WO1995016697A1

WO1995016697A1 - Recombinant bovine conglutinin and fragments thereof

Info

Publication number: WO1995016697A1
Application number: PCT/US1994/014656
Authority: WO
Inventors: Young Moo Lee; Kevin R. Leiby; Thomas B. Okarma; Kedarnath Sastry
Original assignee: Boston University; Applied Immune Sciences Inc
Current assignee: Boston University; Applied Immune Sciences Inc
Priority date: 1993-12-16
Filing date: 1994-12-14
Publication date: 1995-06-22
Anticipated expiration: 1996-06-16
Also published as: AU1440495A

Abstract

Recombinant materials related to bovine conglutinin provide means to prepare convenient reagents for isolation or depletion of moieties containing carbohydrate residues. In particular, these materials are useful in isolating immune complexes or in depleting samples of them.

Description

RECOMBINA T BOVINE CONGLUTININ AND FRAGMENTS THEREOF

This application is a continuation-in-part of U.S. Serial No. 07/921,296 filed 28 July 1992 and now pending, which is a continuation of U.S. Serial No. 07/433,546 filed 8 November 1989 and now abandoned. The contents of these applications are hereby incorporated herein by reference.

Technical Field The invention is directed to defined protein and peptide molecules and recombinant materials for their preparation. More particularly, the invention concerns recombinant bovine conglutinin and fragments thereof.

Background Art

The isolation of conglutinin from bovine plasma was described in 1967 by Lachmann, P.J. Adv Immunol (1967) 6_.:480-527. The activity of this protein was originally described in 1906 as causing agglutination of erythrocytes that have reacted with antibody and complement. Subsequent structural studies have elucidated the structure of this protein. Davis, III, A.E. et al . Biochemistry (1984) 22:2139-2144 describe studies which characterize the isolated single polypeptide chain of conglutinin as having a molecular weight of 48 kD. The conglutinin consists of two domains: a collagenous N-terminal domain and a lectin- type C-terminal domain. This single peptide is associated in vivo as a complex of six disulfide-linked polypeptide chains to provide a complex of molecular weight of about 300 kD. The ultrastructure of the protein was reported on the basis of electron microscopic studies by Strang, A.J. et al. Biochem J (1986) 234:381-389. The homology of various portions of bovine conglutinin with other proteins has been explored by Young, N.M. et al. Biochem Biophys Res Comm (1987) 143 :645-651. Conglutinin is also found in human serum (Baatrup, G. et al . Scand J Immunol (1987) 26 :355-361; Thiel, S. et al . ibid. 461-468; U.S. patent 4,906,734. These conglutinins are members of the mammalian C-type soluble lectin family as defined by Drickamer, K. J Biol Chem (1988) 263 :9557-9560. The complete amino acid sequence of bovine conglutinin has been described by Lee, Y-M et al . J Biol Chem (1991) 266:2715-1723. The protein consists of 351 amino acid residues and contains a 171-residue collagenous domain which separates a short noncollagenous N-terminal region of 25 residues from a 155-residue globular carboxy- terminus which shows a structural relationship with mannose-binding proteins and is responsible for the lectin activity.

The effect of bovine conglutinin in inhibiting bacterial infections was described by Ingram, D.G. Immunolocrv (1959) 4_:334-345; Ingram, D.G. Immunolocrv (1959) 2:322-333; and Friis-Christiansen, P. Scand J Immunol (1990) 21:453-460. It has also been implicated in inhibiting infectivity of Influenza-A virus by Hartley, CA. et al . J Virol (1992) 66:4358- 4363; and Hartshorn, D.L. et al . J Immunol (1993)

151 :6265-6273. The human form of conglutinin has been implicated in the inhibition of Human Immunodeficiency Virus-I by Ushigima, H. et al . Japan J Cancer Res (1992) £2:458-464 and the bovine form of the protein was shown to inhibit interaction of HIV-I envelope glycoprotein gpl60 with CD4 by Andersen, 0. et al . Scand J Immunol (1991) 33 :81-88. Conglutinin has been shown to bind specifically to monocytes and macrophage as well as other cell types through its ability to couple to the Clq receptor present on these cells. This capacity has been noted in articles by Malhotra, R. et al. J Exp Med (1990) 172:955-959; Arvieux, J. et al . Biochem J (1984) 218:547-555; Malhotra, R. et al . Biochem J (1989) 262 :625-631; Tenner, A.J. et al . J Biol Chem (1989) 264 (23) :13923-13928; and Guaan, E. et al . J Biol Chem (1991) 266 (30) :20345-20355. It is suspected that it is the collagen region of this molecule that accounts for the binding through Clq receptor.

Immobilized bovine conglutinin has also been used for the extracorporeal removal of immune complexes as described by Pitt, A.M. et al . International J Artificial Organs (1980) 2:42-49.

The ability of bovine conglutinin and defined fragments thereof to behave as lectins in specifically binding carbohydrate moieties makes this protein and its fragments useful in the design of materials which can be used to selectively deplete samples of, or to purify from biological samples containing them, materials which are characterized by the carbohydrates to which the bovine conglutinin lectin functionality binds. The availability of recombinant materials for production of this characteristic lectin region provides the opportunity to design techniques for these procedures. Disclosure of the Invention

The invention provides recombinant materials which are useful in various separation procedures where the materials to be separated contain carbohydrate functionalities or moieties. The invention materials are related to bovine conglutinin and can be produced recombinantly.

In one aspect, the invention is directed to recombinant materials for the production of conglutinin, especially bovine conglutinin, including purified and isolated DNA molecules containing nucleotide sequences encoding relevant portions of the conglutinin amino acid sequence, expression systems, cells modified to contain the expression systems and methods for recombinant production. The invention is also directed to solid supports to which conglutinin- related fragments have been bound covalently, which are useful in the separation of materials having carbohydrate moieties bound by the lectin portion of conglutinin. The collagenous region is also useful to bind cells displaying Clq receptor.

Brief Description of the Drawings

Figure 1 shows the nucleotide sequence and deduced amino acid sequence of a cDNA encoding bovine conglutinin.

Modes of Carrying Out the Invention

The invention provides recombinant materials for the production of carbohydrate binding portions of the bovine conglutinin protein. Some of these ^' recombinant materials are also useful in regulating the expression of genes encoding conglutinin either in recombinant host cells, in in vi tro systems, or in intact organisms expressing these genes. As the amino acid sequence of the lectin-like portion of bovine conglutinin has been elucidated, the production of the relevant fragments or proteins can be achieved, not only using recombinant techniques, but also by using synthetic methods for the production of peptides including both solution phase and solid phase synthesis. Both of these procedures are well understood in the art, and solid phase synthesis can be conducted using commercially available equipment and reagents. In addition, tandem copies of relevant amino acid sequences in a single protein or peptide can be prepared. Figure 1 shows the nucleotide sequence and deduced amino acid sequence of bovine conglutinin. As shown in Figure 1, the protein is produced including a presequence of 20 amino acids which is cleaved to obtain the mature protein representing amino acids 1- 351 in Figure 1. The residues at positions 26-196 represent a collagen-type domain with the repeating sequence GXY. This portion of the molecule also contains several hydroxylysine residues which are conveniently used for coupling to solid supports. The C-terminal domain beginning at approximately residue 197, appears to be responsible for the lectin-like characteristics of the molecule. In particular, the region at positions 250-351 shares considerable homology with other animal lectins. Accordingly, this region of the molecule, and carbohydrate binding regions thereof, are useful in separation of carbohydrate-bearing moieties. Thus, of particular importance are the residues shown at positions 250-351, or fragments thereof, containing preferably 10 or more, more preferably 15 or more and most preferably 25 or more amino acid residues which demonstrate the capability to bind relevant carbohydrates. Such ability can readily be ascertained by coupling the fragment to a solid support, labeling the moiety whose binding is to be tested, and assessing the ability of the support to retain the label. Such simple techniques for ascertaining affinity are well understood in the art .

Recombinant Production of Conglutinin and Its Fragments

Techniques for recombinant expression of DNA and RNA molecules containing nucleotide sequences encoding proteins of interest are well known in the art. General descriptions of such expression, including host cells and expression system elements for regulation of expression such as promoters, transcription termination signals, and the like are described in Goeddel, D. Gene Expression Technology:

Methods in Enzvmology (1990) 185 : Academic Press, San Diego, California. Methods for preparation of these expression systems and for modifying host cells to contain them can be found, for example, in Sambrook, et al . , Molecular Cloning: A Laboratory Manual. 2nd Ed. (1989) Cold Spring Laboratory Press and in other standard laboratory manuals.

Appropriate control sequences for prokaryotes have been known for more than ten years . These expression systems can use constitutive or inducible promoters, including hybrid promoters such as the commercially available TRC promoter. Suitable

covalently to solid support and tested for the ability of the derivatized support to bind labeled carbohydrate containing moiety. Such tests are simple and straightforward and can be conducted routinely. It is understood that the amino acid sequence shown as residues 250-351 is established to have this capability. Smaller fragments as described above can be conveniently tested for this property. In addition, novel peptides having multiple copies of defined amino acid sequences can be used.

Primers, Probes and Other Oligomers

The availability of the nucleotide sequence shown in Figure 1 and of its complement permit the design of various oligonucleotides useful in recombinant production, therapeutic and diagnostic contexts. Modulation of the expression of the gene encoding bovine conglutinin is useful in regulating the production of the recombinant material as well as modulating immune responses in si tu . In addition, assessing the level of RNA present in the liver which corresponds to the conglutinin molecule is an index to the response of the subject to infection. Thus, oligomers based on the nucleotide sequence disclosed in Figure 1 herein can be used in standard assay methods for detecting the conglutinin-encoding DNA or RNA. By oligomers "based on" the sequence disclosed in Figure 1 is meant oligomers that contain portions of this sequence, that are complementary to the sequence or portions thereof, that represent primers used to amplify portions of the sequence when large amounts of DNA are desirable (such as for genetic manipulation) as well as oligomers designed on the basis of the disclosed sequence which, for example, effect triple helix formation with the relevant portion_. of the duplex representing the conglutinin gene. Relevant design parameters for PCR primers, oligomers capable of hybridizing to single strand targets, and oligomers capable of triple helix formation with DNA duplexes are well known in the art. Thus, oligomers "based on" the DNA of Figure 1 may have the same sequence as a portion of this DNA, the same sequence as the complement or portion thereof, or a different sequence but one which corresponds to that disclosed in Figure 1 through art-known design parameters.

The oligomers having nucleotide sequences based on the nucleotide sequence shown in Figure 1 may be conventional RNA or DNA polymers, or may be modified forms thereof as generally known in the art . For example, the phosphodiester bonds of the oligomers may be substituted by alternative linkages such as phosphorothioates, methylphosphonates and the like. In addition, alternative scaffolding for nucleotide bases has also been disclosed and such modifications are included within the scope of oligomers claimed herein.

Industrial Applicability

Because of the ability of the lectin portion of the conglutinin molecule to bind carbohydrate moieties, the recombinantly reproduced peptides and proteins of the invention find utility in performing separations where the substrates to be separated are marked by the relevant carbohydrates. In particular, immune complexes contain appropriate sugars so that they can be bound to the lectin portion of the conglutinin molecule. While the complete bovine conglutinin has been used in this manner, as described by Pitt, A.M. et al . International J Artificial Organs (1980) 2:42-49, as set forth above, the availability of the amino acid sequence of the relevant portion of the molecule, as well as the recombinant materials related to it permit design of more efficient systems for conducting such separations.

In general, the protein or peptide containing a sequence of amino acids which confers ability to bind the relevant carbohydrates will be bound to a solid support, such as beads, microtiter trays, or other solids used generally in chromatography for use in the invention methods. Such coupling can be achieved through standard derivatization of the support, depending on its chemical nature, and coupling of the derivatized support to, for example, sulfhydryl groups, amino groups, or carboxyl groups of the relevant protein. The peptide or protein is coupled to solid support in an appropriate density for the application desired and optimization of such parameters is well known in the art.

For use in depleting a sample of, for example, an immune complex, the sample is contacted with the derivatized support for a time sufficient, and under conditions which are appropriate to, the binding of the immune complex to the peptide or protein ligand attached to the support. The support is removed, then, from the sample and the remaining unadsorbed sample is depleted in the concentration of the immune complex. If it is then desirable to recover the immune complex, the treated support to which the immune complex is adsorbed is eluted with a suitable eluant . Suitable eluants include, in particular, competitive carbohydrate moieties which displace the immune complex from the solid support, and chelating agents such as EDTA which remove Ca⁺², essential for lectin function. These general methods can be applied to samples containing moieties which are marked by carbohydrates to which the peptide and protein ligands of the invention are bound.

The peptide or protein ligands themselves are susceptible to considerable design parameters. It is believed that the ligand of the bovine conglutinin shown in Figure 1 representing positions 250-351 inclusive is intimately involved in binding of the carbohydrates. Thus, this section of the molecule is particularly relevant to construction of the invention ligands. Smaller portions of this region may also be used as long as their binding characteristics are retained. It may be advantageous to provide peptides or proteins which have more than one carbohydrate binding sequence; for example, a peptide could be constructed which has three contiguous copies of the amino acid sequence represented by positions 250-300 or a peptide which has two such regions and an additional region representing positions 275-325. As set forth above, the capability of the constructed ligand to bind the carbohydrate marked moiety can readily be tested by using either labelled carbohydrate moiety or a competition assay for binding of such moiety.

It may also be useful to assess conglutinin levels produced in individuals as an index to a response to infection. This can be performed on biological samples, for example, using antibodies raised with respect to bovine conglutinin. As is generally understood, such antibodies need not be formed in response to the complete conglutinin molecule, but rather can be formed from suitable epitopes derived from the amino sequence. It may be necessary to couple smaller epitopes to carrier in order to enhance immunogenicity. However, antisera raised using standard immunization protocols, or monoclonal antibodies prepared from peripheral B cells or splenocytes of immunized animals can be used to conduct immunoassays for conglutinin levels in biological samples using standard immunoassay procedures .

The level of conglutinin production can also be assessed by conducting suitable assays for messenger RNA in the liver. As described below, liver appears to be the only tissue in which conglutinin is made. The oligomers of the invention based on the oligonucleotide sequence shown in Figure 1 may be useful as probes in Northern blot assays, although as indicated below, it is preferred to use RNase based assays to detect the presence of mRNA. Oligomers, in particular, antisense oligonucleotide sequences based on the nucleotide sequence of Figure 1 are also useful in this assay, as described in Example 1.

In addition, oligomers designed to modulate the production of conglutinin may be useful in modulating the response of subjects to infection as well as in regulating the production of conglutinin related peptides and proteins using recombinant methods . The following examples are intended to illustrate but not to limit the invention. Example 1 Identification of Tissues Expressing Bovine Conglutinin

Two amino acid stretches (280-291 and 325- 341) in the carbohydrate recognition domains (CRD) from the published primary sequence of bovine conglutinin (Lee, Y.M. et al . J Biol Chem (1991) 266_:2715-2723) were used to synthesize guessmer oligomers. PCR on bovine genomic DNA, using oligomers BC1M and BC2M, amplified a prominent fragment of the expected size (186 bp) . Nucleotide sequencing of the fragment confirmed that this product encoded a portion of the bovine conglutinin CRD exon, and the deduced amino acid sequence matched perfectly with the published bovine conglutinin protein sequence. In more detail, the guessmer oligomers (BC1M

S' GCC CTC GAG AAG AAT GCC TAC CTG TCC ATG AAT GAC and BC2M 5' GCA GAA TTC ATC ATT CCA CTT GCC ATC AGG GAA GAT CTC CAC ACA GTT CTC) primed a PCR on a Perkin-Elmer Cetus DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) using bovine genomic DNA (Novagen, Madison, WI) as the template. The PCR parameters consisted of one initial cycle of denaturation at 95° for 5 min, annealing at 55° for 5 min, and extension at 72° for 3 min followed by 40 cycles of denaturation at 95° for 1 min, annealing at 55° for 1 min, and extension at 72° for 3 min. The final cycle was a 7 min extension at 72°. The reaction mixture (50μl) contained 10 pmol of each primer, 0.2 mM dNTP, 2.5 U Taq polymerase, IX PCR reaction buffer (Perkin Elmer Cetus, Norwalk, CT) , and lug of bovine genomic DNA. A PCR product of 186 bp was obtained.

Northern blot analysis using random primed ³²P-labeled 186 bp double stranded DNA probe to define the tissue (s) expressing bovine conglutinin mRNA was not successful. Therefore an RNase protection assay was substituted. RNA from various bovine tissues was hybridized to a riboprobe obtained by transcription of a 260 bp PstI genomic fragment encoding a portion of the CRD exon inserted in a KS⁺ host vector prepared as follows : the PCR product was labeled using random hexamers with ³²P dCTP, and used to screen a bovine genomic library in cosmid vector pWE15 (Stratagene, La Jolla, CA) . One clone, 16-2 was used for further study. A 260 bp PstI fragment from this genomic clone was subcloned into KS⁺ vector (Stratagene, La Jolla, CA) .

The ³²P riboprobe (Specific activity 7.58 x 10⁸) derived by transcription using T3 polymerase with maxiscript kit (Ambion, Austin, TX) , was used for a RNase protection assay following instructions provided (RPA II kit, Ambion, Austin, TX) . Approximately 1.7 x 10⁵ cpm probe was hybridized with either total RNA from bovine brain, heart, jejunum, lung, rumen and spleen (20 μg each) or poly A+ RNA from bovine kidney and liver (1 μg each) . The jejunum and rumen total RNA were isolated by the modified single step procedure (Chomczynski, P. et al . Anal Biochem (1987) 162 :156- 159) using RNAzol B (Biotex, Houston, TX) and the other bovine RNAs were obtained from Clontech, Palo Alto, CA) . Yeast tRNA (20 μg) served as control. The full length PstI riboprobe along with polylinker sequences was 334 bp long and the expected size of protected fragments was 260 bp. The RNase digests were run on 5% acrylamide-urea gel and were exposed to X-ray film with intensifying screens at -70°C for 96 hours. The results indicated that bovine liver is the only tissue expressing bovine conglutinin mRNA. These results agree with a recent study (Lim, B.L. et al . Immunology (1993) 2ϋ:159- 65) , who, based on the hybridization of a bovine SP-D probe to bovine liver RNA, suggested liver as a possible site for the synthesis of bovine conglutinin.

Example 2 Isolation and Characterization of Bovine Conglutinin cDNA The 186 bp PCR fragment, described in Example

1, was radiolabeled randomly with ³²P following the methods described by Feinberg, A.P. and Vogelstein, B. Anal Biochem (1983) 132 :6-13. and used to screen a bovine genomic library in cosmid vector pWE15 (Stratagene, La Jolla, CA) , following the procedure suggested by the manufacturer. In this method, bacteria harboring cosmids are plated onto nylon membranes placed on petridishes containing agar and nutrients (essential for bacterial growth) and allowed to grow overnight in an incubator at 37°C. These membranes serve as the master plates and replicas of the colonies are made on two more membranes, which are again allowed to grow overnight. After making replicas, the master plates are incubated at room temperature overnight to allow additional growth of the bacterial colonies and are then stored in the refrigerator. The replica membranes with bacterial colonies are lysed and processed, following the protocol suggested by the manufacturer of the membranes (Hybond N⁺, Amersham, Arlington Heights, IL) . The objective of this process is to immobilize the cosmid and bacterial DNA onto nylon membranes. The nylon membranes are next immersed in prehybridization solution which consists of 5X SSPE [IX SSPE is 0.18M NaCl, lOmM sodium phosphate, pH7.7 (Na₂HP0₄ is added to NaH₂P0₄ to bring sodium phosphate to pH7.7) , lmM EDTA] ; 5X Denhardt's reagent [IX Denhardt's is 0.02% ficoll (mw=400, 000) , 0.02% polyvinylpyrrolidone (mw=40, 000) , 0.02% bovine serum albumin] ; 0.lmg/ml sonicated salmon sperm DNA; 0.5% (w/v) sodium dodecyl sulfate (SDS) ) to block nonspecific binding at 65° for 2 hours. The heat denatured ³²P labeled 186bp PCR fragment was then added to prehybridization solution and the incubation is continued overnight in a shaking water bath at 65°C. The membranes were next washed for 30 min in 2X SSC (IX SSC is 0.15M NaCl/0.015M sodium citrate) /0.1% (w/v) SDS at room temperature, followed by 30 min in IX SSC/0.1% (w/v) SDS at 65°C. The nylon membranes were exposed to X-ray film overnight (approx. 16 hours) at -70°C, with intensifying screens to detect colonies harboring the bovine conglutinin gene cosmid. After picking the colonies on the master plate corresponding to the positive signal and purifying them through two additional rounds of screening, eight genomic bovine conglutinin cosmid clones were obtained (which hybridized to the 186bp radiolabeled PCR fragment) by screening approximately 1.2 X 10⁶ bacterial colonies from the library.

One of the genomic cosmid clones isolated, called 16-2, was studied further and a 550bp X oI-Ba_τ_HI genomic fragment (encoding the amino terminal end of the bovine conglutinin) was subcloned from 16-2 cosmid into KS⁺ vector and was used as a probe to screen lambda phages from a 5' stretch bovine liver cDNA library (Clontech, Palo Alto, CA) . The lambda phage library of bovine liver cDNA was screened following the procedures recommended by the manufacturer (Clontech) . The method for screening the phage library is essentially similar to the procedure described above for the isolation of genomic clone, except that the cDNA is inserted into a phage, which multiplies inside bacterial cells, eventually killing them, causing appearance of 'plaques' . Of the 1.2 X 10⁶ phage clones screened, only one clone was found to hybridize to the probe. Restriction analysis of the clone insert revealed three EcoRI fragments of approximately 1.6, 0.6 and 0.5 kb, respectively. The two small EcoRI fragments were cloning artifacts since they had 100% identity to mouse and human 28 S rRNA. The 1.6 kb EcoRI fragment that encodes bovine conglutinin was sequenced in its entirety on both the strands, by subcloning various fragments into plasmid vectors. The isolated bovine conglutinin cDNA clone has a 230 bp 5' untranslated region, a 1116 bp coding region which is then followed by a 173 bp 3' untranslated region. The complete nt sequence of the isolated bovine conglutinin cDNA clone is shown in Figure 1.

There are three in-frame stop codons and three ATG codons in the 5' untranslated region, with only the first and third ATG having at -3 position a purine, the most highly conserved nt in all eukaryotic initiation sequences (Kozak, M. J Cell Biol (1989) 108 :229-241) . The third and the longest ORF from the 5' end of the cDNA clone encodes a signal peptide and an amino acid sequence which is identical to the previously determined bovine conglutinin amino acid sequence, except two differences in the collagen domain: His¹⁵³, Lys¹⁹⁰ in place of Arg and Ser respectively (Lee, et al . (1991) supra) . There are two polyadenylation signal sequences in the 3' untranslated region of the cDNA clone, with the poly(A) tail being found 16 bp after the second polyadenylation sequence.

Claims

1. An isolated and purified DNA molecule which comprises the nucleotide sequence encoding the amino acid sequence numbered 250-351 inclusive in Figure 1 herein.

2. The DNA molecule of claim 1 which comprises the nucleotide sequence encoding the amino acid sequence numbered 197-351 inclusive in Figure 1.

3. The DNA molecule of claim 1 which comprises the nucleotide sequence encoding the amino acid sequence numbered 1-351 inclusive of Figure 1.

4. The DNA molecule of claim 1 which comprises the nucleotide sequence encoding the amino acid sequence numbered -20 to 351 inclusive of Figure 1.

5. A DNA molecule which comprises an expression system for the production of a protein with an amino acid sequence comprising positions 250-351 inclusive of Figure 1, or a carbohydrate-binding fragment thereof, wherein said expression system comprises a nucleotide sequence encoding said protein operably linked to control sequences for its expression.

6. The DNA molecule of claim 5 wherein said nucleotide sequence encodes a protein having the amino acid sequence of positions 197-351 inclusive of Figure 1.

7. The DNA molecule of claim 5 wherein said nucleotide sequence encodes a protein having the amino acid sequence of positions 1-351 inclusive of Figure 1.

8. The DNA molecule of claim 5 wherein said nucleotide sequence encodes a protein having the amino acid sequence of positions -20 to 351 inclusive of Figure 1.

9. A recombinant host cell modified to contain the DNA molecule of claim 5.

10. A recombinant host cell modified to contain the DNA molecule of claim 6.

11. A recombinant host cell modified to contain the DNA molecule of claim 7.

12. A recombinant host cell modified to contain the DNA molecule of claim 8.

13. A method to produce a recombinant bovine conglutinin peptide or protein which method comprises culturing the cells of claim 9 under conditions to effect expression so as to produce said peptide or protein; and recovering the peptide or protein from the culture.

14. A method to produce a recombinant bovine conglutinin peptide or protein which method comprises culturing the cell of claim 10 under conditions to effect expression so as to produce said peptide or protein; and recovering the peptide or protein from the culture.

15. A method to produce a recombinant bovine conglutinin peptide or protein which method comprises culturing the cells of claim 11 under conditions to effect expression so as to produce said peptide or protein; and recovering the peptide or protein from the culture.

16. A method to produce a recombinant bovine conglutinin peptide or protein which method comprises culturing the cells of claim 12 under conditions to effect expression so as to produce said peptide or protein; and recovering the peptide or protein from the culture.

17. An oligomer based on the nucleotide sequence of Figure 1.

18. The oligomer of claim 17 wherein said oligomer is a complement of at least a portion of the nucleotide sequence of Figure 1.

19. The oligomer of claim 17 wherein said oligomer is designed to form a triple helix with a duplex formed by at least a portion of the nucleotide sequence of claim 1 and its complement.

20. A solid support to which is covalently bound a peptide comprising the amino acid sequence shown as positions 250-351 in Figure 1.

21. The support of claim 20 wherein said peptide comprises at least two copies of said sequence,

22. The solid support of claim 20 wherein the peptide comprises the amino acid sequence of residues 197-351 in Figure 1.

23. The support of claim 22 wherein said peptide comprises at least two copies of said sequence.

24. The solid support of claim 20 wherein the peptide comprises the amino acid sequence of residues 1-351 in Figure 1.

25. The support of claim 24 wherein said peptide comprises at least two copies of said sequence,

26. The solid support of claim 25 wherein the peptides comprises the amino acid sequence of residues -20 to 351 in Figure 1.

27. The support of claim 26 wherein said peptide comprises at least two copies of said sequence,

28. A method to modulate the production of conglutinin which comprises contacting liver cells which produce conglutinin with an effective amount of the oligomer of claim 18.

29. A method to modulate the production of conglutinin which comprises contacting liver cells which produce conglutinin with an effective amount of the oligomer of claim 19.

30. A method to deplete a sample of substances containing a carbohydrate moiety, which carbohydrate moiety binds the lectin region of bovine conglutinin which method comprises contacting said sample containing said carbohydrate with the solid support of claim 20 under conditions wherein said carbohydrate is bound, removing the solid support from the sample, and recovering the remaining sample which is depleted in said substances.

31. A method to enrich a sample in substances containing a carbohydrate which binds the lectin region of conglutinin, which method comprises contacting said sample with the solid support of claim 20 under conditions wherein said carbohydrate binds the solid support; removing the solid support from the sample,• and recovering the substances bound to the solid support to obtain a composition enriched in said substance.