for further experiments.

To obtain a modification of pS311, in which the BamHI site located downstream of the stop codon was converted to a Sail site to facilitate further cloning, the following method was used. pS311 was linearized by partial BamHI digestion. The linearized fragment was isolated and a synthetic DNA linker that converts BamHI to a Sail site (5'-GATCGTCGAC-3'), thereby destroying the BamHI site, was inserted. Since there were two potential positions for integration of the synthetic ( linker the resulting plasmids were analyzed by restriction enzyme cleavage. A plasmid with the linker inserted at the desired position downstream of exon 11 was isolated and designated pS313.

To obtain the expression vector construct that harbours CEL genomic sequences and encodes the truncated CEL variant, the plasmid pS314 which was designed to mediate stage and tissue specific expression in the mammmary gland cells under lactation periods was used. Plasmid pS314 contains a genomic fragment from the murine whey acidic protein (WAP) gene (Campbell et al. 1984) cloned as a Notl fragment. The genomic fragment bAU ORIGINAL ft

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-2 8has approximately 4.5 kb upstream regulatory sequences (URS), the entire transcribed exon/intron region and about 3 kb cf sequence downstream of the last exon. A unique Kpnl site is located in the first exon 24 bp upstream of the natural WAP translation initiation codon. Another unique restriction enzyme site is the Sail site located in exon 3. In pS314, this Sail site was destroyed by digestion, fill in using Klenow and religation. Instead, a new Sail site was introduced directly downstream of the Kpnl site in exon 1. This was performed by Kpnl digestion and introduction of annealed synthetic oligomers SYM 2401 5'-CGTCGACGTAC3', and SYM 2402 5'-GTCGACGGTAC-3', at this position (Fig. 8) The human CEL genomic sequence was inserted between these sites, Kpnl and Sail, by the following strategy. First, pS314 was digested with Kpnl and Sail and a fragment representing the cleaved plasmid was electrophoretically isolated. Second, pS312 was digested with Kpnl and BamHI and a approximately 4.7 kb fragment representing the 5'part of the human CEL gene was isolated. Third, pS313 was digested with BamHI and Sail and the 3'-part of the human CEL gene was isolated. These three fragments were ligated, transformed into competent E. coli bacteria and transformants were isolated after ampicillin selection. Plasmids were prepared from several transformants and carefully analyzed by restriction enzyme mapping and sequence analysis. One plasmid representing the desired expression vector was defined and designated pS317.

In order to construct a genomic CEL expression vector encoding full-length CEL pS317 was modified as follows (Fig. 5). First, a pTZ18R plasmid (Pharmacia) containing a 5.2 kb BamHI fragment of the human CEL gene extending from the fifth intron to downstream of the eleventh exon, pS451, was digested with Bindlll and Sacl. This digestion generated a fragment of about 1.7 kb that extends from the Bindlll site located in intron

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-2 99 to the Sacl site located in exon 11. Second, the plasmid pS313 was digested with Sacl and Sail, and a 71 bp fragment containing the 3 'part of exon 11 and the generated Sail site was isolated. Third, the rest of the WAP/CEL recombinant gene and the plasmid sequences was isolated as a Sail/Hindlll fragment of about 20 kb from pS317. These three fragments were ligated and transformed into bacteria. Plasmids were prepared from several transformants. The plasmids were digested with various restriction enzymes and subjected to sequence analysis. One plasmid containing the desired recombinant gene was identified. This final expression vector was designated pS452 (Fig.7).

To remove the prokaryotic plasmid sequences, pS452 was digested with Notl. The recombinant vector element consisting of murine WAP sequence flanking the human CEL genomic fragment was then isolated by agarose electrophoresis. The isolated fragment was further purified using electroelution, before it was injected into mouse embryos .

The recombinant WAP/CEL gene for expression in mammary gland of transgenic animals is shown in Figure 8.

DEPOSITS

The following plasmids have been deposited in accordance with the Budapest Treaty at DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen):

Plasmid	Deposit No.	Date of deposit
pS309	DSM 7101	12 June 1992
pS310	DSM 7102
pS451	DSM 7498	26 February 1993
pS452	DSM 7499

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EXAMPLE 3: GENERATION OF TRANSGENIC ANIMALS

A Noel fragment was isolated from the plasmid pS452 according to Example 2. This DNA fragment contained the murine WAP promoter linked to a genomic sequence encoding human BSSL/CEL. The isolated fragment, at a concentration of 3 ng/μΐ, was injected into the pronucleus of 350 C57Bl/6JxCBA/2J-f2 embryos obtained from donor mice primed with 5 IU pregnant mare's serum gonadotropin for superovulation. The C57Bl/6JxCBA/2J-f animals were obtained from BomholtgArd Breeding and Research Centre LID, Ry, Denmark. After collection of the embryos from the oviduct, they were separated from the cumulus cells by treatment with hyaluronidase in the medium M2 (Hogan et al. , 1986) . After washing the embryos were transferred to the medium M16 (Hogan et al., 1986) and kept in an incubator with 5% CO₂atmosphere. The injections were performed in a microdrop of M2 under light paraffin oil using

Narishigi hydraulic micromanipulators and a Nikon inverted microscope equipped with Nomarski optics.

After injection, healthy looking embryos were implanted into pseudopregnant C57Bl/6JxCBA/2J-frecipients given 0.37 ml of 2.5% Avertin intraperitoneally. Mice that had integrated the transgene were identified with PCR analysis of DNA from tail biopsy specimens obtained three weeks after birth of the animals. Positive results were confirmed with Southern blot analysis.

EXAMPLE 4: EXPRESSION OF BSSL/CEL IN TRANSGENIC MICE

Transgenic mice were identified by analysis of DNA which has been prepared from excised tail samples. The tissue samples were incubated with proteinase K and phenol/chloroform extracted. The isolated DNA was used in polymerase chain reactions with primers which amplify specific fragments if the heterologous bad ORIGINAL

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-31introduced DNA representing the expression vector fragment is present. The animals were also analyzed by DNA hybridization experiments to confirm PCR data and to test for possible rearrangements, structure of the integrated vector elements and to obtain information about the copy number of integrated vector elements.

In one set of experiments, 18 mice were analyzed with the two methods and the results demonstrated that 1 mouse was carrying the heterologous DNA vector element derived from pS452. The result from the PCR analysis and the hybridization experiments were identical (Fig.

9).

The mouse identified to carry vector DNA element (founder animal) was then mated and the FI litter was analyzed for transgene by the same procedures.

Female lactating animals were injected with 2 IU oxytocin intraperitoneally and 10 minutes later anaesthetized with 0.40 ml of 2.5% Avertin intraperitoneally. A milk collecting device was attached to the nipple via a siliconized tubing and milk was collected into a 1.5 ml Eppendorf tube by gentle massage of the mammary gland. The amount of milk ( varied, dependent on the day of lactation, between 0.1 and 0.5 ml per mouse and collection.

Analyze for the presence of recombinant human BSSL/CEL was done by SDS-PAGE, transfer to nitrocellulose membranes and incubation with polyclonal antibodies generated against native human BSSL/CEL. The obtained results demonstrated expression of recombinant human BSSL/CEL in milk from transgenic mice. Figure 10 demonstrates presence of recombinant human BSSL/CEL in milk from transgenic mice: the band at about 116.5.

Stable lines of transgenic animals are generated.

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-32In a similar manner, other transgenic animals such as cows or sheep capable of expressing human BSSL/CEL may be prepared.

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-34Taggart. R.T., Mohandas, Τ., Shows, T.B. and Bell, G.I. (1985): Proc. Natl. Acad. Sci. U.S.A. 82, 6240-6244.

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Hjerten, S., Petterson, U. and Chattopadhyaya, J. (1984): Biochim. Biophys. Acta 782, 120-126.

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Table 1. Correlation of CEL sequences with human chromosomes in 16 human-rodent somatic cell hybrids.

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HX 1147

-38FIGURE LEGENDS

Figure 1

The CEL gene locus. Localization and restriction enzyme 5 map of the two partly overlapping clones, XESSLl and

XBSSL5A are shown. The exon-intron organization and used restriction enzyme site are shown below. Exons are represented by boxes numbered 1-11. Asp=Asp700,

B=BamHI, E=EcoRI, S=SacI, Sa = SalI, Sp=Sphl and X=Xbal.

Positions and orientation of Alu repetitive elements are shown by bold arrows, a-h represent different subcloned fragments.

Figure 2

Primer extension analysis of RNA from human lactating mammary gland, pancreas and adipose tissue. An endradiolabeled 26-mer oligonucleotide, which is complementary to nt positions 33 to 58 of the CEL gene, was used to prime reverse transcription of the RNA.

Lane A is a molecular size marker (a sequencing ladder), lane B pancreatic RNA, lane C adipose tissue RNA and lane D lactating mammary gland RNA.

Figure 3

Dotplot analysis of the human CEL and rat CEL gene 5'flanking regions. The homology regions are labeled A-H and the sequences representing these parts are written, upper is human and lower is rat.

Figure 4

Analysis of 5'-flanking sequence of the human CEL gene. The putative recognition sequences are either highlighted underline or underline representing the complementary strand. Bold letters show the locations of the homologies to the rCEL (regions A-H). The TATAbox is underlined with dots.

BAD ORIGINAL fi

AP Ο Ο Ο 4 1 1

HX 1147

-39There are two sequences that both show a 80% similarity to the consensus sequence of the glucocorticoid receptor binding site, GGTACANNNTGTTCT, (Beato, Μ. , 1989), the first one on the complementary strand at nt position -231 (IA) and the second one at nt position

-811 (IB). Moreover, at nt position -861 (2) there is a sequence that shows 87% similarity to the consensus sequence of the estrogen receptor binding site,

AGGTCANNNTGACCT, (Beato, Μ. , 1989).

Lubon and Henninghausen (1987) have analyzed the promoter and 5'-flanking sequences of the whey acidic protein (WAP) gene and established the binding sites for nuclear proteins of lactating mammary gland cells.

One of them, an 11 bp conserved sequence, AAGAAGGAAGT, is present in a number of milkprotein genes studied e.g. the rat α-lactalbumin gene (Qasba et al., 1984) and the rat α-casein gene (Yu-Lee et al., 1986). In the CEL gene's 5'-flanking region, on the complementary strand at nt position -1299 (3) there is a sequence that shows 82% similarity to this conserved sequence.

In a study of the β-casein gene's regulation, a tissue specific mammary gland factor (MGF) was found in nuclear extracts from pregnant or lactating mice and its recognition sequence was identified (ANTTCTTGGNA). In the human CEL gene's 5'-flanking region there are two sequences, one on the complementary strand at nt position -368 (4A) and the other at nt position -1095 (4B), they both show 82% similarity to the consensus sequence of the MGF binding site. Beside these two putative MGF binding sites in the 5'-flanking region there is a sequence on the complementary strand at nt 275 in intron I, AGTTCTTGGCA, which shows 100% identity to the consensus sequence of the MGF binding site.

Furthermore, there are four sequences which all show 65% similarity to the consensus sequence of rat

BAD ORIGINAL ft

HX 1147

AP 0 00 41 1

-4 0pancreas-specific enhancer element,

GTCACCTGTGCTTTTCCCTG, (Boulet et al. , 1986), one at nt position -359 (5A), the second at nt position -718 (5B), the third at nt position -1140 (50 and the last at nt position -1277 (5D).

Figure 5

Method for production of the plasmid pS452. For further details, see Example 2.

Figure 6

Schematic structure cf the plasmid pS312.

Figure 7

Schematic structure of the plasmid pS452.

Figure 8

Physical map representing the physical introduction of human BSSL/CEL genomic structure in the first exon of the WAP gene as described in Example 2.

Figure 9

A. Schematic representation of the localization of PCR-primers used for identification of transgenic animals. The 5'-primer is positioned within the WAP sequence starting at the position -148 bp upstream of the fusion between the WAP and BSSL/CEL. The 3'-primer is localized in the first BSSL/CEL intron ending 398 bp downstream of the fusion point.

B. The sequences of the PCR primers used.

C. Agarose gel showing a typical analysis of the PCR analysis of the potential founder animals. M: molecular weight markers. Lane 1: control PCR-product generated from the plasmid pS452. Lanes 2-13: PCR reactions done with DNA preparations from potential founder animals.

BADORIGINAL $

ΑΡ Ο Ο Ο 4 1 1

Ηλ 1147 —41—

Fiqure 10

Immunoblot analysis of milk from a mouse line transgenic for the recombinant murine WAP/human CEL gene of pS452. The proteins were separated on SDS-PAGE, transferred to Immobilon membranes (Millipore) and visualized with polyclonal rabbit antibodies generated using highly purified human native CEL, followed by alkaline phosphatase labelled swine anti-rabbit IgG (Dakopatts). Lane 1, Low molecular weight markers, 106,

80, 49.5, 32.5, 27.5, and 18.5 kDa, respectively. Lane

2, High molecular weight markers, 205, 116.5, 80 and

49.5 kDa, respectively. Lane 3, 25 ng purified nonrecombinant CEL from human milk. Lane 4, 2 μΐ milk sample from a CEL transgenic mouse diluted 1:10. Lanes

5 and 6, 2 μΐ milk samples from two different non-CEL transgenic mice, diluted 1:10, as control samples.

BAD ORIGINAL ft

AP Ο Ο Ο 4 1 1

ΗΧ 1147 —42—

SEQUENCE LISTING i! GENERAL INFORMATION:

(l) APPLICANT:

(A) NAME: A3 ASTRA (Bi STREET: Kvarnbergagatan li (C) CITY: Sodertalje (E) COUNTRY: Sweden (F) POSTAL CODE (ZIP): S-151 35 (G) TELEPHONE: +46-8-553 26000 (H) TELEFAX: +46-8-553 28820 (I) TELEX: 19237 astra s (ii) TITLE OF INVENTION: New DNA Sequences (iii) NUMBER OF SEQUENCES : 1 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: SE 9201809-2 (B) FILING DATE: ll-JUN-1992 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: SE 9201326-6 (B) FILING DATE: 12-JUN-1992 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: SE 9202088-2 (B) FILING DATE: 03-JUL-1992 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: SE 9300902-5 (B) FILING DATE: 19-MAR-1993 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11531 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: Mammary gland (ix) FEATURE:

(A) NAME/KEY : CDS (B) LOCATION: join (1653..1727, 4071..4221, 4307..4429, 4707

..4904, 6193..6323, 6501..6608, 6751..6868, 8335 ..8521, 8719..8922, 10124..10321, 10650..11394) (ix) FEATURE:

(A) NAME/KEY: mat_peptide (B) LOCATION: join(1722..1727, 4071..4221, 4307..4429, 4707

..4904, 6193..6323, 6501..6608, 6751..6868, 8335 ..8521, 8719..8922, 10124..10321, 10650..11391)

BAD ORIGINAL ft

AP ο Ο Ο 4 1 1

HX 1147

-43(D) OTHER INFORMATION: /EC_number= 3.1.1.1 /product= Bile Salt-Stimulated Lipase (ix) FEATURE:

(A) NAME/KEY: 5 ' UTR (B) LOCATION: 1..1640 (ix) FEATURE:

	(A) NAME/KEY: (B) LOCATION:	TATA_signal
1611.	. 1617
(ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 1641.	.1727
(ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	exon 4071.	.4221
( ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 4307 .	.4429
(ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 47 07.	.4904
(ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	exon 6193 .	.6323
( ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	exon 6501.	.6608
(ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 6751.	.6868
(ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	exon 8335.	.8521
(ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 8719.	.8922
(ix)	FEATURE : (A) NAME/KEY: (B) LOCATION:	exon 10124	. . 10321
(ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	exon 10650	. . 11490
(ix)	FEATURE: (A) NAME/KEY: (B) LOCATION:	3 'UTR 11491	..11531
(xi)	SEQUENCE DESCRIPTION	: SEQ ID NO: 1

GGATCCCTCG	AACCCAGGAG	TTCAAGACTG	CAGTGAGCTA	TGATTGTGCC	ACTGCACTCT	60
AGCCTGGGTG	ACAGAGACCC	TGTCTCAAAA	AAACAAACAA	ACAAAAAACC	TCTGTGGACT	120
CCGGGTGATA	ATGACATGTC	AATGTGGATT	CATCAGGTGT	TAACAGCTGT	ACCCCCTGGT	180

BAD ORIGINAL ft

AP Ο Ο Ο 4 1 1

ΗΧ 1147

	-44-
GGGGGATGTT	GATAACGGGG	GAGACTGGAG	TGGGGCGAGG	ACATACGGGA	AATCTCTGTA	240
ATCTTCCTCT	AATTTTGCTG	TGAACCTAAA	GCTGCTCTAA	AAATGTACAT	AGATATAAAC	300
TGGGGCCTTC		GCCCTGCCCC	AGCCCTCCCC	CACCTCCTTC	CTCTCCCTGC	360
TGCCTCCCCT	CTGCCCTCCC	CTTTCCTCCT	TAGCCACTGT	AAATGACACT	GCAGCAAAGG	420
TCTGAGGCAA	ATGCCTTTGC	CCTGGGGCGC	CCCAGCCACC	TGCAGGCCCC	TTATTTCCTG	430
TGGCCGAGCT	CCTCCTCCCA	CCCTCCAGTC	CTTTCCCCAG	CCTCCCTCGC	CCACTAGGCC	540
TCCTGAATTG	CTGGCACCGG	CTGTGGTCGA	CAGACAGAGG	GACAGACGTG	GCTCTGCAGG	600
TCCACTCGGT	CCCTGGCACC	GGCCGCAGGG	GTGGCAGAAC	GGGAGTGTGG	TTGGTGTGGG	660
AAGCACAGGC	CCCAGTGTCT	CCTGGGGGAC	TGTTGGGTGG	GAAGGCTCTG	GCTGCCCTCA	720
CCCTGTTCCC	ATCACTGCAG	AGGGCTGTGC	GGTGGCTGGA	GCTGCCACTG	AGTGTCTCGG	780
TGAGGGTGAC	CTCACACTGG	CTGAGCTTAA	AGGCCCCATC	TGAAGACTTT	GTTCGTGGTG	840
TTCTTTCACT	TCTCAGAGCC	TTTCCTGGCT	CCAGGATTAA	TACCTGTTCA	CAGAAAATAC	900
GAGTCGCCTC	CTCCTCCACA	ACCTCACACG	ACCTTCTCCC	TTCCCTCCCG	CTGGCCTCTT	960
TCCCTCCCCT	TCTGTCACTC	TGCCTGGGCA	TGCCCCAGGG	CCTCGGCTGG	GCCCTTTGTT	1020
TCCACAGGGA	AACCTACATG	GTTGGGCTAG	ATGCCTCCGC	ACCCCCCCAC	CCACACCCCC	1080
TGAGCCTCTA	GTCCTCCCTC	CCAGGACACA	TCAGGCTGGA	TGGTGACACT	TCCACACCCT	1140
TGAGTGGGAC	TGCCTTGTGC	TGCTCTGGGA	TTCGCACCCA	GCTTGGACTA	CCCGCTCCAC	1200
GGGCCCCAGG	AAAAGCTCGT	ACAGATAAGG	TCAGCCACAT	GAGTGGAGGG	CCTGCAGCAT	1260
GCTGCCCTTT	CTGTCCCAGA	AGTCACGTGC	TCGGTCCCCT	CTGAAGCCCC	TTTGGGGACC	1320
TAGGGGACAA	GCAGGGCATG	GAGACATGGA	GACAAAGTAT	GCCCTTTTCT	CTGACAGTGA	1380
CACCAAGCCC	TGTGAACAAA	CCAGAAGGCA	GGGCACTGTG	CACCCTGCCC	GGCCCCACCA	1440
TCCCCCTTAC	CACCCGCCAC	CTTGCCACCT	GCCTCTGCTC	CCAGGTAAGT	GGTAACCTGC	1500
ACAGGTGCAC	TGTGGGTTTG	GGGAAAACTG	GATCTCCCTG	CACCTGAGGG	GGTAGAGGGG	1560
AGGGAGTGCC	TGAGAGCTCA	TGAACAAGCA	TGTGACCTTG	GATCCAGCTC	CATAAATACC	1620
CGAGGCCCAG	GGGGAGGGCC	ACCCAGAGGC	TG ATG CTC Met Leu -23	ACC ATG GGG CGC CTG Thr Met Gly Arg Leu -20	1673
CAA CTG GTT GTG TTG GGC CTC ACC TGC TGC TGG Gin Leu Val Val Leu Gly Leu Thr Cys Cys Trp -15 -10	GCA GTG GCG AGT GCC Ala Val Ala Ser Ala -5	1721
GCG AAG GTAAGAGCCC AGCAGAGGGG CAGGTCCTGC TGCTCTCTCG CTCAATCAGA Ala Lys 1	1777
TCTGGAAACT	TCGGGCCAGG	CTGAGAAAGA	GCCCAGCACA	GCCCCGCAGC	AGATCCCGGG	1837
CACTCACGCT	CATTTCTATG	GGGACAGGTG	CCAGGTAGAA	CACAGGATGC	CCAATTCCAT	1897
TTGAATTTCA	GATAAACTGC	CAAGAACTGC	TGTGTAAGTA	TGTCCCATGC	AATATTTGAA	1957
ACAAATTTCT	ATGGGCCGGG	CGCAGTGGCT	CACACCTGCA	ATCCCACCAG	TTTGGGAGGC	2017

BAD ORIGINAL

AP Ο Ο Ο 4 1 1

HX, 1147

	-45-
CGAGGTGGGT	GGATCACTTG	AGGTCAGGAG	TTGGAGACCA	GCCTGGCCAA	CATGGTGAAA	2077
CCCCGTCTCT	ACTAAAAATA	CAAATATTAA	TCGGGCGTGG	TGGTGGGTGC	CTGTAATCCC	2137
AGCTACTCGG	GAGGCTGAGG	CAGGAGAACC	GCTTGAAGCT	GGGAGGTGGA	GATTGCGGTG	2197
AGCTGAGATC	ACGCTACTGC	ACTCCAGCCT	GGGTGACAGG	GCGAGACTCT	GTCTCAAAAA	2257
ATAGAAAAAG	AAAAAAATGA	AACATACTAA	AAAACAATTC	ACTGTTTACC	TGAAATTCAA	2317
ATGTAACTGG	GCCTCTTGAA	TTTACATTTG	CTAATCCTGG	TGATTCCACC	TACCAACCTC	2377
TCTGTTGTTC	CCATTTTACA	GAAGGGGAAA	CGGGCCCAGG	GGCAGGGAGT	GTGGAGAGCA	2437
GGCAGACGGG	TGGAGAGAAG	CAGGCAGGCA	GTTTGCCCAG	CATGGCACAG	CTGCTGCCTC	2497
CTATTCCTGT	GCAGGAAGCT	GAAAGCCGGG	CTACTCCACA	CCCGGGTCCG	GGTCCCTCCA	2557
GAAAGAGAGC	CGGCAGGCAG	GAGCTCTCTC	GAGGCATCCA	TAAATTCTAC	CCTCTCTGCC	2617
TGTGAAGGAG	AAGCCACAGA	AACCCCAAGC	CCCACAGGAA	GCCGGTGTCG	GTGCCCGGCC	2677
CAGTCCCTGC	CCCCAGCAGG	AGTCACACAG	GGGACCCCAG	ATCCCAACCA	CGCTGTTCTG	2737
CTGCCTGCGG	TGTCTCAGGC	CCTGGGGACT	CCTGTCTCCA	CCTCTGCTGC	CTGCTCTCCA	2797
CACTCCCTGG	CCCTGGGACC	GGGAGGTTTG	GGCAGTGGTC	TTGGGCTCCT	GACTCAAAGG	2857
AGAGGTCACC	TTCTTCTTGG	GCGAGCTCTT	CTTGGGGTGC	TGAGAGGCCT	TCGGCAGGTC	2917
ATCACGACCC	CTCCCCATTT	CCCCACCCTG	AGGCCCTCTG	GCCAGTCTCA	ATTGCACAGG	2977
GATCACGCCA	CTGGCACAAG	GAGACACAGA	TGCCTCGCAG	GGGATGCCCA	CGATGCCTGC	3037
ATGTGTTGCT	TCTGGTTCCT	TTCCTCCAGT	TCCAACCGCC	GCACTCTCCC	ACACCAGTGT	3097
GACAGGGGGC	CCATCACCCT	AGACTTCAGA	GGGCTGCTGG	GACCCTGGCT	GGGCCTGGGG	3157
GTGTAGGGCC	ACCCTGCCCT	TCCCCACCTG	GAACCTGGCA	CAGGTGACAG	CCAGCAAGCA	3217
ATGACCTGGT	CCCACCATGC	ACCACGGGAA	GAGGGAGCTG	CTGCCCAAGA	TGGACAGGAG	3277
GTGGCACTGG	GGCAGACAGC	TGCTTCTCAA	CAGGGTGACT	TCAAGCCCAA	AAGCTGCCCA	3337
GCCTCAGTTC	CGTCAGGGAC	AGAGGGTGGA	TGAGCACCAA	CCTCCAGGCC	CCTCGTGGGG	3397
GTGGACAGCT	TGGTGCACAG	AGGCCATTTT	CATGGCACAG	GGAAGCGTGG	CGGGGGTGGG	3457
AGGTGTGGTC	CCTAGGGGGT	TCTTTACCAG	CAGGGGGCTC	AGGAACTGTG	GGGACTTGGG	3517
CATGGGGCCA	TCGACTTTGT	GCCCAGCCAG	CTAGGCCCTG	TGCAGGGAGA	TGGGAGGAGG	3577
GAAAAGCAGG	CCCCACCCCT	CAGAAAGGAG	GAAGGTTGGT	GTGAAACATC	CCGGGTACAC	3637
TGAGCATTGG	GTACACTCCT	CCCGGGAGCT	GGACAGGCCT	CCCATGTGAT	GGCAAACAGG	3697
CCGACAGGAG	ACACGGCTGT	TGCTCGTCTT	CCACATGGGG	AAACTGAGGA	TCGGAGTCAA	3757
AGCTGGGCGG	CCATAGCCAG	AACCCAAACC	TCCATCCCAC	CTCTTGGCCG	GCTTCCCTAG	3817
TGGGAACACT	GGTTGAACCA	GTTTCCTCTA	AGATTCTGGG	AGCAGGACAC	CCCCAGGGAT	3877
AAGGAGAGGA	ACAGGAATCC	TAAAGCCCTG	AGCATTGCAG	GGCAGGGGGT	GCTGCCTGGG	3937
TCTCCTGTGC	AGAGCTGTCC	TGCTTTGAAG	CTGTCTTTGC	CTCTGGGCAC	GCGGAGTCGG	3997
CTTGCCTTGC	CCCCTCCGGA	TTCAGGCCGA	TGGGGCTTGA	GCCCCCCTGA	CCCTGCCCGT	4057

bad ORIGINAL

AP Ο Ο Ο 4 1 1

ΗΧ 1147 —46—

GTCTCCCTCG CAG CTG GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA 410 6

Leu Gly Ala Val Tyr Thr Glu Glv Gly Phe Val Glu

10

GGC Gly 15

GTC Va l·

ΛΛ i rtb Γί

AAG Lys

AAG Lys

CTC Leu 20

GGC Gly

CTC Leu

CTG Leu

GGT Gly

GAC Asp 25

TCT Ser

GTG Val

GAC Asp

ATC lie

TTC Phe 30

4154

AAG

GGC

ATC

CCC

TTC

GCA

GCT

CCC

ACC

AAG

GCC

CTG

GAA

AAT

CCT

CAG

4202

Lys

Gly

lie

Pro

Phe

Ala

Ala

Pro

Thr

Lys

Ala

Leu

Glu

Asn

Pro

Gin

35

40

45

CCA CAT CCT GGC TGG CAA G GTGGGAGTGG GTGGTGCCGG ACTGGCCCTG 42 51

Pro His Pro Gly Trp Gin

CGGCGGGGCG GGTGAGGGCG GCTGCCTTCC TCATGCCAAC TCCTGCCACC TGCAG GG 4308

Gly

ACC Thr

CTG Leu 55

Λ ' Lys

GCC Ala

AAG Lys

AAC Asn

TTC Phe 60

AAG Lys

AAG Lys

AGA TGC Arg Cys

CTG Leu 65

CAG Gin

GCC Ala

ACC Thr

ATC He

4356

ACC

CAG

GAC

AGC

ACC

TAC

GGG

GAT

GAA

GAC

TGC

CTG

TAC

CTC

AAC

ATT

4404

Thr

Gin

Asp

Ser

Thr

Tyr

Gly

Asp

Glu

Asp

Cys

Leu

Tyr

Leu

Asn

lie

70

75

80

85

TGG

GTG

CCC

CAG

GGC

AGG

AAG

CAA

G GTCTGCCTCC CCTCTACTCC

4449

Trp

Val

Pro

Gin

Gly

Arg

Lys

Gin

CCAAGGGACC CTCCCATGCA GCCACTGCCC CGGGTCTACT CCTGGCTTGA GTCTGGGGGC 4509

TGCAAAGCTG AACTTCCATG AAATCCCACA GAGGCGGGGA GGGGAGCGCC CACTGCCGTT 4569

GCCCAGCCTG GGGCAGGGCA GCGCCTTGGA GCACCTCCCT GTCTTGGCCC CAGGCACCTG 4629

CTGCACAGGG ACAGGGGACC GGCTGGAGAC AGGGCCAGGC GGGGCGTCTG GGGTCACCAG 4689

CCGCTCCCCC ATCTCAG TC TCC CGG GAC CTG CCC GTT ATG ATC TGG ATC 4733

Val Ser Arg Asp Leu Pro Val Met lie Trp He

100

TAT GGA GGC

GCC Ala

TTC Phe

CTC ATG GGG

TCC GGC Ser Gly

CAT GGG GCC

AAC Asn

TTC Phe

CTC Leu 120

4786

Tyr 105

Gly Gly

Leu Met 110

Gly

His 115

Gly

Ala

AAC

AAC

TAC

CTG

TAT

GAC

GGC

GAG

GAG

ATC

GCC

ACA

CGC

GGA

AAC

GTC

4834

Asn

Asn

Tyr

Leu

Tyr

Asp

Gly

Glu

Glu

He

Ala

Thr

Arg

Gly

Asn

Val

125

130

135

ATC

GTG

GTC

ACC

TTC

AAC

TAC

CGT

GTC

GGC

CCC

CTT

GGG

TTC

CTC

AGC

4832

lie

Val

Val

Thr

Phe

Asn

Tyr

Arg

Val

Gly

Pro

Leu

Gly

Phe

Leu

Ser

140

145

150

ACT

GGG

GAC

GCC

AAT

CTG

CCA

G GTGCGTGGGT GCCTTCGGCC CTGAGGTGGG

4934

Thr

Gly

Asp

Ala

Asn

Leu

Pro

155

GCGACCAGCA TGCTGAGCCC AGCAGGGAGA TTTTCCTCAG CACCCCTCAC CCCAAACAAC 4994

CAGTGGCGGT TCACAGAAAG ACCCGGAAGC TGGAGTAGAA TCATGAGATG CAGGAGGCCC 5054

TTGGTAGCTG TAGTAAAATA AAAGATGCTG CAGAGGCCGG GAGAGATGGC TCACGCCTGT 5114

AATCCCAGCA CTTTAGGAGG CCCACACAGG TGGGTCACTT GAGCGCAGAA GTTCAAGACC 5174

BAD ORIGINAL

AH υ υ Ο 4 1 1

HX 1147

	—47—
AGCCTGAAAA	TCACTGGGAG ACCCCCATCT CTACACAAAA	ATTAAAAATT	AGCTGGGGAC	5234
TGGGCGCGGC	GGCTCACCTC TGTAATCCCA GCACGTTGGG	AGCCCAAGGT	GGGTAGATCA	5294
CCTGAGGTCA	GGAGTTTGAG ACCAGCCTGA CTAAAATGGA	GAAACCTCTT	CTCTACTAAA	5354
AATACAAAAT	TAGCCAGGCG 7GGTGGCGCT TGCCTGTAAT	CCCAGCTACT	CGGGAGGCTG	5414
AGGCAGGAGA	ATCGCTTGAA C7CAGGAGGC GGAGGTTGCG	GTGAGCCGAG	ATCATGCCAC	5474
TGCACTCCAG	CCTGGAGAAC AA3AGTAAAA CTCTGTCTCA	AAAAAAAAAA	AAAAAAAAAA	5534
ATAGCCAGGC	GTGGTATCTC ATGCCTCTGT CCTCAGCTAC	CTGGGAGGCA	GAGGTGGAAG	5594
GATCGCTTGA	GCCCAGGGGT TCAAAGCTGC AGTGAGCCGT	GGTCGTGCCA	CTGCACTCCA	5654
GCCTGGGCGA	CAGAGTGAGG CCCCATCTCA AAAATAAGAG	GCTGTGGGAC	AGACAGACAG	5714
GCAGACAGGC	TGAGGCTCAG AGAGAAACCA GGAGAGCAGA	GCTGAGTGAG	AGACAGAGAA	5774
CAATACCTTG	AGGCAGAGAC AGCTGTGGAC ACAGAAGTGG	CAGGACACAG	ACAGGAGGGA	5834
CTGGGGCAGG	GGCAGGAGAG GTGCATGGGC CTGACCATCC	TGCCCCCGAC	AAACACCACC	5894
CCCTCCAGCA	CCACACCAAC CCAACCTCCT GGGGACCCAC	CCCATACAGC	ACCGCACCCG	5954
ACTCAGCCTC	CTGGGACCCA CCCACTCCAG CAACCAACGT	GACCTAGTCT	CCTGGGACCC	6014
ACCCCCTCCA	GCACCCTACC C3ACCCAGCT TCTTAGGGAC	CCACCATTTG	CCAACTGGGC	6074
TCTGCCATGG	CCCCAACTCT GTTGAGGGCA TTTCCACCCC	ACCTATGCTG	ATCTCCCCTC	6134
CTGGAGGCCA	GGCCTGGGCC ACTGGTCTCT AGCACCCCCT	CCCCTGCCCT	GCCCCCAG GT Gly 160	6194
AAC TAT GGC	CTT CGG GAT CAG CAC ATG GCC ATT	GCT TGG GTG	1 AAG AGG	6242
Asn Tyr Gly	Leu Arg Asp Gin His Met Ala lie 165 170	Ala Trp Val	Lys Arg 175
AAT ATC GCG	GCC TTC GGG GGG GAC CCC AAC AAC	ATC ACG CTC	TTC GGG	6290
Asn lie Ala	Ala Phe Gly Gly Asp Pro Asn Asn 180 185	He Thr Leu 190	, Phe Gly
GAG TCT GCT Glu Ser Ala 195	GGA GGT GCC AGC GTC TCT CTG CAG Gly Gly Ala Ser Val Ser Leu Gin 200	GTCTCGGGAT	CCCTGTGGGG	6343
AGGGCCTGCC	CCACAGGTTG AGAGGAAGCT CAAACGGGAA	GGGGAGGGTG	GGAGGAGGAG	6403
CGTGGAGCTG	GGGCTGTGGT GCTGGGGTGT CCTTGTCCCA	GCGTGGGGTG	GGCAGAGTGG	6463
GGAGCGGCCT	TGGTGACGGG ATTTCTGGGT CCCGTAG ACC	: CTC TCC CCC TAC AAC	6518

Thr Leu Ser Pro Tyr Asn

	205
AAG GGC CTC ATC CGG CGA GCC ATC AGC CAG AGC	GGC GTG GCC CTG AGT 6566
Lys Gly Leu lie Arg Arg Ala He Ser Gin Ser	Gly Val Ala Leu Ser
210 215 220	225
CCC TGG GTC ATC CAG AAA AAC CCA CTC TTC TGG	GCC AAA AAG 6608
Pro Trp Val He Gin Lys Asn Pro Leu Phe Trp	Ala Lys Lys
230 235
GTAAACGGAG GAGGGCAGGG CTGGGCGGGG TGGGGGCTGT	CCACATTTCC GTTCTTTATC 6668
CTGGACCCCA TCCTTGCCTT CAAATGGTTC TGAGCCCTGA	GCTCCGGCCT CACCTACCTG 6728

bad original

AP Ο Ο Ο 4 1 1

ΗΧ 1147 —48—

CTGGCCTTGG TTCTGCCCCC AG GTG GCT GAG AAG GTG GGT TGC CCT GTG GGT 67 8 0

Val Ala Glu Lys Val Gly Cys Pro Val Gly 240 245

GAT Asp 250

GCC Ala

GCC Ala

AGG Arg

ATG Met

GCC Ala 255

CAG Gin

TGT Cys

CTG AAG GTT ACT GAT

CCC Pro

CGA Arg

GCC Alci 265

6328

Leu

Lys

Val 260

Thr

Asp

CTG

ACG

CTG

GCC

TAT

AAG

GTG

CCG

CTG

GCA

GGC

CTG

GAG

T <

3TGAGTAGCT

6878

Leu

Thr

Leu

Ala

Tyr

Lys

Val

Pro

Leu

Ala

Gly

Leu

Glu

270 275

GCTCGGGTTG	GCCCATGGGG	TCTCGAGGTG GGGGTTGAGG GGGGTACTGC CAGGGAGTAC	6938
TCCGGAGGAG	AGAGGAAGGT AAGGCCCCAG	GCCAGAGCTG CGGTCTTGTC CTGTCACCAA CTAGCTGGTG	6998
TCTCCCCTCG	CTGTAAGGGA GAGGGGGTGC CGTTTCTTCT TTTTTTTTGA	7058
GATGGAGTCT	CACTGTTGCC	CAGGCTGGAG TGCAGTGTCA CGATCTCAGC TCACTGCAAC	7113
CTCCACCTCC	TGGGTTCAAG	TGATTCTCTG ACTCAACCTC CCATGTAGCT GGGACTACAG	7178
GCACATGCCA	CCATGCCCAG	ATAATTTTTC TGTGTGTTTA GTAGGGATGG AGTTTCATCG	7238
TGTTAGCTAG	GATGATCTCG	GTCTTGGGAC CTCATGATCT GCCCACCTCG GCCTCCCAAA	7298
GTGCTGGAAT	TACAGGCGTG	AGCCACTGTG CCCGGCCCCT TCTTTATTCT TATCTCCCAT	7358
GAGTTACAGA	CTCCCCTTTG	AGAAGCTGAT GAACATTTGG GGCCCCCTCC CCCACCTCAT	7418
GCATTCATAT	GCAGTCATTT	GCATATAATT TTAGGGAGAC TCATAGACCT CAGACCAAGA	7478
GCCTTTGTGC	TAGATGACCG	TTCATTCATT CGTTCATTCA TTCAGCAAAC ATTTACTGAA	7538
CCGTAGCACT	GGGGCCCAGC	CTCCAGCTCC ACTATTCTGT ACCCCGGGAA GGCCTGGGGA	7598
CCCATTCCAC	AAACACCTCT	GCATGTCAGC CTTACCAGCT TGCTACGCTA AGGCTGTCCC	7658
TCACTCATTC	TTCTATGGCA	ACATGCCATG AAGCCAAGTC ATCTGCACGT TTACCTGACA	7718
TGAGCTCAAC	TGCACGGGCT	GGACAAGCCC AAACAAAGCA ACCCCCACGG CCCCGCTAGA	7778
AGCAAAACCT	GCTGTGCTGG	GCCCAGTGAC AGCCAGGCCC CGCCTGCCTC AGCAGCCACT	7838
GGGTCCTCTA	GGGGCCCGTC	CAGGGGTCTG GAGTACAATG CAGACCTCCC ACCATTTTTG	7898
GCTGATGGAC	TGGAACCCAG	CCCTGAGAGA GGGAGCTCCT TCTCCATCAG TTCCCTCAGT	7958
GGCTTCTAAG	TTTCCTCCTT	CCTGCTTCAG GCCCAGCAAA GAGAGAGAGG AGAGGGAGGG	8018
GCTGCCGCTG	AAGAGGACAG	ATCTGGCCCT AGACAGTGAC TCTCAGCCTG GGGACGTGTG	8078
GCAGGGCCTG	GAGACATCTG	TGATTGTCAC AGCTGGGGAG GGGGTGCTCC TGGCACCTCG	8138
TGGGTCGAGG	CCGGGGATGC	TCTAAACATC CTACAGGGCA CAGGATGCCC CTGATGGTGC	8198
AGAATCAACC	CTGCCCCAAG	TGTCCATAGA TCAGAGAAGG GAGGACATAG CCAATTCCAG	8258
CCCTGAGAGG	CAAGGGGCGG	CTCAGGGGAA ACTGGGAGGT ACAAGAACCT GCTAACCTGC	8318
TGGCTCTCCC	ACCCAG AC Tyr	CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT Pro Met Leu His Tyr Val Gly Phe Val Pro	8366

280 285

GTC ATT GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC 8414

Val lie Asp Gly Asp Phe lie Pro Ala Asp Pro lie Asn Leu Tyr Ala

290 295 300 305

BAD ORIGINAL

AP Ο Ο Ο 4 1 1

ΗΧ 1147

AAC Asn

GCC Ala

GCC Ala

GAC Asp

ATC lie 310

GAC Asp

TAT Tyr

ATA He

GCA Ala

GGC Gly 315

ACC Thr

AAC Asn

AAC Asn

ATG Met

GAC Asp 320

GGC Gly

3462

CAC

ATC

TTC

GCC

AGC

ATC

GAC

ATC

CCT

GCC

ATC

AAC

J-lAG

GGC

AAC

AnG

3510

His

He

Phe

Ala

Ser

He

Asp

Met

Pro

Ala

He

Asn

Lys

Gly

Asn

Lys

325 330

AAA GTC ACG GA GTAAGCAGGG GGCACAGGAC TCAGGGGCGA CCCGTGCGGG 3561

Lys Val Thr Glu

340

AGGGCCGCCG GGAAAGCACT GGCGAGGGGG CCAGCCTGGA GGAGGAAGGC ATTGAGTGGA 8621

GGACTGGGAG TGAGGAAGTT AGCACCGGTC GGGGTGAGTA TGCACACACC TTCCTGTTGG 8681

CACAGGCTGA GTGTCAGTGC CTACTTGATT CCCCCAG G GAG GAC TTC TAC AAG 8734

Glu Asp Phe Tyr Lys

345

CTG GTC AGT GAG

TTC ACA ATC ACC AAG

GGG CTC AGA GGC GCC AAG ACG

8782

Leu

Val

Ser

Glu 350

Phe

Thr

lie

Thr

Lys 355

Gly

Leu

Arg Gly

Ala 360

Lys

Thr

ACC

TTT

GAT

GTC

TAC

ACC

GAG

TCC

TGG

GCC

CAG

GAC

CCA

TCC

CAG

GAG

8830

Thr

Phe

Asp

Val

Tyr

Thr

Glu

Ser

Trp

Ala

Gin

Asp

Pro

Ser

Gin

Glu

365

370

375

AAT

AAG

AAG

AAG

ACT

GTG

GTG

GAC

TTT

GAG

ACC

GAT

GTC

CTC

TTC

CTG

8878

Asn

Lys

Lys

Lys

Thr

Val

Val

Asp

Phe

Glu

Thr

Asp

Val

Leu

Phe

Leu

380

385

390

GTG

CCC

ACC

GAG

ATT

GCC

CTA

GCC

CAG

CAC

AGA

GCC

AAT

GCC

AA

8922

Val

Pro

Thr

Glu

lie

Ala

Leu

Ala

Gin

His

Arg

Ala

Asn

Ala

Lys

395 400 405

GTGAGGATCT	GGGCAGCGGG	TGGCTCCTGG	GGGCCTTCCT	GGGGTGCTGC	ACCTTCCAGC	8982
CGAGGCCTCG	CTGTGGGTGG	CTCTCAGGTG	TCTGGGTTGT	CTGGGAAAGT	GGTGCTTGAG	9042
TCCCCACCTG	TGCCTGCCTG	ATCCACTTTG	CTGAGGCCTG	GCAAGACTTG	AGGGCCTCTT	9102
TTTACCTCCC	AGCCTACAGG	GCTTTACAAA	CCCTATGATC	CTCTGCCCTG	CTCAGCCCTG	9162
CACCCCATGG	TCCTTCCCAC	TGGAGAGTTC	TTGAGCTACC	TTCCATCCCC	CATGCTGTGT	9222
GCACTGAGAG	AACACTGGAC	AATAGTTTCT	ATCCACTGAC	TCTTATGGGC	CTCAACTTTG	9282
CCCATAATTT	CAGCCCACCA	CCACATTAAA	AATCTTCATG	TAATAATAGC	CAATTATAAT	9342
AAAAAATAAG	GCCAGACACA	GTAGCTCATG	CCTGTAATCC	CAGCACATTG	GGAGGTCAAG	9402
GTGGGAGGAT	CACTTGAGGT	CAGGAGTCTG	AGACTAGTCT	GGCCAACATG	GCAAAACCCC	9462
ATCTCTACTA	AAAATACAAA	AATTATCCAG	GCATGGTGGT	GCATGCCTAT	AATCCTAGCT	9522
ACTCAGGAGG	CTGAGGTAGC	AGAATTGATT	GACCCAGGGA	GGTGGAGGTT	GCAGTGAGCC	9582
GAGATTACGC	CACTGCACTC	CAGCAGGGGC	AACAGAGTGA	GACTGTGTCT	CGAATAAATA	9642
AGTAAATAAA	TAATAAAAAT	AAAAAATAAG	TTAGGAATAC	GAAAAAGATA	GGAAGATAAA	9702
AGTATACCTA	GAAGTCTAGG	ATGAAAGCTT	TGCAGCAACT	AAGCAGTACA	TTTAGCTGTG	9762
AGCCTCCTTT	CAGTCAAGGC	AAAAAGGGAA	ACAGTTGAGG	GCCTATACCT	TGTCCAATCT	9822
AATTGAAGAA	TGCACATTCA	CTTGGAGAGC	AAAATATTTC	TTGATACTGA	ATTCTAGAAG	9882

BAD original

AP 0 0 0 4 11

HX 1147 —50—

GAAGGTGCCT CACAATGTTT TGTGGAGGTG AAGTATAAAT TCAGCTGAAA TTGTGGAACC 9942

CATGAATCCA TGAATTTGGT TCTCAGCTTT CCCTTCCCTG GGTGTAAGAA GCCCCATCTC 10002

TTCATGTGAA TTCCCCAGAC ACTTCCCTGC CCACTGCCCG GGACCTCCCT CCAAGTCCGG 10062

TCTCTGGGCT GATCGGTCCC CAGTGAGCAC CCTGCCTACT TGGGTGGTCT CTCCCCTCCA 10122

G G AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC CAT CCC TCT CGG ATG 10169

Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met

410 415 420

CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT GCA GAT GAC ATT CAG 10217

Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gin

425 430 435 440

TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG GGC TAC CGG CCC CAA 10265

Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gin

445 450 455

GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC TGG ACC AAC TTT GCC 10313

Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn Phe Ala

460 465 470

AAA ACA GG GTAAGACGTG GGTTGAGTGC AGGGCGGAGG GCCACAGCCG 10361

Lys Thr Gly

475

AGAAGGGCCT CCCACCACGA GGCCTTGTTC CCTCATTTGC CAGTGGAGGG ACTTTGGGCA 10421

AGTCACTTAA CCTCCCCCTG CATCGGAATC CATGTGTGTT TGAGGATGAG AGTTACTGGC 10431

AGAGCCCCAA GCCCATGCAC GTGCACAGCC AGTGCCCAGT ATGCAGTGAG GGGCATGGTG 10541

CCCAGGGCCA GCTCAGAGGG CGGGGATGGC TCAGGCGTGC AGGTGGAGAG CAGGGCTTCA 10601

GCCCCCTGGG AGTCCCCAGC CCCTGCACAG CCTCTTCTCA CTCTGCAG G GAC CCC 10656

Asp Pro

AAC ATG GGC GAC TCG GCT GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG 10704

Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr

480 485 490

GAA AAC AGC GGC TAC CTG GAG ATC ACC AAG AAG ATG GGC AGC AGC TCC 10752

Glu Asn Ser Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser

495 500 505

ATG AAG CGG AGC CTG AGA ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC 10800

Met Lys Arg Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr

510 515 520 525

TAT CTG GCG CTG CCC ACA GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC 10848

Tyr Leu Ala Leu Pro Thr Val Thr Asp Gin Glu Ala Thr Pro Val Pro

530 535 540

CCC ACA GGG GAC TCC GAG GCC ACT CCC GTG CCC CCC ACG GGT GAC TCC 10896

Pro Thr Gly Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser

545 550 555

GAG ACC GCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG 10944

Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val

560 565 570

CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC 10992

Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp

575 580 585

BAD ORIGINAL fl

AP Ο Ο Ο 411

ΗΧ 1147

TCC Ser 590

GGG Gly

GCC Ala

CCC Pro

CCC Pro

GTG Val 595

CCG Pro

CCC Pro

ACG GGT

GAC Asp 600

TCC GGG GCC

CCC Pro

CCC Pro 605

11040

Thr

Gly

Ser

Gly

Ala

GTG

CCG

CCC

ACG

GGT

GAC

TCC

GGG

GCC

CCC

CCC

GTG

CCG

CCC

ACG

GGT

1103S

7a 1

Pro

Pro

Thr

Gly 610

Asp

Ser

Gly

Ala

Pro 615

Pro

Val

Pro

Pro

Thr 620

Gly

GAC

TCC

GGG

GCC

CCC

CCC

GTG

CCG

CCC

ACG

GGT

GAC

TCC

GGC

GCC

CCC

11135

Asp

Ser

Gly

Ala 625

Pro

Pro

Val

Pro

Pro 630

Thr

Gly

Asp

Ser

Gly 635

Ala

Pro

CCC

GTG

CCG

CCC

ACG

GGT

GAC

GCC

GGG

CCC

CCC

CCC

GTG

CCG

CCC

ACG

11184

Pro

Val

Pro 640

Pro

Thr

Gly

Asp

Ala 645

Gly

Pro

Pro

Pro

Val 650

Pro

Pro

Thr

GGT

GAC

TCC

GGC

GCC

CCC

CCC

GTG

CCG

CCC

ACG

GGT

GAC

TCC

GGG

GCC

11232

Gly

Asp 655

Ser

Gly

Ala

Pro

Pro 660

Val

Pro

Pro

Thr

Gly 665

Asp

Ser

Gly

Ala

CCC

CCC

GTG

ACC

CCC

ACG

GGT

GAC

TCC

GAG

ACC

GCC

CCC

GTG

CCG

CCC

11280

Pro 670

Pro

Val

Thr

Pro

Thr 675

Gly

Asp

Ser

Glu

Thr 680

Ala

Pro

Val

Pro

Pro 685

ACG

GGT

GAC

TCC

GGG

GCC

CCC

CCT

GTG

CCC

CCC

ACG

GGT

GAC

TCT

GAG

11328

Thr

Gly

Asp

Ser

Gly 690

Ala

Pro

Pro

Val

Pro 695

Pro

Thr

Gly

Asp

Ser 700

Glu

GCT

GCC

CCT

GTG

CCC

CCC

ACA

GAT

GAC

TCC

AAG

GAA

GCT

CAG

ATG

CCT

11376

Ala

Ala

Pro

Val

Pro

Pro

Thr

Asp

Asp

Ser

Lys

Glu

Ala

Gin

Met

Pro

705 710 715

GCA GTC ATT AGG ΤΤΤ TAGCGTCCCA TGAGCCTTGG TATCAAGAGG CCACAAGAGT 11431

Ala Val lie Arg Phe

720

GGGACCCCAG GGGCTCCCCT CCCATCTTGA GCTCTTCCTG AATAAAGCCT CATACCCCTG 11491

TCGGTGTCTT TCTTTGCTCC CAAGGCTAAG CTGCAGGATC 11531

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Claims (31)

1. A DNA molecule encoding for biologically functional BSSL/CEL and containing intron sequences .

2. A DNA molecule according to claim 1 which is shown in SEQ ID NO: 1 in the Sequence Listing.

3. An analogue of the DNA molecule according to claim 2 which contains intron sequences and which hybridizes with the DNA sequence shown in SEQ ID NO: 1 in the Sequence Listing or a specific parr thereof under stringent hybridization conditions.

4. A replicable expression vector which carries and is capable of mediating the expression of a DNA molecule according to any one of claims 1-3, encoding human BSSL/CEL.

5. A vector according to claim 4 capable of encoding biologically funtional BSSL/CEL and containing regulatory elements directing expression to the mammary gland of a non-human mammal.

6. A vector according to claim 4 which is the vector pS452 (DSM 7499).

7. A cell derived from a multicellular organism and harbouring a vector according to any one of claims 4-6.

8. A process for production of human BSSL/CEL, comprising (a) inserting a DNA molecule as defined in any one of claims 1-3 in a vector which is able to replicate in a specific host cell; (b) introducing the resulting recombinant vector into a host cell; (c) growing the

BAD ORIGINAL fi

AP Ο Ο Ο 4 1 1

HX 1147 —53— resulting cell in or on a culture medium for expression of the polypeptide; and (d) recovering the polypeptide.

5

9. A mammalian expression system, comprising a hybrid gene which is expressible in the mammary gland of an adult female of a non-human mammal harbouring said hybrid gene, so that human BSSL/CEL is produced when the hybrid gene is

10 expressed, said hybrid gene being produced by inserting a DNA molecule according to any one of claims 1-3 into a gene regulating a milk protein gene of a non-human mammal.

10. A hybrid gene as defined in claim 9.

11. A mammalian cell harbouring an expression system as defined in claim 9.

12. A non-human mammalian cell according to claim 11 which is an embryo cell.

13. A process of producing a transgenic non-human mammal capable of expressing human BSSL/CEL,

25 comprising (a) introducing an expression system as defined in claim 9 into a fertilized egg or a cell of an embryo of a non-human mammal so as to incorporate the expression system into the germline of the mammal and (b) developing the

30 resulting introduced fertilized egg or embryo into an adult female non-human mammal.

14. A process of producing a transgenic non-human mammal capable of expressing human BSSL/CEL and

35 substantially incapable of expressing BSSL/CEL from the mammal itself, comprising (a) destroying the mammalian BSSL/CEL expressing capability of the mammal so that substantially no mammalian

BAD ORIGINAL ft

AP Ο Ο Ο 41 1

HX 1147

-54BSSL/CEL is expressed and inserting an expression system as defined in claim 9 into the germline of the mammal in such a manner that human BSSL/CEL is expressed in the mammal; and/or (b) replacing

5 the mammalian BSSL/CEL gene or part thereof with an expression system as defined in claim 9.

15. A transgenic non-human mammal harbouring in its genome a DNA molecule according to any one of

10 claims 1-3.

Ιό. A transgenic non-human mammal according to claim 15 in which the DNA molecule is present in the germline of the mammal.

17. A transgenic non-human mammal according to claim 15 or 16 in which the DNA molecule is present in a milk protein gene of the mammal.

20

18. A transgenic non-human mammal prepared by the process of claim 13 or 14.

19. A transgenic non-human mammal according to any one of claims 15-18 which is selected from the

25 group consisting of mice, rats, rabbits, sheep, pigs and cattle.

20. Progeny of a transgenic non-human mammal according to any one of claims 15-19.

21. Milk obtained from a transgenic non-human mammal according to any one of claims 15-20.

22. An infant formula comprising milk as defined in

35 claim 21.

23. A process for production of an infant formula by supplementing an infant food formula with a

BAD ORIGINAL &

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HX 1147 —55— polypeptide encoded by a DNA molecule according to any one of claims 1-3.

24. Use of a DNA molecule according to any one of

5 claims 1-3 for the production of human BSSL/CEL.

25. The use according to claim 24 for production of a transgenic non-human mammal expressing human BSSL/CEL.

26. The use according to claim 24 for production of milk, containing human ESSL/CEL, derived from a transgenic non-human mammal.

15

27. The use according to claim 24 for production of an infant formula comprising milk, containing human BSSL/CEL, derived from a transgenic nonhuman mammal.

28. The use of a DNA molecule according to any oneof claims 1-3 in the manufacture of a medicament for the treatment of a pathological condition related to exocrine pancreatic insufficiency.

25

29. The use according to claim 28 in the manufacture of a medicament for the treatment of cystic fibrosis .

30. The use according to claim 28 in the manufacture

30 of a medicament for the treatment of chronic pancreatitis .

31. The use according to claim 28 in the manufacture of a medicament for the treatment of fat

35 malabsorption.

BAD ORIGINAL

AP Ο Ο Ο 4 1 1

HX 1147

32 .

5 33 .

—56—

The use according to claim 28 in the manufacture cf a medicament for the treatment of malabsorption of fat soluble vitamins.

The use according to claim 28 in the manufacture of a medicament for the treatment of fat malabsorption due to physiological reasons.