WO1996018734A1 - Production of recombinant secretory component - Google Patents

Abstract

The current invention relates to a recombinant secretory component (rSC) obtainable from a Chinese Hamster Ovary (CHO) cell that can be crystallized and, hence, is susceptible to systematic studies of its 3-dimensional structure. The crystallized form may be used directly or indirectly (e.g. via the derived structure) for lead finding, screening and binding studies.

Description

PRODUCTION OF RECOMBINANT SECRETORY COMPONENT

The current invention relates to a new form of a recombinant secretory component (rSC), obtainable from a recombinant Chinese Hamster Ovary (CHO) cell line, that can be crystallized and, hence, is susceptible to systematic studies of its 3-dimensional structure. The crystallisable form may be used directly (e.g. to stabilize immunoglobulins) or indirectly (e.g. via the derived structure) for lead finding, screening and binding studies.

In vivo, the secretory component (SC) is synthesized as the extracellular part of an integral membrane glycoprotein of specific cells which are responsible for the transport of polymeric immunoglobu'ⁱn (Ig) to external mucosal surfaces. Such cells include epithelial cells found in several tissues including those in the lining of the respiratory, gastrointestinal, biliary and urogenital tracts and in the salivary, lacrimal, and mammalian gland and also hepatocytes. This membrane protein is termed polymeric immunogobulin receptor (plgR) and specifically binds polymeric IgA (poly-lgA) and IgM (poly-lgM) on the basal side of the cells. During transport of the plgR/poly-lg complex from the basal to the apical (extracorporal) side of the cell, the plgR is cleaved thus forming the SC. Most of the SC is released from the cell as part of the SC/poly-lg complex, but also free SC is found in external secretions. In the SC/poly-lg complex, SC is thought to stabilize the quaternary structure of poly-lg and to increase resistance of the complex to various proteolytic enzymes. This resistance, for example to digestive proteases, is an important if not essential prerequisite for the protective function of secretory immunoglobulins.

Accordingly, SC usually occurs in three molecular forms:

- as a membrane protein on the surface of epithelial cells and hepatocytes;

- as a component chain of secretory IgA and soluble IgM; and

- as a free glycoprotein in several external secretions.

Based on cDNA (SEQ ID NO 1 ), the deduced amino acid sequence of human plgR (SEQ ID NO 2) has a length of 764 residues and shows overall similarity of 56% and 64% with the rabbit and rat counterparts, respectively (Krajci et al., Human Genetics (1991 ), 87, 642- 648). Free SC isolated from human milk is a 78-kDa single chain glycoprotein with a content of up to 20% carbohydrate. The primary sequence analysis indicates that SC consists of five domains, each approximately 100 amino acids in length. These domains are homologous in size and sequence to the domains characteristic of the immunoglobulin superfamily of proteins. As described above, plgR mediates the transport of IgA and IgM into mucosal secretions. These mucosal antibodies are of enormous importance in the immune response. They are capable of inactivating pathogens that have not yet entered the body and those that do not enter the body but which cause disease by secreting toxins which are taken up by the body. Accordingly, antagonists and, preferable, agonists of SC are of great pharmaceutical value for the modulation of mucosal immune response.

For effective drug design it is important to know the 3-dimensional structure of a receptor. Despite various efforts, neither the plgR nor native SC have been crystallized successfully yet.

The glycosylation pattern of a recombinant glycoprotein, such as rSC, is determined by certain ill-understood determinants in the amino acid sequence of the protein, and by the availability of competing processing enzyme activities which determine the final structure of the glycans. Thus different glycosylation variants, termed glycoforms, of the same polypeptide can be produced by different cell lines, depending on the distribution of competing oligosaccharide processing enzyme activities in these cell lines. The type of glycosylation of the glycoprotein influences functional and structural parameters of the protein. For example, glycosylation often enhances stability of the polypeptide in the presence of proteolytic enzymes. On the other hand, substantial glycosylation, as especially in the case of SC, often interferes with attempts to crystallize the protein.

Recombinant glycoproteins expressed in CHO cells are known to contain carbohydrate chains terminally substituted with sialic acids. The most prominent sialic acids, α 2-3-linked N,O-acetylneuraminic acids (NeuAc) and α2-3-linked N-glycolylneuraminic acid (NeuGc), are present in a ratio of about of about 97:3 (Hokke et al., FEBS Lett. (1990), 275, 9-14). The occurrence of N-acetylneuraminate mono-oxygenase (EC 1.14.99.18) activity, responsible for conversion of CMP-Neu5Ac into CMP-Neu5GC acting as precursor for incorporation of NeuδGc in the glycoprotein, has not been demonstrated to come to expression in normal adult human tissue. Extensive studies have shown that when normal adult humans are exposed to sera of animal species, immunogenic responses may occur. The so-called Hanganutziu-Deicher (serum sickness) antibodies are directed towards glycoconjugates containing terminal Neu5Gc, α2-3-linked to β-Gal. Surprisingly we found that CHO SSF 3 cells incorporates NeuGc in the carbohydrate chains of rSC in amounts much lower than thus far observed for a typical glycoprotein produced by any other CHO cells. The ratio of NeuGc for rSC produced in CHO SFF3 cells was below 0.25% of total sialic acid whereas a typical recombinant glycoprotein produced in CHO cells contains about 3%. The reduced content in NeuGc, usually not present in human glycoproteins, makes rSC produced by CHO SSF 3 cells more human-like and thus less antigenic and safer for human applications.

A further surprising fact is, that it is possible to produce glycoforms of recombinant SC (rSC), which are both active in binding polymeric immunoglobulin and can be crystallized. The inventive glycosylated rSC can be crystallized, e.g., by the 'hanging drop method' and provides an ideal starting point for the evaluation of the 3-dimensional structure of SC and facilitate the search for SC antagonists and, preferably, SC agonists including muteins of the SC polypeptide itself.

Detailed description of the invention

The current invention relates to a recombinant secretory component (rSC) or a functional fragment thereof, obtainable by a process comprising culturing a CHO SSF 3 cell transfected with a vector comprising a DNA coding for said secretory component or a functional fragment thereof, and isolating the expressed protein from the culture medium.

The rSC as defined above has, for example, a content of N-glycolylneuraminic acid (NeuGc) that is below 0.5% in respect to total sialic acid. Hence, the inventive rSC exhibit, for example, decreased antigenicity in human.

The inventive process preferably comprises the following steps: a) constructing a vector capable of expressing the secretory component or a functional fragment thereof; b) transfecting a CHO SSF 3 cell with said vector; c) culturing the transfected cells; and d) isolating the secretory component or a fragment thereof from the culture medium.

Secretory component (SC)

The plgR is a receptor capable to bind to poly-lg, especially IgA and IgM. Human plgR usually has nucleotide sequence as depicted in SEQ ID NO 1 , and an amino acid sequence basically as depicted in SEQ ID NO 2. The plgR is cleaved during transport of the plgR/poly-lg complex from the basal to the apical (extracorporal) side of the cell thus forming the SC. A functional fragment of rSC is a derivative of plgR that has one or more amino acid deletions or modifications, that is capable of binding to poly-lg, especially poly- IgA, and/or that reacts in the presence of antagonists and, preferable, agonists comparable to natural SC. A preferred fragment is, for example, a soluble fragment that lacks a membrane anchoring domain that resides, e.g., between amino acids 639-661 of the plgR, and/or the intracellular C-terminal domain between amino acids 662-764. Especially preferred are fragments as depicted in SEQ ID NO 3 and SEQ ID NO 4.

The DNA fragments coding for said rSC or the fragment thereof may be modified, e.g., in so far as to adapt the codons to the preferred codon usage of the host, or the DNA sequence immediately in front or behind of the coding sequence may be modified in order to enhance the transcription, stabilize the mRNA produced, or to ease the genetic modifications, e.g., by an insertion of restriction sites.

Plasmids

The DNA coding for the secretory component or a functional fragment thereof, as described above, usually is comprised in a polypeptide expression cassette capable of expressing said DNA. In a preferred expression cassette according to the invention, a promoter is operably linked to said DNA that is transcribed under the control of this promoter, and to a terminator.

The promoter can be of almost any origin. It is for example possible to use a tightly regulated promoter or the promoter that is naturally adjacent to said DNA. Preferred are promoter that are active in CHO cells like viral promoters such as the 'early' promoter of SV40, the immediate early promoter of a cytomegalovirus (mouse, simian or human), or cellular promoters such the promoter of the β-actin gene, the metallothionein gene, or the heat shock genes. Especially preferred is the SV40 early promoter for the NEO gene, the mouse or human cytomegalovirus immediately early promoter for the SC and the human adenovirus type 2 promoter for the DHFR gene.

A DNA sequence containing the transcription termination signals is preferably the 3' flanking sequence of a gene which contains proper signals for transcription termination and polyadenylation for the desired host. Suitable signals are, for example, the polyadenylation signal of cellular genes such as the human growth hormone or the rabbit β-globin gene, or of viral genes the as those of the SV40 'early' and 'late' genes. The plasmids may also contain fragments of DNA that increase the stability of the plasmid in the desired host or that ease the integration of the plasmid DNA or the essential part thereof into the chromosome of the desired host. Examples for suitable DNA fragments are the long terminal repeats of retroviruses, in case the recombinant genes are to be transferred as retrovirus particles, viral origins of replication, such as SV40, EBV, AAV, vaccinia, papillomavirus, Semliki forest virus etc., or DNA segments containing recognition sites for site-specific recombinases such as CRE and FLP.

The promoter, the DNA sequence coding for the secretory component or a functional fragment thereof and the DNA sequence containing transcription termination signals are operably linked to each other, 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 SC gene and the transcription termination signals effect proper termination of transcription and polyadenylation. The junction of these sequences may, for example, be effected by means of synthetic oligodeoxynucleotide linkers carrying the recognition sequence of a specific endonuclease.

The expression cassettes according to the invention may be maintained in the desired host in form of a stable episome or plasmid or as part of the chromosome, wherein the latter case is preferred.

However, it is likewise possible that the expression plasmids according to the invention include one or more, especially one or two, selective genetic markers for the host used for the construction, amplification and test of the plasmid, such a marker and an origin of replication for a bacterial host, especially Escherichia coli.

As to the selective gene markers, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers are, for example, those expressing resistance to an antibiotic or another antimetabolite or, in the case of auxotrophic host mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotics tetracyclin, ampicillin, G418, hygromycin, puromycin or bleomycin or provide for prototrophy in an (conditionally) auxotrophic mutant, for example the thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene and the E. coli gpt, HisD or Trp genes. These selectable marker genes can also be provided by cotransfection of the expression gene with a physically unlinked selection gene. After such a cotransfection the enzymatic machinery will with great probability cointegrate the two gene sets such that elimination of untransfected cells is possible.

As the amplification of the expression plasmids is usually done in a prokaryote, such as E. coli., a prokaryote, e.g. E. coli, genetic marker and a prokaryote, e.g. E. coli, replication origin are included advantageously. These can be obtained from corresponding prokaryotic plasmids, for example E. coli plasmids, such as pBR322, pTZ18R, or a pUC plasmid, for example pUC18 or pUC19, which contain both prokaryotic, e.g. £ coli, replication origin and genetic marker conferring resistance to antibiotics, such as ampicillin and tetracyclin.

Apart from the polypeptide expression cassette, replication origin(s) and genetic marker(s) the expression plasmids according to the invention can contain optionally additional expression cassettes, such as 1 to 3 additional polypeptide expression cassettes, which may be the same or different.

Examples for suitable vectors are mammalian cell expression vectors based, for example, on pEUK-C1 (Clontech Inc., Palo Alto, California, USA ), pcDNAlneo (Invitrogen Corp. San Diego California, USA) pCGA28 (Asselbergs et al. Fibrinolysis (1993), 7, 1 -14) or PCGA93D-PPREN (Asselbergs etal., Biotech. (1994), 32, 191-202).

The expression plasmids according to the invention are prepared by methods known in the art, for example by linking the polypeptide expression cassette, the DNA fragments containing selective genetic markers for the host used in the test and optionally for a bacterial host, the origin(s) of replication, and the optionally additional polypeptide expression cassettes in the predetermined order using conventional chemical or biological in vitro synthesis procedures. Preferentially, the plasmids are constructed and prepared using recombinant DNA techniques. For the preparation by recombinant DNA techniques suitable DNA fragments are ligated in vitro in conventional manner. The ligation mixture is then transformed into a suitable prokaryotic or eukaryotic host depending on the nature of the regulatory elements used, and a transformant containing the desired vector is selected according to conventional procedures. The plasmids can be multiplicated by means of the transformed hosts and can be isolated in conventional manner. The choice of the host depends on the regulatory sequences located on the vector. For the construction and multiplication of the vector a prokaryotic host, e.g., E. coli, is preferred. Hosts, transfection and culturing

A suitable host for the production of rSC is a CHO SSF 3 cell (Gandor, C.R. (1993) Establishment and characterization of growth-factor-prototrophic Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, Zurich: Dissertation Nr 10087, Swiss Federal Institute of Technology.) or a cell that is derived therefrom and produces the same glycoforms of rSC.

The suitable host, as defined above, can be transfected by the standard methods in genetic engineering, as for example with the aid of cationic lipid vesicles, electroporation or particle gun. To increase the amount of rSC produced, it is advantageous to use a high copy plasmid or the plasmid DNA is integrated into the genome in several copies. The latter can be achieved, for example, via an amplification with methotrexate as described for example in (Asselbergs et al. J. Biotechnol. (1994), 32, 191-202,; Asselbergs et al. J. Biotechnol. (1992), 23, 143-151 ; Asselbergs etal. J. Mol. Biol. (1986), 189, 401-411 and Kaufman et al. Mol. Cell Biol. (1985.), 5, 1750-1759,).

The modified CHO SSF 3 cell can be cultured by standard methods in cell culture. In a preferred embodiment of the invention the cells are cultured in a serum-free medium and more preferred in a serum- and protein-free medium.

Surprisingly, it has been found, that the addition of Plutonic to the culture medium has a productivity enhancing effect. Although, the mean doubling time is about 20% lower than in the absence of Pluronic the daily yield of rSC is several times higher. This selective effect on the cell specific productivity is surprising and has not been observed yet. The amount of Pluronic , especially Pluronic F-68 , added to the culture medium, is preferably about 0.005 to 0.5% (w/v) and more preferred 0.01 to 0.1 % (w/v).

Isolation

The rSC produced by the inventive method is secreted predominantly in to the culture medium. It can be isolated therefrom by conventional means. During the isolation conventional additives like protein stabilizers, inhibitors of proteinases and the like may be added. For example, the first step consists usually in separating the cells from the culture fluid by means of centrifugation or filtration. In the presence of additional proteins and impurities, the resulting supernatant can be enriched for rSC. Representative purification schemes include, e.g., treatment with polyethyleneimine as to remove most of the non- proteinaceous material, and precipitation of proteins by saturating the solution with ammonium sulfate or the like, ultrafiltration, diafiltration, gel electrophoresis, carrier-free electrophoresis, chromatographic processes such as ion exchange chromatography, size exclusion chromatography, partition chromatography, affinity chromatography, HPLC, reverse phase HPLC, treatment with Sephadex , dialysis, or by other processes, especially those known from the literature. Those skilled in the art would appreciate that a combination of purification schemes can be used. In general, only a few purification steps are required in order to obtain a rSC product which is essentially free of contaminants.

Crystallization

A further embodiment of the invention is a method for the crystallization of the secretory component (SC) according to the invention comprising placing a solution of said secretory component in a vessel containing a precipitating agent buffer, wherein the solution and the buffer are separated. Common methods and details for the crystallization of protein according to the 'hanging drop' method are, for example, described in Mc Pherson, A (1982): Preparation and Analysis of protein crystals. John Wiley and Sons, NY.

To crytallize the protein the solution of the secretory component or the functional fragment thereof can contain a precipitating agent buffer. It is, for example, preferred to mix the solution comprising the inventive rSC and the precipitating agent buffer in an amount of 1:2 to 2:1 or, preferred, in about equal amounts.

The precipitating agent buffer usually contains in addition to the compounds used to establish a certain pH, one or more hygroscopic compounds and preservatives. Examples for suitable ingredients are NaN₃, Na-citrate, HEPES, ammonium phosphate, and/or Li₂SO₄. Preferred buffers comprise for example a mixture of Na-citrate, ammonium phosphate and NaN₃; or a mixture of HEPES, Li₂SO₄ and NaN₃.

In a further preferred method the solution comprising rSC is placed in a hanging manner over the precipitating agent buffer (hanging drop method).

The crystallization is carried out preferred at temperatures from 3°C to 30°C, more preferred from 5°C to 25°C, and especially preferred at room temperature. Use of the isolated rSC

The isolated and crystallized rSC as described above can be used to identify the 3- dimensional structure of the whole protein or at least of the areas responsible for binding and secretion of poly-lg. Conventional methods for the identification of the 3-dimensional structure are, for example, X-ray studies or NMR studies. The data received with these or comparable methods may be used directly or indirectly for the identification of antagonists or, preferably, agonists of the rSC mediated IgA transport. A commonly used method in this respect is, for example, computer aided drug design or molecular modeling.

The highly enriched rSC also may be used directly for binding studies and in the screening of compounds for their ability to influence poly-lg binding. For these tests rSC according to the invention may be, for example, immobilized on a solid carrier like a micro titer plate or on beads; or may bear one or more identifiable maker like biotin or a radioactive, fluorescent or chemoluminescent group.

A further embodiment of the invention concerns the antagonist or, preferably, agonist identified with the inventive rSC, or with the aid of the 3-dimensional structure derived therefrom, for use in a method of treatment.

The inventive rSC has valuable pharmaceutical properties because of its lower immunogenicity in respect to previously known SC. This lower immunogenicity is based, e.g., on a low content of NeuGc (0.25% or less of the total sialic acid content). Accordingly, a further embodiment of the invention concerns the use of the inventive rSC as defined above in a method of treatment, e.g., in the stabilization of poly Ig, especially poly IgA.

EXAMPLES:

The following examples illustrate the invention and should not be construed as a limitation thereof.

Standard methods in genetic engineering like cleavage with restriction enzymes, ligations, transformation and annealing are carried out essentially as described in Sambrook et al., Molecular Cloning: A laboratory manual, 2^nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989. Example 1 : human polymeric immunoαlobuline receptor cDNA

The cDNA sequence of the human polymeric immunoglobuline receptor (plgR, SEQ ID NO:1) is known (Krajci et al., Biochem. Biophys. Res. Commun. (1989), 158, 783-789; Krajci et al., Hum. Genet. (1991), 87, 642-648; Piskurich et al., Mol. Immunol. (1993), 30, 413-421 ; SEQ ID NO 1). Such cDNA can be generated using standard methods in genetic engineering, e.g., by reverse transcription of mRNA from samples of tissue expressing plgR (Krajci et al., 1989, opus cit.) or from a publicly available cell line such as HT29 (ATCC HTC-38, Piskurich et al., Mol. Immunol. (1993) 30, 413 -421). The cloned cDNA can be identified by hybridization with PCR fragments generated from the cDNA mixture with primers designed using the cDNA sequences in the public domain.

The cDNA is cloned in a plasmid vector, which can be multiplied in E. coli. Plasmid DNA is prepared according conventional procedures (Sambrook et al., Molecular Cloning: A laboratory manual, 2^nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) and the nucleotide sequence of the cDNA sequence is determined. The natural coding sequence of the precursor of the plgR protein (SEQ ID NO 2) starts with an ATG methionine codon and ends with a TAG stop codon. Within the coding sequence significant features are a) the presence of a hydrophobic leader sequence immediately following the initiation codon (amino acids 1 -18) and b) a second stretch of hydrophobic amino acids (639-661 ) corresponding to the transmembrane portion, which separates the extracellular amino-terminal part from the intracellular carboxyterminal portion of plgR.

The leader peptide is cleaved off from the precursor polypeptide within the cell prior to presentation of the receptor protein on the cell membrane.

Example 2: PCR mutaαenesis of human plgR cDNA

An artificial DNA molecule encoding secretory component is generated by creation of a stop codon immediately before the transmembrane segment using PCR mutagenesis. For this purpose the information contained in DNA sequence encoding pre-plgR is sufficient, but optionally, vector DNA sequences lying upstream of the ATG initiation codon can be used in the creation of a DNA molecule coding for secretory component.

The polymerase chain reaction (PCR) reaction is done with two synthetic oligonucleotide primers, here termed forward and backward primer (terms relating to the direction of transcription primed by the respective oligonucleotides relative to the direction of the plgR translational reading frame).

The forward PCR primer is chosen such that the functional properties of the leader peptide are preserved. The simplest way to achieve this is, is not altering the natural leader peptide sequence. The primer sequence upstream of the ATG codon is further chosen such that a convenient restriction site, which is compatible with a restriction site in a suitable expression vector, is created. One preferred restriction site is that of the restriction endonuclease Hindlll, AAGCTT. A preferred DNA molecule for PCR modification is plgR cDNA cloned in vector pCB6. In this vector the plgR cDNA is cloned downstream of the major immediate early promoter of the human cytomegalovirus. In this case, a primer hybridizing to the cytomegalovirus promoter segment can be used, preserving restriction sites already present in the vector between the priming site and the position of the ATG codon. A preferred oligonucleotide primer is 5' PCR primer 1 (SEQ ID NO 5).

A second criterion used in the design of the oligonucleotide is that the sequence around the ATG codon is such that the initiation of protein synthesis at the ATG is efficient. The DNA sequence found upstream of the ATG codon in natural mRNAs is not always optimal as under natural conditions only limited amounts of a protein are needed. The criteria for optimal initiation efficiency are known (Kozak et al., Nucleic Acids Res. (1987), 15, 8125- 8148; Peabody D.S. in Setlow J.K. ed. Genetic Engineering Vol. 12, pp. 99-76). It is preferred that the nucleotide immediately following the ATG is G. This is not the case in the natural plgR cDNA, but can be achieved be designing the forward PCR primer accordingly. However, this can only be achieved by changing the codon of the second amino acid, leucine, to one starting with G, coding either for valine, alanine, aspartate, glutamate or glycine. The preferred amino acid is valine. This is a conservative substitution as both amino acids belong to the class of hydrophobic amino acids. The functional properties of the peptide leader sequence in promoting secretion of the protein are preserved. One preferred DNA sequence immediately upstream of the ATGG is CC, thus situating the ATG codon within the recognition site of the restriction endonuclease Ncol, CCATGG). This Ncol site is compatible with Ncol sites is many efficient expression vectors. Another preferred DNA sequence immediately upstream of the ATGG is GCCACC. A preferred 5' PCR primer, which creates also a Hindlll site upstream of the ATG, is 5' PCR primer 2 (SEQ ID NO 6).

The backward primer is designed with two main criteria in mind. Firstly, it serves to generate a stop codon at position 1906 just upstream of the hydrophobic transmembrane segment of the plgR protein. The primary translation product will thus terminate with Ser- Glu-Glu-Gln-Gly-Gly-COOH. Secondly, it serves to create downstream of the new stop codon a restriction endonuclease site compatible with a restriction site in a suitable expression vector. One preferred stop codon is TGA and a preferred restriction site is that of Xbal, TCTAGA as created by preferred 3' PCR primer 1 (SEQ ID NO 7).

Example 3: Polymerase chain reaction

A DNA fragment is transcribed by a heat-stable DNA polymerase using a specific forward and 3' PCR primer as described in example 2 and with plgR cDNA cloned in a plasmid vector as template. The PCR reaction is done (according to Sambrook et al., Molecular Cloning: A laboratory manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) in a buffered solution containing the four deoxyribonucleotide triphosphates and the temperature is of the incubation controlled such that multiple cycles of primer-driven transcription of the SC-coding fragment occur. This produces a DNA fragment terminating with DNA sequences of the two oligonucleotide primers. A Hindlll- Xbal fragment encoding SC is generated from this DNA fragment by first separating the SC DNA fragment from the primers and other components of the PCR reaction and then digesting this DNA to completion with Hindlll and Xbal. Subsequently, the 1.9 kb Hindlll- Xbal fragment purified from the restriction enzyme reaction and is ready for ligation to the restricted expression vector DNA.

The PCR product generated in this way with 5' PCR primer 1 (SEQ ID NO 5) and 3' PCR primer 1 (SEQ ID NO 7) is called SC DNA fragment 1 (SEQ ID NO 3). The PCR product generated in this way with 5' PCR primer 2 (SEQ ID NO 6) and 3' PCR primer 1 (SEQ ID NO 7) is called SC DNA fragment 2 (SEQ ID NO 4).

Example 4: Construction of SC expression vectors with the oeneticin resistance gene

The artificial DNA segment encoding secretory component (SC) is inserted between restriction endonuclease sites in a suitable mammalian cell expression vector. One preferred expression vector is pCB6, of which the main features are listed in Table 1 and that can be synthesized easily with standard methods in genetic engineering:

The vector pCB6 or pCB6 containing the plgR cDNA (pCB6plgR) is cut to completion with Hindlll and Xbal and the 6.1 kb fragment is isolated. This DNA fragment is ligated using DNA ligase (from bacteriophage T4 or another enzyme with ligation activity) to SC DNA fragment 1. This ligation product is used to transform E. coli DH5α and ampicillin resistant strains are isolated. A plasmid DNA is isolated from such an ampicillin resistant strain is termed pCB6-SC and is structured as pCB6, but with the 1.9 kb SC DNA fragment 1 (Example 3) replacing the small Hindlll-Xbal fragment of pCB6.

An alternative expression vector is constructed from pCB6-SC, by replacing the major immediate early promoter from the human cytomegalovirus, by the more powerful major immediate early promoter from the mouse cytomegalovirus (mCMV). This promoter is obtained together with the beginning of the ampicillin resistance gene as a 1.1 kb Pvul- Hindlll fragment from plasmid pCGA93D-PPREN (Asselbergs et al. J. Biotechnol. (1994), 32, 191-202). In parallel, pCB6 is cut with Pvul and Hindlll and the largest fragment (6.1 kb), which contains all the structural elements of the expression vector (see table 1 ) except of the human cytomegalovirus promoter and the beginning of the pTZ18R-derived ampicillin resistance gene, is purified. The 1.1 kb and the 6.1 kb fragment are ligated and after transformation of E. coli with the ligation product ampicillin resistant colonies containing plasmid pMC-SC are obtained. pMC-SC is functionally equivalent to pCB-6 (see table 1) except that the human cytomegalovirus promoter is replaced by the murine one. Example 5 : Construction of SC expression plasmid pCGA93D-SC

The artificial DNA segment encoding secretory component (SC) is inserted between restriction endonuclease sites in a suitable mammalian cell expression vector. One preferred expression vector is pCGA93D-PPREN which is constructed according to Asselbergs et al., J. Biotech. (1994), 32, 191-202, and of which the main features are listed in Table 2:

The vector pCGA93D-PPREN is cut to completion with BamHI and the large 4.9 kb fragment is isolated, self-ligated and used to transform E. coli DH5α (the plasmid is termed pINTERMEDI ). From an ampicillin resistant strain plasmid pINTERMEDI (4.9 kb) is purified. In pINTERMEDI the Hindlll and Xbal sites flanking the prorenin cDNA are unique restriction sites. pINTERMEDI is cut to completion with Hindlll and Xbal and the 3.5 kb fragment is isolated. This DNA fragment is ligated using DNA ligase (from bacteriophage T4 or another enzyme with ligation activity) to SC DNA fragment 2 (Example 3). This ligation product is used to transform E. co// DH5α and ampicillin resistant strains are isolated. A plasmid DNA is isolated from such an ampicillin resistant strain is termed plNTERMED2 (6.8 kb). plNTERMED2 is cut to completion with Sail and Xbal and the large fragment (6.5 kb) is isolated. In parallel pCGA93D-PPREN is cut to completion with Sail and Xbal and the 3.3 kb fragment is isolated. The two DNA fragments are ligated and the product is used to transform E. coli DH5α. From an ampicillin and tetracyclin resistant strain thus obtained pCGA93D-SC is isolated. This plasmid is structured like pCGA93D-PPREN, but with the SC coding DNA replacing the preprorenin coding DNA. In pCGA93D-SC the SC coding DNA starts with an Ncol site and the DNA sequence around the ATG initiation codon of SC allows efficient initiation of translation of the recombinant SC mRNA.

Example 6 : Expression of human secretory component in CHO SSF3 cell transfected with pCB6-SC or pMC-SC

CHO SSF 3 cells are known (Gandor C.R., Establishment and characterization of growth- factor-prototrophic Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, Zurich: Dissertation #10087, Swiss Federal Institute of Technology, 1993). A cell stock is maintained in FMX-8 medium (Dr. F. Messi Cell Culture Technologies, Rohrstrasse 29, CH-8152 Glattbrugg/Zϋrich) without further additives. If transfection is going to be performed without serum, one to ten million cells are pelleted at low speed in a centrifuge and the cells are resuspended at 200O00 cells/ml in fresh FMX-8 medium of 37°C. In the alternative procedure, a dense culture of CHO SSF 3 cells is diluted 10-fold in FMX-8 medium with 4% fetal calf serum and plated in 3 cm diameter tissue culture Petri dishes. Cultured in this way the CHO SSF 3 cells form a cell monolayer, which adheres to the plastic of the Petri dish. When this monolayer is 30-50% confluent, they can be used for transfection.

The solutions for transfection are prepared in a polystyrene vessels to prevent adsorption of the cationic lipid used and the complex formed of the cationic lipid and DNA to the vessel wall. The pCB6-SC (example 3) DNA is prepared for transfection as follows: 4 μg plasmid DNA is dissolved in 0.1 ml FMX-8 medium. Separately 14 μl cationic lipid solution (lipofectin, GIBCO) is diluted in 0.1 ml FMX-8 medium. Subsequently, the DNA solution and the lipofectin solution are carefully mixed and incubated at room temperature for 15 min to allow formation of a lipofectin-DNA complex.

For the serum-free procedure, the lipofectin-DNA complex is mixed with the CHO SSF 3 cell suspension (end volume 0.5 ml) and placed in a CO₂ incubator (5% CO₂) at 37°C. After a 16h 1 ml FMX-8 is added to the cells and the incubation continued. After another 24h the cells are diluted 10-fold in fresh FMX-8 + 0.25 mg/ml geneticin, divided in 96-well microtiter plate and incubated at 37°C until after approximately 3 weeks incubation dense cell growth is detected in some of the wells. An aliquot of the medium from such wells is assayed for the presence of human SC. Cells from the wells in which SC is detected are transferred to larger culture vessels and expanded until sufficient cells are obtained to inoculate a stirred tank bioreactor.

In the alternative procedure the serum-containing medium is suctioned off, cells are rinsed with FMX-8 without serum and 0.3 ml FMX-8 medium without serum is added. To this medium the lipofectin-DNA complex solution is added and the cells are incubated for 5 h in a CO₂ incubator (5% CO₂) at 37°C, after which 1 ml of FMX-8 with 4% serum is added. 24h later the cells are trypsinized with undiluted 0.25 % porcine trypsin (JRH Biosciences Lenexa, Kansas U.S. A) as described in Asselbergs et al., J. Biotechnol. (1992). 26, 301 - 313, diluted 20-fold in FMX-8 with 4% serum and 1 mg/ml geneticin and plated in Petri dishes. After 2-3 weeks colonies of geneticin-resistant cells have developed, which are individually scraped off and transferred to a 24-well microtiter plate. FMX-8 without serum with 0.25 mg/ml geneticin is added to the cells. After a week a dense culture of mostly non- adherent cells has developed. The concentration of SC in the conditioned medium from each well is measured by ELISA (as described in example 10) and cells from the wells in which a high amount SC is detected are transferred to larger culture vessels and expanded until sufficient cells are obtained to inoculate a stirred tank bioreactor.

One cell line CHO SSF 3 producing human SC is designated SSF3-HSC-1 and is used for production of human SC in stirred tank bioreactor. The cell population produces more than 10 μg SC per million cells day.

Using the same procedures cells, which are transfected with pMC-SC instead of pCB6-SC, are obtained. One pMC-SC -transfected CHO SSF3 cell line producing human SC is designated CHO-SSF3/pMC-SC3 (DSM ACC2203) and is used for production of human SC in a stirred bioreactor. The cell population produces more than 10 mg SC per million cells per day.

Example 7: Expression of human secretory component in CHO SSF 3 cells transfected with pCGA93D-SC

CHO SSF 3 cells and lipofectin-DNA complex are prepared as described above. (Example 6), except that instead of plasmid pCB6-SC plasmid pCGA993D-SC (Example 5) is used.

For the serum-free procedure, the lipofectin-DNA complex is mixed with the CHO SSF 3 cell suspension <end volume 0.5 ml) and placed in a CO₂ incubator (5% CO₂) at 37°C. After a 16h 1 ml FMX-8-minus (FMX-8 lacking glycine, hypoxanthine and thymidine) is added to the cells and the incubation continued. After another 24h the cells are diluted 10-fold in fresh FMX-8-minus + 5 nM methotrexate, divided in 96-well microtiter plate and incubated at 37°C until after approximately 3 weeks incubation dense cell growth is detected in some of the wells. An aliquot of the medium from such wells is assayed for the presence of human SC. Cells from the wells in which SC is detected are transferred to larger culture vessels and expanded until sufficient cells are obtained for selection of cell lines with increased methotrexate resistance (Example 8) or to inoculate a stirred tank bioreactor.

In the alternative procedure the serum-containing medium is suctioned off, the cells are rinsed with FMX-8-medium without serum and 0.3 ml FMX-8-medium without serum is added. To this medium the lipofectin-DNA complex solution is added and the cells are incubated for 5 h in a CO₂ incubator (5% CO₂) at 37°C, after which 1 ml of FMX-8-minus with 4% dialyzed serum is added. 24h later the cells are trypsinized, diluted 20-fold in FMX- 8-minus with 4% dialyzed serum and 5 nM methotrexate and plated in Petri dishes. After 2- 3 weeks colonies of methotrexate-resistant cells have developed, which are individually scraped off and transferred to a 24-well microtiter plate. FMX-8-minus without serum with 5 nM methotrexate is added to the cells. After a week a dense culture of mostly non- adherent cells has developed. The concentration of SC in the conditioned medium from each well is measured and cells from the wells in which a high amount SC is detected are transferred to larger culture vessels and expanded until sufficient cells are obtained for selection of cell lines with increased methotrexate resistance (Example 8) or to inoculate a stirred tank bioreactor. One CHO SSF 3 cell line producing human SC is designated SSF3-HSC-M1 and is used for production of human SC in stirred tank bioreactor. The cell population produces more than 10 μg SC per million cells day.

Example 8: Methotrexate selection of cell lines with increased number of copies of DCGA93D-SC

It is possible to select for spontaneous amplification of the plasmid DNA integrated into the chromosome of the transfected CHO SSF 3 cells. To achieve this, the transfected CHO SSF 3 cells selected to be resistant to 5 nM methotrexate are subcultured in gradually increasing concentrations of methotrexate. Like the transfection, this procedure can be done in protein-free medium or in medium containing serum. The latter method has the advantage that it is easier to isolate individual colonies of cells resistant to the higher methotrexate concentration, but cells have to readapted to growth in protein-free medium. This is done by gradually diluting out the serum over a two week growth period.

For the serum-free procedure, the cells cultured in FMX-8-minus (FMX-8 lacking glycine, hypoxanthine and thymidine) plus 5 nM methotrexate are diluted to a cell density of approximately 500 cells/ml in medium with the new methotrexate concentration and divided over several 96-well microtiter culture plates. It is known that the frequency of amplification is approximately one in 10000 and that small increments of the methotrexate concentration favor the development of resistance due to gene amplification rather than other gene alterations (Kaufman R.J., Methods in Enzymology (1990), 185, 537-566). Therefore, the selection is initiated at about twice the initial concentration of 5 nM methotrexate. In about one in 20 wells a dense culture of cells with increased methotrexate resistance develops over a period of 2-3 weeks. Such cells are transferred to larger culture vessels and expanded until sufficient cells are obtained to test the specific production of secretory component. Cells with higher specific SC production are obtained with a frequency of 20- 40% of the more resistant subcultures. This selection procedure is repeated several times, each time raising the methotrexate concentration 1.5-2.5-fold. When a concentration of 50- 150 nM methotrexate is reached a cell population producing more than 10 μg SC per million cells day is obtained. These cells are then transferred to an appropriate bioreactor for large scale SC production

In the alternative procedure, the cells are cultured adherent to plastic in FMX-8-minus medium with 4% dialyzed fetal calf serum initially with 5 nM methotrexate. The cells are trypsinized and replated at 20000 cells/ml in several 10 cm diameter Petri dishes (10 ml medium total) in the same medium but with the higher concentration of methotrexate. Like stated above, the selection is initiated at about twice the initial concentration of 5 nM methotrexate. After about 3 weeks colonies of more resistant cells are developed which are individually scaped off and transferred to multiwell dishes with medium with the same methotrexate concentration. When sufficient cells are obtained the specific production of SC is measured. Cells with higher specific SC production are obtained with a frequency of 20-40% of the more resistant subcultures. This selection procedure is repeated several times, each time raising the methotrexate concentration 1.5-2.5-fold. When a concentration of 50-150 nM methotrexate is reached a cell population producing more than 10 μg SC per million cells day is obtained. The cell line thus obtained is readapted to growth in protein- free FMX-8 minus by gradually over a period of 1 -2 weeks lowering the serum concentration. These cells are then transferred to an appropriate bioreactor for large scale SC production.

Example 9: ELISA assay for human SC in conditioned medium of transfected CHO SSF 3 cells

Polystyrene microtiter plates are coated overnight at 4°C with 100 μl/well of 10 μg/ml rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) dissolved in PBS without MgCI₂ and CaCI₂ (GIBCO) containing 0.1 mg/ml of the bacteriostatic sodium ethyl mercurithiosalicylate. Subsequently, the plates are rinsed three times with in washing solution: PBS lacking MgCI₂ and CaCI₂ containing 0.05% Tween-20 (EIA-grade, BIORAD) and 0.1 mg/ml sodium ethyl mercurithiosalicylate. Non-specific protein binding sites on the polystyrene are neutralized by incubation with blocking buffer: PBS containing 2% bovine serum albumin (BSA), 0.5% rabbit serum and 0.05% Tween-20. Subsequently, the plates are rinsed three times with washing solution. A 100 μl sample of an SC-containing solution (conditioned medium, column fraction from a purfication etc.) diluted in blocking solution is added to each well and incubated overnight at 4°C. Subsequently, the plates are rinsed three times with washing solution. Next, 50 μl of a solution containing 250 ng/ml biotinylated anti-human SC immunoglobulin in blocking buffer is added followed by an incubation of 1 h at room temperature. This biotinylated antibody is prepared by treatment of rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) with aminohexanoyl-biotin-n-hydroxysuccinimide ester (Zymed) according to the instructions of the manufacturer. Subsequently, the plates are rinsed three times with washing solution. Next, 50 μl of avidin crosslinked to horseradish peroxidase (Zymed) 1000-fold diluted in blocking buffer is added followed by an incubation of 1 h at room temperature. After washing the plate three times with washing solution, 150 μl well of enzyme color substrate solution, 21 mg/ml citric acid and 35.6 mg/ml Na₂HPO₄ at pH 4.5 containing 1.5 mg/ml O-phenyldiamine (SIGMA) and 1 μl/ml 30% H₂O₂, is added. After 15 min. at 37°C the enzyme reaction is stopped by addition of 50 μl/well of 0.5 N H₂SO and the absorption measured at 492 nm. Relative concentration (titer) of SC is calculated as an arbitrary unit (AU) i.e. the absorbance measured multiplied by the volume and dilution factor of the sample. Alternatively, the assay is standardized by testing a dilution series of purified recombinant human SC in parallel.

Example 10: ELISA assay for binding of SC to human IgA

Polystyrene microtiter plates are coated overnight at 4°C with 100 μl/well of either 10 μg/ml human serum albumin (negative control, Sigma A-6003), human IgG (Sigma I-4506), IgM (Sigma I-8640), IgA (Sigma, I-0633) or rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) dissolved in PBS without MgCI₂ and CaCI₂ (GIBCO) containing 0.1 mg/ml of the bacteriostatic sodium ethyl mercurithiosalicylate. Subsequently, the plates are rinsed three times with in washing solution : PBS lacking MgCI₂ and CaCI₂ containing 0.05% Tween-20 (EIA-grade, BIORAD) and 0.1 mg/ml sodium ethyl mercurithiosalicylate. Non-specific protein binding sites on the polystyrene are neutralized by incubation with blocking buffer: PBS containing 2% bovine serum albumin (BSA), 0.5% rabbit serum and 0.05% Tween-20. Subsequently, the plates are rinsed three times with washing solution. A 100 μl sample of an SC-containing solution diluted in blocking solution is added to each well and incubated overnight at 4°C. Subsequently, the plates are rinsed three times with washing solution. Next, 50 μl of a solution containing 250 ng/ml biotinylated anti-human SC immunoglobulin in blocking buffer is added followed by an incubation of 1 h at room temperature. This biotinylated antibody is prepared by treatment of rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) with aminohexanoyl-biotin-n-hydroxysuccinimide ester (Zymed) according to the instructions of the manufacturer. Subsequently, the plates are rinsed three times with washing solution. Next, 50 μl of avidin crosslinked to horseradish peroxidase (Zymed) 1000-fold diluted in blocking buffer is added followed by an incubation of 1 h at room temperature. After washing the plate three times with washing solution, 150 μl/well of enzyme color substrate solution, 21 mg/ml citric acid and 35.6 mg/ml Na₂HP0₄ at pH 4.5 containing 15 mg/ml O-phenyldiamine (SIGMA) and 1 μl/ml 30% H₂O₂, is added. After 15 min. at 37°C the enzyme reaction is stopped by addition of 50 μl/well of 0.5 N H₂SO₄ and the absorption measured at 492 nm. Relative concentration (titer) of SC is calculated as an arbitrary unit (AU) i.e. the absorbance measured multiplied by the volume and dilution factor of the sample. Alternatively, the assay is standardized by testing a dilution series of purified recombinant human SC in parallel.

Example 11 : Production of SC of IgA in small pilot scale suspension culture in serum and protein free cultivated CHO SSF 3 cells.

All cell cultivations are performed as suspended repeated step-wise-fed-batch cultivations in 10 L glass-bioreactors with marine type impellers. After having reached maximal cell density and working volume, indicated by beginning stationary growth phase, 90% of the cell suspension is harvested and the remaining 10% are diluted by a factor 1 :10 with fresh medium. Tne process is controlled by on-line control loops for temperature, pH and pO₂. Cell concentration, cell viability and product concentration (end concentration) are off-line determined. The basal medium consists of FMX-8 (Dr. F. Messi AG, Zurich, Switzerland). The cells however are proved to be shear sensitive with respect to their production kinetics. The growth kinetics, however, are only slightly affected (see below). Medium is therefore supplied with Pluronic F-68 (P-1300, SIGMA) as productivity enhancer. The two different medium configurations are compared below.

Example 11.1 : Batch cultivation with FMX-8 medium

After inoculation of the bioreactor with 1.2 x 10⁵ cells/ml and 2.5 L working volume the cells are grown up to 9.8 x 10⁵ cells/ml. During growth phase the working volume is increased twice by the addition of 3 L of fresh medium after 4 d and 2 L of fresh medium after 7 d respectively. This batch cultivation results in a final working volume of 8 L after 9 d of culture and with a final concentration of 109 mg/L of SC of IgA. The mean doubling time of the cells is 2.82 d and the daily yield of the SC of IgA is 12.1 mg/L d.

Example 11.2: Batch cultivation with FMX-8 medium, supplied with Pluronic F-68

The FMX-8 medium is supplied with 0.05% (w/v) Pluronic F-68 as productivity enhancer. After inoculation of the bioreactor with 2.3 x 10⁵ cells/ml (from Example 8)and 2.5 L working volume the cells are grown up to 1.1 x 10⁶ cells/ml. During growth phase the working volume is increased 3 times by the addition of 2.5 L of fresh medium after 1 d, 3 L after 3 d and 1 L after 4 d respectively. This batch cultivation results in a final working volume of 9 L after 7 d of culture and with a final cell concentration of 1 .1 x 10⁶ cells/ml and a final concentration of 229 mg/L of SC of IgA. With 2.26 d the mean doubling time is only 20% lower than without Pluronic supplementation. On the contrary, the daily yield of the SC of IgA is with 32.7 mg/LJd 3 times higher than without Pluronic supplementation. Example 12: Human secretory component produced by Chinese hamster ovary cells in the absence and presence of Pluronic.

After removal of the residual cellular biomass human secretory component (hSC) is isolated from the FMX-8-based cell culture medium. Protease inhibitors, like PMSF (0.1 M in 2- propanol) and E-64 (7 mM in 50% (v/v) aqueous ethanol), and NaCI are added to give final concentrations of 1 mM, 2.8 μM and 0.5 M, respectively, and the pH is adjusted to 5.6 with 4 N HCI. After addition of CaCI₂ and MnCI₂ to give a final concentration of 1 mM each, 150 ml Concanavalin A Sepharose (Pharmacia) slurry are added and suspended overnight at 4°C. The immobilized lectin is collected above a glassfilter, washed with 5 mM sodiumacetate pH 5.6, containing 0.5 M NaCI, 1 mM CaCI₂, and 1 mM MnCI₂ (binding buffer), and packed into a 2.6 cm x 30 cm column-housing. Concanavalin A-bound protein is eluted with binding buffer, containing 0.5 M methyl-α-D-mannopyranoside. The hSC- containing fraction are concentrated and dialyzed against 50 mM sodiumacetate pH 5.5, containing 0.15 M NaCI and 0.02 (w/v) NaN₃, by ultrafiltration using an YM10® membrane (AMICON) in an Amicon cell. Further fractionated is achieved by gel-permeation chromatography on a column (1.6 cm x 61 cm) of Sephacryl S-300 (Pharmacia). The column is eluted with 50 mM sodiumacetate pH 5.5, containing 0.15 M NaCI, and the effluent monitored at 278 nm. The major peak, containing hSC as monitored by SDS/PAGE, is collected and concentrated by ultrafiltration using an YM10 membrane in an Amicon cell to a concentration of about 20 mg/ml. Approximately 35 mg of hSC are isolated per liter of cell culture medium as measured using the Bio-Rad protein assay using IgG as a standard. The final preparation shows an apparent purity in excess of 95%, and is stored at 4^SC in the presence of 0.02% (w/v) NaN₃.

Example 13: Crystallization of recombinant soluble polymeric Ig receptor (hSC)

The crystallization experiments are performed using the hanging drop method.

2 μl of the SC solution from example 12 at a concentration of about 5 mg/ml in 10 mM sodium acetate buffer at pH 5.5 containing 100 mM NaCI is mixed with an equal volume of precipitating agent buffer. The precipitating agent buffer can be :

A) 0.1 M Na-citrate, 1.0 M ammonium phosphate and 0.02% NaN₃; or

B) 0.1 M HEPES, 1.5 M (Li)₂SO₄ and 0.02% NaN₃ The protein and precipitating agent solutions are mixed on a glass cover slide. 1000 μl of the respective precipitating buffer solution (reservoir) is placed into wells of Linbro plates. The glass cover slide with the hanging drop of protein solution is placed over the well. Equilibration of the concentration of precipitating agent between reservoir and hanging drops via the vapor phase occurs within a few weeks at room temperature. During this period plate-like crystals of hSC appear in the drops.

Example 14: Sialic acid analysis of recombinant soluble polymeric Ig receptor (hSC)

The analysis of sialic acids is carried out essentially as described in Harra et al., Anal. Biochem. (1989), 179, 162-166. An aliquot of 236 μg recombinant soluble polymeric Ig receptor (hSC) in 10 μl 0.05 M sodiumacetate pH 5.5, containing 0.15 M sodiumchloride is dried under reduced pressure and solved in 200 μl 2 M acetic acid, heated for 3 h at 80°C. Released sialic acids are converted into fluorescent derivatives by the addition of 200 μl 7.0 mM 1 ,2-diamino-4,5-methylenedioxybenzene (DMB, Sigma) in 1.4 M acetic acid, containing 0.75 M β-mercaptoethanol and 18 mM sodium dithionate at 50°C for 2.5 h. N- acetylneuraminic acid (Neu5Ac, Sigma), N-glycolylneuraminic acid (Neu5Gc, Sigma), human serumtransferrin (hST, Serva), bovine glycoprotein fraction VI (bGP, Miles Laboratories) and a mixture of Neu5Ac, Neu5Gc, N-acetyl-7-O-acetylneuraminic acid (Neu5,7Ac₂), N-glycolyl-9-O-acetylneuraminic acid (Neu9Ac₅Gc), N-acetyl-9-O-acetyl- neuraminic acid (Neu5,9Ac₂), N-acetyl-7(8),9-di-O-acetylneuraminic acid (Neu5,7(8),9Ac₃) (Oxford Glycosystems) are taken through the procedure as standards. HPLC analysis is carried out on a Waters Novapak C ⁸ 4-μm (60 A) column (3.9 x 150 mm) fitted into a Waters 840 chromatography system equipped with two model 510 HPLC pumps, a WISP model 712 sample processor, a model 490 programmable multi-wavelength detector and a Kratos GM 970 fluorescence detector operating at an excitation wavelength of 373 nm, detecting emission at wavelength >418 nm using a cut-off filter. Simultaneously, the absorbance of the eluent is monitored at a wavelength of 373 nm. Elutions are performed isocratically using acetonitrile : methanol : water (6.4 : 4.9 : 88.7, v/v/v) as eluent at a flow rate of 0.7 ml/min.

The HPLC profiles of the DMB sialic acids derived from hST and bGP show only NeuδAc for hST (as described in Spik et al., FEBS Lett. (1975), 50, 296-299 and Hokke et al., FEBS Lett. (1990), 275, 9-14) and a mixture of NeuδGc and NeuδAc in a ratio of 1.0 : 1.0 for bGP. The HPLC pattern of the DMB sialic acids derived from hSC show peaks at the elution positions of NeuδGc and NeuδAc, respectively. The content of Neu5Ac in hSC is more than 99.9% and that of NeuδGc less than 0.1%.

DEPOSITIONS

The following microorganism strains were deposited at the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg 1 b, D-38124 Braunschweig (accession numbers and deposition dates given):

CHO SSF 3 / p' ΛC-SC3 DSM ACC2203 deposited on Dec. 15, 1994

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: CIBA-GEIGY AG

(B) STREET: Klybeckstr. 141

(C) CITY: Basel

(E) COUNTRY: Switzerland

,F) POSTAL CODE (ZIP) : 4002

(G) TELEPHONE: +41 61 69 11 11

<H) TELEFAX: + 41 61 696 79 76

(I) TELEX: 962 991

(ii) TITLE OF INVENTION: Production of Recombinant Secretory Coπponent

(iii) NUMBER OF SEQUENCES: 7

(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 #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2405 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION:111..2402

(D) OTHER INFORMATION: /produc = "original plgR"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGGTAGGCGT GTACGGTGGG AGGTCTATAT AGCAGAGCTC GTTTAGTGAA CCGTCAGAAT 60

TAATTCAGAT CTGGTACCAC GCGTATCGAT AAGCTTGAAT TCCACCAGCA ATG CTG 116

Met Leu

1

CTC TTC GTG CTC ACC TGC CTG CTG GCG GTC TTC CCA GCC ATC TCC ACG 164 Leu Phe Val Leu T r Cys Leu Leu Ala Val Phe Pro Ala lie Ser Thr

5 10 15

AAG AGT CCC ATA TTT GGT CCC GAG GAG GTG AAT AGT GTG GAA GGT AAC 212 Lys Ser Pro He Phe Gly Pro Glu Glu Val Asn Ser Val Glu Gly Asn 20 25 30

TCA GTG TCC ATC ACG TGC TAC TAC CCA CCC ACC TCT GTC AAC CGG CAC 260 Ser Val Ser He Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn Arg His 35 40 45 50

ACC CGG AAG TAC TGG TGC CGG CAG GGA GCT AGA GGT GGC TGC ATA ACC 308 Thr Arg Lys Tyr Trp Cys Arg Gin Gly Ala Arg Gly Gly Cys He Thr 55 60 65

CTC ATC TCC TCG GAG GGC TAC GTC TCC AGC AAA TAT GCA GGC AGG GCT 356 Leu He Ser Ser Glu Gly Tyr Val Ser Ser Lys Tyr Ala Gly Arg Ala 70 75 80

AAC CTC ACC AAC TTC CCG GAG AAC GGC ACA TTC GTG GTG AAC ATT GCC 404 Asn Leu Thr Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn He Ala 85 90 95 CAG CTG AGC CAG GAT GAC TCC GGG CGC TAC AAG TGT GGC CTG GGC ATC 452 Gin Leu Ser Gin Asp Asp Ser Gly Arg Tyr Lys Cys Gly Leu Gly He 100 105 110

AAT AGC CGA GGC CTG TCC TTT GAT GTC AGC CTG GAG GTC AGC CAG GGT 500 Asn Ser Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser Gin Gly 115 120 125 130

CCT GGG CTC CTA AAT GAC ACT AAA GTC TAC ACA GTG GAC CTC GGC AGA 548 Pro Gly Lei¹ Leu Asn Asp Thr Lys Val Tyr Thr Val Asp Leu Gly Arg 135 140 145

ACG GTG ACC ATC AAC TCC CCT TTC AAG ACT GAG AAT GCT CAA AAG AGG 596 Thr Val Thr He Asn Cys Pro Phe Lys Thr Glu Asn Ala Gin Lys Arg 150 155 160

AAG TCC TTC TAC AAG CAG ATA GGC CTG TAC CCT GTG CTG GTC ATC GAC 644 Lys Ser Leu Tyr Lys Gin He Gly Leu Tyr Pro Val Leu Val He Asp 165 170 175

TCC AGT GGT TAT GTG AAT CCC AAC TAT ACA GGA AGA ATA CGC CTT GAT 692 Ser Ser Gly Tyr Val Asn Pro Asn Tyr Thr Gly Arg He Arg Leu Asp 180 185 190

ATT CAG GGT ACT GGC CAG TTA CTG TTC AGC GTT GTC ATC AAC CAA CTC 740 He Gin Gly Thr Gly Gin Leu Leu Phe Ser Val Val He Asn Gin Leu 195 200 205 210

AGG CTC AGC GAT GCT GGG CAG TAT CTC TGC CAG GCT GGG GAT GAT TCC 788 Arg Leu Ser Asp Ala Gly Gin Tyr Leu Cys Gin Ala Gly Asp Asp Ser 215 220 225

AAT AGT AAT AAG AAG AAT GCT GAC CTC CAA GTG CTA AAG CCC GAG CCC 836 Asn Ser Asn Lys Lys Asn Ala Asp Leu Gin Val Leu Lys Pro Glu Pro 230 235 240 GAG CTG GTT TAT GAA GAC CTG AGG GGC TCA GTG ACC TTC CAC TGT GCC 884 Glu Leu Val Tyr Glu Asp Leu Arg Gly Ser Val Thr Phe His Cys Ala 245 250 255

CTG GGC CCT GAG GTG GCA AAC GTC GCC AAA TTT CTG TGC CGA CAG AGC 932 Leu Gly Pro Glu Val Ala Asn Val Ala Lys Phe Leu Cys Arg Gin Ser 260 265 270

AGT GGG GAA AAC TGT GAC GTC GTC GTC AAC ACC CTG GGG AAG AGG GCC 980 Ser Gly Glu Asn Cys Asp Val Val Val Asn Thr Leu Gly Lys Arg Ala 275 280 285 290

CCA GCC TTT GAG GGC AGG ATC CTG CTC AAC CCC CAG GAC AAG GAT GGC 1028 Pro Ala Phe Glu Gly Arg He Leu Leu Asn Pro Gin Asp Lys Asp Gly 295 300 305

TCA TTC AGT GTG GTG ATC ACA GGC CTG AGG AAG GAG GAT GCA GGG CGC 1076 Ser Phe Ser Val Val He Thr Gly Leu .Arg Lys Glu Asp Ala Gly Arg 310 315 320

TAC CTG TGT GGA GCC CAT TCG GAT GGT CAG CTG CAG GAA GGC TCG CCT 1124 Tyr Leu Cys Gly Ala His Ser Asp Gly Gin Leu Gin Glu Gly Ser Pro

325 330 335

ATC CAG GCC TGG CAA CTC TTC GTC AAT GAG GAG TCC ACG ATT CCC CGC 1172 He Gin Ala Trp Gin Leu Phe Val Asn Glu Glu Ser Thr He Pro Arg 340 345 350

AGC CCC ACT GTG GTG AAG GGG GTG GCA GGA AGC TCT GTG GCC GTG CTC 1220 Ser Pro Thr Val Val Lys Gly Val Ala Gly Ser Ser Val Ala Val Leu 355 360 365 370

TGC CCC TAC AAC CGT AAG GAA AGC AAA AGC ATC AAG TAC TGG TGT CTC 1268 Cys Pro Tyr Asn Arg Lys Glu Ser Lys Ser He Lys Tyr Trp Cys Leu 375 380 385 TCG GAA GGG GCC CAG AAT GGC CGC TGC CCC CTG CTG GTG GAC AGC GAG 1316 Trp Glu Gly Ala Gin Asn Gly Arg Cys Pro Leu Leu Val .Asp Ser Glu 390 395 400

GGG TGG GTT AAG GCC CAG TAC GAG GGC CGC CTC TCC CTG CTG GAG GAG 1364 Gly Trp Val Lys Ala Gin Tyr Glu Gly Arg Leu Ser Leu Leu Glu Glu 405 410 415

CCA GGC AAC GGC ACC TTC ACT GTC ATC CTC AAC CAG CTC ACC AGC CGG 1412 Pro Gly Asn Gly Thr Phe Thr Val He Leu Asn Gin Leu Thr Ser Arg 420 425 430

GAC GCC GGC TTC TAC TGG TGT CTG ACC AAC GGC GAT ACT CTC TCG AGG 1460 Asp Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Thr Leu Trp Arg 435 440 445 450

ACC ACC GTG GAG ATC AAG ATT ATC GAA GGA GAA CCA AAC CTC AAG GTA 1508 Thr Thr Val Glu He Lys He He Glu Gly Glu Pro Asn Leu Lys Val 455 460 465

CCA GGG AAT GTC ACG GCT GTC CTC GGA GAG ACT CTC AAG GTC CCC TGT 1556 Pro Gly Asn Val Thr Ala Val Leu Gly Glu Thr Leu Lys Val Pro Cys 470 475 480

CAC TTT CCA TGC AAA TTC TCC TCG TAC GAG AAA TAC TGG TGC AAG TGG 1604 His Phe Pro Cys Lys Phe Ser Ser Tyr Glu Lys Tyr Trp Cys Lys Trp 485 490 495

AAT AAC ACG GGC TGC CAG GCC CTG CCC AGC CAA GAC GAA GGC CCC AGC 1652 Asn Asn Thr Gly Cys Gin Ala Leu Pro Ser Gin Asp Glu Gly Pro Ser 500 505 510

AAG GCC TTC GTC AAC TGT GAC GAG AAC AGC CGG CTT GTC TCC CTG ACC 1700 Lys Ala Phe Val Asn Cys Asp Glu Asn Ser Arg Leu Val Ser Leu Thr 515 520 525 530 CTG AAC CTG GTG ACC AGG GCT GAT GAG GGC TGG TAC TGG TGT GGA GTG 1748 Leu Asn Leu Val Thr Arg Ala Asp Glu Gly Trp Tyr Trp Cys Gly Val 535 540 545

AAG CAG GGC CAC TTC TAT GGA GAG ACT GCA GCC GTC TAT GTG GCA GTT 1796 Lys Gin Gly His Phe Tyr Gly Glu Thr Ala Ala Val Tyr Val Ala Val 550 555 560

GAA GAG AGG AAG GCA GCG GGG TCC CGC GAT GTC AGC CTA GCG AAG GCA 1844 Glu Glu Arg Lys Ala Ala Gly Ser Arg Asp Val Ser Leu Ala Lys Ala 565 570 575

GAC GCT GCT CCT GAT GAG AAG GTG CTA GAC TCT GGT TTT CGG GAG ATT 1892 Asp Ala Ala Pro Asp Glu Lys Val Leu Asp Ser Gly Phe Arg Glu He 580 585 590

GAG AAC AAA GCC ATT CAG GAT CCC AGG CTT TTT GCA GAG GAA AAG GCG 1940 Glu Asn Lys Ala He Gin Asp Pro Arg Leu Phe Ala Glu Glu Lys Ala 595 600 605 610

GTG GCA GAT ACA AGA GAT CAA GCC GAT GGG AGC AGA GCA TCT GTG GAT 1988 Val Ala Asp Thr Arg Asp Gin Ala Asp Gly Ser Arg Ala Ser Val Asp 615 620 625

TCC GGC AGC TCT GAG GAA CAA GGT GGA AGC TCC AGA GCG CTG GTC TCC 2036 Ser Gly Ser Ser Glu Glu Gin Gly Gly Ser Ser Arg Ala Leu Val Ser 630 635 640

ACC CTG GTG CCC CTG GGC CTG GTG CTG GCA GTG GGA GCC GTG GCT G G 2084 Thr Leu Val Pro Leu Gly Leu Val Leu Ala Val Gly Ala Val Ala Val 645 650 655

GGG GTG GCC AGA GCC CGG CAC AGG AAG AAC GTC GAC CGA GTT TCA ATC 2132 Gly Val Ala Arg Ala Arg His Arg Lys Asn Val Asp Arg Val Ser He 660 665 670 AGA AGC TAC AGG ACA GAC ATT AGC ATG TCA GAC TTC GAG AAC TCC AGG 2180 Arg Ser Tyr Arg Thr Asp He Ser Met Ser Asp Phe Glu Asn Ser Arg 675 680 685 690

GAA TTT GGA GCC AAT GAC AAC ATC GGA GCC TCT TCG ATC ACT CAG GAG 2228 Glu Phe Gly Ala Asn Asp Asn Met Gly Ala Ser Ser He Thr Gin Glu 695 700 705

ACA TCC CTC GGA GGA AAA GAA GAG TTT GTT GCC ACC ACT GAG AGC ACC 2276 Thr Ser Leu Gly Gly Lys Glu Glu Phe Val Ala Thr Thr Glu Ser Thr 710 715 720

ACA GAG ACC AAA GAA CCC AAG AAG GCA AAA AGG TCA TCC AAG GAG GAA 2324 Thr Glu Thr Lys Glu Pro Lys Lys Ala Lys Arg Ser Ser Lys Glu Glu 725 730 735

GCC GAG ATG GCC TAC AAA GAC TTC CTG CTC CAG TCC AGC ACC GTC GCC 2372 Ala Glu Met Ala Tyr Lys Asp Phe Leu Leu Gin Ser Ser Thr Val Ala 740 745 750

GCC GAG GCC CAG GAC GGC CCC CAG GAA GCC TAG 2405

Ala Glu Ala Gin Asp Gly Pro Gin Glu Ala 755 760

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 764 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Leu Leu Phe Val Leu Thr Cys Leu Leu Ala Val Phe Pro Ala He 1 5 10 15 Ser Thr Lys Ser Pro He Phe Gly Pro Glu Glu Val Asn Ser Val Glu 20 25 30

Gly Asn Ser Val Ser He Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn 35 40 45

Arg His Thr Arg Lys Tyr Trp Cys Arg Gin Gly Ala Arg Gly Gly Cys 50 55 60

He Thr Leu He Ser Ser Glu Gly Tyr Val Ser Ser Lys Tyr Ala Gly 65 70 75 80

Arg Ala Asn Leu Thr Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn 85 90 95

He Ala Gin Leu Ser Gin Asp Asp Ser Gly Arg Tyr Lys Cys Gly Leu 100 105 110

Gly He Asn Ser Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser 115 120 125

Gin Gly Pro Gly Leu Leu Asn Asp Thr Lys Val Tyr Thr Val Asp Leu 130 135 140

Gly Arg Thr Val Thr He Asn Cys Pro Phe Lys Thr Glu Asn Ala Gin 145 150 155 160

Lys Arg Lys Ser Leu Tyr Lys Gin He Gly Leu Tyr Pro Val Leu Val 165 170 175

He Asp Ser Ser Gly Tyr Val Asn Pro Asn Tyr Thr Gly Arg He Arg 180 185 190

Leu Asp He Gin Gly Thr Gly Gin Leu Leu Phe Ser Val Val He .Asn 195 200 205 Gln Leu Arg Leu Ser Asp Ala Gly Gin Tyr Leu Cys Gin Ala Gly Asp 210 215 220

Asp Ser Asn Ser Asn Lys Lys Asn Ala Asp Leu Gin Val Leu Lys Pro 225 230 235 240

Glu Pro Glu Leu Val Tyr Glu Asp Leu .Arg Gly Ser Val Thr Phe His 245 250 255

Cys Ala Leu Gly Pro Glu Val Ala Asn Val Ala Lys Phe Leu Cys Arg 260 265 270

Gin Ser Ser Gly Glu Asn Cys Asp Val Val Val Asn Thr Leu Gly Lys 275 280 285

Arg Ala Pro Ala Phe Glu Gly Arg He Leu Leu Asn Pro Gin Asp Lys 290 295 300

Asp Gly Ser Phe Ser Val Val He Thr Gly Leu Arg Lys Glu Asp Ala 305 310 315 320

Gly Arg Tyr Leu Cys Gly Ala His Ser Asp Gly Gin Leu Gin Glu Gly

325 330 335

Ser Pro He Gin Ala Trp Gin Leu Phe Val Asn Glu Glu Ser Thr He 340 345 350

Pro Arg Ser Pro Thr Val Val Lys Gly Val Ala Gly Ser Ser Val Ala 355 360 365

Val Leu Cys Pro Tyr Asn Arg Lys Glu Ser Lys Ser He Lys Tyr Trp 370 375 380

Cys Leu Trp Glu Gly Ala Gin Asn Gly Arg Cys Pro Leu Leu Val Asp 385 390 395 400 Ser Glu Gly Trp Val Lys Ala Gin Tyr Glu Gly Arg Leu Ser Leu Leu 405 410 415

Glu Glu Pro Gly Asn Gly Thr Phe Thr Val He Leu Asn Gin Leu Thr 420 425 430

Ser Arg Asp Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Thr Leu 435 440 445

Trp Arg Thr Thr Val Glu He Lys He He Glu Gly Glu Pro Asn Leu 450 455 460

Lys Val Pro Gly Asn Val Thr Ala Val Leu Gly Glu Thr Leu Lys Val 465 470 475 480

Pro Cys His Phe Pro Cys Lys Phe Ser Ser Tyr Glu Lys Tyr Trp Cys

485 490 495

Lys Trp Asn Asn Thr Gly Cys Gin Ala Leu Pro Ser Gin Asp Glu Gly 500 505 510

Pro Ser Lys Ala Phe Val Asn Cys Asp Glu Asn Ser Arg Leu Val Ser

515 520 525

Leu Thr Leu Asn Leu Val Thr Arg Ala Asp Glu Gly Trp Tyr Trp Cys 530 535 540

Gly Val Lys Gin Gly His Phe Tyr Gly Glu Thr Ala Ala Val Tyr Val 545 550 555 560

Ala Val Glu Glu Arg Lys Ala Ala Gly Ser Arg Asp Val Ser Leu Ala 565 570 575

Lys Ala Asp Ala Ala Pro Asp Glu Lys Val Leu Asp Ser Gly Phe Arg 580 585 590 Glu He Glu Asn Lys Ala He Gin Asp Pro Arg Leu Phe Ala Glu Glu 595 600 605

Lys Ala Val Ala Asp Thr Arg Asp Gin Ala Asp Gly Ser Arg Ala Ser 610 615 620

Val Asp Ser Gly Ser Ser Glu Glu Gin Gly Gly Ser Ser Arg Ala Leu 625 630 635 640

Val Ser Thr Leu Val Pro Leu Gly Leu Val Leu Ala Val Gly Ala Val 645 650 655

Ala Val Gly Val Ala Arg Ala Arg His Arg Lys Asn Val Asp Arg Val 660 665 670

Ser He Arg Ser Tyr Arg Thr Asp He Ser Met Ser Asp Phe Glu Asn 675 680 685

Ser Arg Glu Phe Gly Ala Asn Asp Asn Met Gly Ala Ser Ser He Thr 690 695 700

Gin Glu Thr Ser Leu Gly Gly Lys Glu Glu Phe Val Ala Thr Thr Glu 705 710 715 720

Ser Thr Thr Glu Thr Lys Glu Pro Lys Lys Ala Lys Arg Ser Ser Lys 725 730 735

Glu Glu Ala Glu Met Ala Tyr Lys Asp Phe Leu Leu Gin Ser Ser Thr 740 745 750

Val Ala Ala Glu Ala Gin Asp Gly Pro Gin Glu Ala 755 760

(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2031 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "PCR-modified SEQ ID NO:l"

(ix) FEATURE:

(A) NAME/KEY: mat_pep ide

(B) LOCATION:111..2015

(D) OTHER INFORMATION: /product= "transcript from SC DNA fragment 1"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:1..33

(D) OTHER INFORMATION:/product= "from PCR with 5' primer 1 (SEQ ID NO 5)"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:coιτplement (1997..2031)

(D) OTHER INFORMATION: /product= "from PCR with 3' primer (SEQ ID NO 7)"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:1..2031

(D) OTHER INFORMATION: /produc = "SC DNA fragment 1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CGGTAGGCGT GTACGGTGGG AGGTCTATAT AGCAGAGCTC GTTTAGTGAA CCGTCAGAAT 60 TAATTCAGAT CTGGTACCAC GCGTATCGAT AAGCTTGAAT TCCACCAGCA ATGCTGCTCT 120

TCGTGCTCAC CTGCCTGCTG GCGGTCTTCC CAGCCATCTC CACGAAGAGT CCCATATTTG 180

GTCCCGAGGA GGTGAATAGT GTGGAAGGTA ACTCAGTGTC CATCACGTGC TACTACCCAC 240

CCACCTCTGT CAACCGGCAC ACCCGGAAGT ACTGGTGCCG GCAGGGAGCT AGAGGTGGCT 300

GCATAACCCT CATCTCCTCG GAGGGCTACG TCTCCAGCAA ATATGCAGGC AGGGCTAACC 360

TCACCAACTT CCCGGAGAAC GGCACATTCG TGGTGAACAT TGCCCAGCTG AGCCAGGATG 420

ACTCCGGGCG CTACAAGTGT GGCCTGGGCA TCAATAGCCG AGGCCTGTCC TTTGATGTCA 480

GCCTGGAGGT CAGCCAGGGT CCTGGGCTCC TAAATGACAC TAAAGTCTAC ACAGTGGACC 540

TGGGCAGAAC GGTGACCATC AACTGCCCTT TCAAGACTGA GAATGCTCAA AAGAGGAAGT 600

CCTTGTACAA GCAGATAGGC CTCTACCCTC TGCTGGTCAT CGACTCCAGT GGTTATGTGA 660

ATCCCAACTA TACAGGAAGA ATACGCCTTG ATATTCAGGG TACTGGCCAG TTACTGTTCA 720

GCGTTGTCAT CAACCAACTC AGGCTCAGCG ATGCTGGGCA GTATCTCTGC CAGGCTGGGG 780

ATGATTCCAA TAGTAATAAG AAGAATGCTG ACCTCCAAGT GCTAAAGCCC GAGCCCGAGC 840

TGGTTTATGA AGACCTGAGG GGCTCAGTGA CCTTCCACTG TGCCCTGGGC CCTGAGGTGG 900

CAAACGTGGC CAAATTTCTG TGCCGACAGA GCAGTGGGGA AAACTGTGAC GTGGTCGTCA 960

ACACCCTGGG GAAGAGGGCC CCAGCCTTTG AGGGCAGGAT CCTGCTCAAC CCCCAGGACA 1020

AGGATGGCTC ATTCAGTGTG GTGATCACAG GCCTGAGGAA GGAGGATGCA GGGCGCTACC 1080

TGTGTGGAGC CCATTCGGAT GGTCAGCTGC AGGAAGGCTC GCCTATCCAG GCCTGGCAAC 1140 TCTTCGTCAA TGAGGAGTCC ACGATTCCCC GCAGCCCCAC TGTGGTGAAG GGGGTGGCAG 1200

GAAGCTCTGT GGCCGTGCTC TGCCCCTACA ACCGTAAGGA AAGCAAAAGC ATCAAGTACT 1260

GGTGTCTCTG GGAAGGGGCC CAGAATGGCC GCTGCCCCCT GCTGGTGGAC AGCGAGGGGT 1320

GGGTTAAGGC CCAGTACGAG GGCCGCCTCT CCCTGCTGGA GGAGCCAGGC AACGGCACCT 1380

TCACTGTCAT CCTCAACCAG CTCACCAGCC GGGACGCCGG CTTCTACTGG TGTCTGACCA 1440

ACGGCGATAC TCTCTGGAGG ACCACCGTGG AGATCAAGAT TATCGAAGGA GAACCAAACC 1500

TCAAGGTACC AGGGAATGTC ACGGCTGTGC TGGGAGAGAC TCTCAAGGTC CCCTGTCACT 1560

TTCCATGCAA ATTCTCCTCG TACGAGAAAT ACTGGTGCAA GTGGAATAAC ACGGGCTGCC 1620

AGGCCCTGCC CAGCCAAGAC GAAGGCCCCA GCAAGGCCTT CGTGAACTGT GACGAGAACA 1680

GCCGGCTTGT CTCCCTGACC CTGAACCTGG TGACCAGGGC TGATGAGGGC TCGTACTGGT 1740

GTGGAGTGAA GCAGGGCCAC TTCTATGGAG AGACTGCAGC CGTCTATGTG GCAGTTGAAG 1800

AGAGGAAGGC AGCGGGGTCC CGCGATGTCA GCCTAGCGAA GGCAGACGCT GCTCCTGATG 1860

AGAAGGTGCT AGACTCTGGT TTTCGGGAGA TTGAGAACAA AGCCATTCAG GATCCCAGGC 1920

TTTTTGCAGA GGAAAAGGCG GTGGCAGATA CAAGAGATCA AGCCGATGGG AGCAGAGCAT 1980

CTGTCGATTC CGGCAGCTCT GAGGAACAAG GTGGATGATC TAGAGCGCTG G 2031

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1936 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "PCR-modified SEQ ID NO:l"

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION:16..1920

(D) OTHER INFORMATION: /product= "transcript from SC DNA fragment 2"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:1..46

(D) OTHER INFORMATION: /product= "from PCR with 5' primer 2 (SEQ ID NO 6) "

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:complement (1902..1936)

(D) OTHER INFORMATION:/product= "from PCR with 3' primer (SEQ ID NO 7) "

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION:1..1936

(D) OTHER INFORMATION:/product= "SC DNA fragment 2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GGGAAGCTTG CCACCATGGT GCTCTTCGTC CTCACCTCCC TGCTGGCGGT CTTCCCAGCC 60

ATCTCCACGA AGAGTCCCAT ATTTGGTCCC GAGGAGGTGA ATAGTGTGGA AGGTAACTCA 120

GTGTCCATCA CGTGCTACTA CCCACCCACC TCTGTCAACC GGCACACCCG GAAGTACTGG 180 TGCCGGCAGG GAGCTAGAGG TGGCTGCATA ACCCTCATCT CCTCGGAGGG CTACGTCTCC 240

AGCAAATATG CAGGCAGGGC TAACCTCACC AACTTCCCGG AGAACGGCAC ATTCGTGGTC 300

AACATTCCCC AGCTGAGCCA GGATGACTCC GGGCGCTACA AGTGTGGCCT GGGCATCAAT 360

AGCCGAGGCC TGTCCTTTGA TCTCAGCCTC GAGGTCAGCC AGGGTCCTCG GCTCCTAAAT 420

GACACTAAAG TCTACACAGT GGACCTGGGC AGAACGGTGA CCATCAACTG CCCTTTCAAG 480

ACTGAGAATG CTCAAAAGAG GAAGTCCTTG TACAAGCAGA TAGGCCTGTA CCCTCTGCTG 540

GTCATCGACT CCAGTGGTTA TGTGAATCCC AACTATACAG GAAGAATACG CCTTGATATT 600

CAGGGTACTG GCCAGTTACT GTTCAGCGTT GTCATCAACC AACTCAGGCT CAGCGATGCT 660

GGGCAGTATC TCTGCCAGGC TGGGGATGAT TCCAATAGTA ATAAGAAGAA TGCTGACCTC 720

CAAGTGCTAA AGCCCGAGCC CGAGCTGGTT TATGAAGACC TGAGGGGCTC AGTGACCTTC 780

{^CTGTGCCC TGGGCCCTGA GGTGGCAAAC GTGGCCAAAT TTCTGTGCCG ACAGAGCAGT 840

GGGGAAAACT GTGACGTGGT CGTCAACACC CTGGGGAAGA GGGCCCCAGC CTTTGAGGGC 900

AGGATCCTGC TCAACCCCCA GGACAAGGAT GGCTCATTCA GTGTGGTGAT CACAGGCCTG 960

AGGAAGGAGG ATGCAGGGCG CTACCTGTGT GGAGCCCATT CGGATGGTCA GCTGCAGGAA 1020

GGCTCGCCTA TCCAGGCCTG GC. AACTCTTC GTCAATGAGG AGTCCACGAT TCCCCGCAGC 1080

CCCACTGTGG TGAAGGGGGT GGCAGGAAGC TCTGTGGCCG TGCTCTGCCC CTACAACCGT 1140

AAGGAAAGCA AAAGCATCAA GTACTGGTGT CIOTGGGAAG GGGCCCAGAA TGGCCGCTGC 1200

CCCCTGCTGG TGGACAGCGA GGGGTGGGTT AAGGCCCAGT ACGAGGGCCG CCTCTCCCTG 1260 CTGGAGGAGC CAGGCAACGG CACCTTCACT GTCATCCTCA ACCAGCTCAC CAGCCGGGAC 1320

GCCGGCTTCT ACTCGTGTCT GACCAACGGC GATACTCTCT GGAGGACCAC CGTGGAGATC 1380

AAGATTATCG AAGGAGAACC AAACCTCAAG GTACCAGGGA ATCTCACGGC TGTGCTGGGA 1440

GAGACTCTCA AGGTCCCCTC TCACTTTCCA TGCAAATTCT CCTCGTACGA GAAATACTGG 1500

TGCAAGTGGA ATAACACGGG CTGCCAGGCC CTGCCCAGCC AAGACGAAGG CCCCAGCAAG 1560

GCCTTCGTGA ACTGTGACGA GAACAGCCGG CTTGTCTCCC TGACCCTGAA CCTGGTGACC 1620

AGGGCTGATG AGGGCTGGTA CTGGTGTGGA GTGAAGCAGG GCCACTTCTA TCGAGAGACT 1680

GCAGCCGTCT ATGTGGCAGT TGAAGAGAGG AAGGCAGCGG GGTCCCGCGA TGTCAGCCTA 1740

GCGAAGGCAG ACGCTGCTCC TGATGAGAAG GTCCTAGACT CTGGTTTTCG GGAGATTGAG 1800

AACAAAGCCA TTCAGGATCC CAGGCTTTTT GCAGAGGAAA AGGCGGTGGC AGATACAAGA 1860

GATCAAGCCG ATCGGAGCAG AGCATCTGTG GATTCCGGCA GCTCTGAGGA ACAAGGTCGA 1920

TGATCTAGAG CGCTGG 1936

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic PCR primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CGGTAGGCGT GTACGGTGGG AGGTCTATAT AGC 33

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic PCR primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GGGAAAGCTT CCACCATGGT GCTCTTCGTG CTCACCTCCC TGCTGG 46

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic PCR primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCAGCGCTCT AGATCATCCA CCTTGTTGTT CCTCAGAG 38

Claims

CLAIMS:

1. Recombinant secretory component (rSC) or a functional fragment thereof, obtainable by a process comprising culturing a CHO SSF 3 cell transfected with a vector comprising a DNA coding for said secretory component or a fragment thereof, and isolating the expressed protein from the culture medium.

2. Secretory component according to claim 1 having a content of N-glycolylneuraminic acid that is below 0.5% in respect to total sialic acid.

3. Process for the production of a secretory component or a functional fragment thereof according to claim 1 comprising a) constructing a vector capable of expressing the secretory component or a functional fragment thereof; b) transfecting a CHO SSF 3 cell with said vector; c) culturing the transfected cells; and d) isolating the secretory component or a fragment thereof from the culture medium.

4. Process according to claim 3, wherein the vector is capable of expressing a soluble fragment of the secretory component.

5. Process according to claim 3, wherein the vector comprises a functional fragment of the secretory component as depicted in SEQ ID NO 3 or SEQ ID NO 4.

6. Process according to claim 3, wherein the vector is a mammalian cell expression vector.

7. Process according to claim 3, wherein the vector is based on pCB6 or pCGA93D- PPREN.

8. Process according to claim 3, wherein the vector is pCB6-SC, pMC-SC or pCGA93D- SC.

9. Process according to claim 3, wherein the vector integrates into the chromosome of the CHO SSF 3 cells.

10. Process according to claim 9, wherein transfected CHO SSF 3 cells with amplified integrated vector DNA are selected.

11. Process according to claim 3, wherein the cells are cultured in serum-free medium.

(B)

12. Process according to claim 3, wherein Pluronic is added to the culture medium to increase productivity.

13. Method for the crystallization of a secretory component or a functional fragment thereof according to claim 1 , comprising placing a solution of said secretory component or a functional fragment thereof in a vessel containing a precipitating agent buffer, wherein the solution and the buffer are separated.

14. Method according to claim 13, wherein the solution of said secretory component or the functional fragment thereof contains precipitating agent buffer.

15. Method according to claim 13, wherein the precipitating agent buffer comprises Na- citrate, HEPES, NaN₃, ammonium phosphate and/or Li₂SO₄.

16. Method according to claim 13, wherein the precipitating agent buffer comprises Na- citrate, NaN₃ and ammonium phosphate; or HEPES, NaN₃ and Li₂SO .

17. Method according to claim 13, wherein said secretory component or the functional fragment thereof is placed in a hanging manner over the precipitating agent buffer.

18. Use of rSC according to claim 1 in a method of treatment.

19. Use of the crystallized rSC according to claim 1 for the determination of the 3- dimensional structure.

20. Use of rSC according to claim 1 for binding studies.

21. Use of rSC according to claim 1 in the screening of compounds for their ability to influence poly-lg binding.

22. Use of rSC according to claim 1 for the identification of antagonists or agonists of the SC mediated IgA transport.

23. A modified rSC according to claim 1 that is immobilized on a solid carrier or that bears one or more identifiable maker like biotin or a radioactive, fluorescent or chemoluminescent group.

24. Use of an antagonist or agonist identified using rSC according to claim 1 , in a method of treatment.

25. Use of an antagonist or agonist identified using the 3-dimensional structure according to claim 19, in a method of treatment.