Abstract:
The invention relates to the cytoplasmic expression of antibodies, antibody fragments and antibody fragment fusion molecules in  E. coli.  In particular, antibody fragment fusion molecules having an antibody moiety which is directed against tumors and an enzyme moiety which cleaves a nontoxic prodrug to give the toxic drug can be advantageously prepared in this way while retaining their respective functional properties.

Description:
The present application is a divisional of U.S. application Ser. No. 08/630,820 (now U.S. Pat. No. 6,008,023), filed Apr. 10, 1996, which claims priority under 35 U.S.C. §119(b) to German application No. 195 13 676.4, filed on Apr. 11, 1995. 
    
    
     BACKGROUND OF THE INVENTION 
     The expression of functional antibodies and antibody fragments in  E. coli  is known in the prior art, but these methods require the use of signal sequences which direct polypeptide transport into the periplasm. When expression takes place in the  E. coli  periplasm, the expression yields are in the range of a few μg per liter of culture medium (Ayala et al., Bio Techniques 13, pp. 790-799, 1992). In addition, refolding experiments are often required in order to obtain functionally active antibody fragments (such as Fab) or antigen-binding regions (such as a single chain Fv(sFv)). There is a need therefore, to develop improved methods for expressing functionally active antibodies and antibody fragments. The prior art does not teach recombinant production of antibodies or antibody fragments which can be isolated from the cytoplasm in functional form. Such molecules would be useful in the production of therapeutic agents. 
     Bagshawe describes a method for generating cytotoxic agents that are directed towards cancer sites, termed Antibody Directed Enzyme Prodrug Therapy (ADEPT). Bagshawe, Br. J. Cancer, vol. 60, pp. 275-281, 1989. Using ADEPT, an antibody or antibody fragment that specifically binds to a cancer cell is fused to an enzyme that is capable of converting a non-toxic drug into a toxic drug. Only those cells to which the fusion protein is bound will be killed upon administration of the precursor of the toxic drug. 
     The β-glucuronidase of  Escherichia coli  has been well characterized biochemically and genetically. The gene (uid A) has been cloned by Jefferson et al. (PNAS vol. 83, pp. 8447-8451, 1986) and employed as a reporter gene for heterologous control regions. 
     β-Glucuronidase (β-D-glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) is an acid hydrolase which catalyzes the cleavage of β-glucuronides. As a result of the mammalian glucuronidases having been intensively investigated, a variety of substances are available for histological, spectrophotometric and fluorometric analyses. This enzyme has gained new, additional importance in its use for fusion proteins for targeted tumor therapy. In this connection, human glucuronidase is used in the form of a fusion protein which contains antibodies/antibody fragments or antigen-binding regions (Bosslet et al., Br. J, Cancer, 65, 234-238, 1992). As an alternative to the human enzyme, it is also possible to use the homologous  E. coli  β-glucuronidase. One of the advantages of the  E. coli  β-glucuronidase is that its catalytic activity at physiological pH is significantly higher than that of the human β-glucuronidase. 
     In the past, it has only been possible to express antibody fragment-enzyme fusion molecules periplasmically in  E. coli.  The enzyme moiety which is used in this context is therefore always composed of periplasmic  E. coli  enzymes such as β-lactamase (Goshorn et al., Canc. Res. 53, 2123-2117, 1993). 
     An  E. coli  strain which is deficient in thioredoxin reductase (TRR), for example the strain AD 494, is capable of forming disulfide bridges in the cytoplasm and thus enzymes which are naturally secretory, for example alkaline phosphatase, can be expressed intracellularly See Derman et al.,  Science,  262:1744-1747, 1993. Derman describes the selection and isolation of TRR-deficient  E. coli  mutants. 
     The prior art does not teach expression of an antibody fragment-enzyme fusion molecule using a cytoplasmic  E. coli  enzyme, such as β-glucuronidase, which is functionally active—i.e., which retains both enzymatic activity and antigen-binding ability of the antibody moiety. As a rule, functionally active expression of most antibodies or antibody fragment molecules requires defined signal sequences for exporting the expressed molecules via the endoplasmic reticulum into the culture medium (animal cells and yeast) or into the periplasm ( E. coli ). It is only in the endoplasmic reticulum or in the periplasm that the necessary oxidative conditions pertain for forming the disulfide bridges which are important for functional activity. In addition, the secretory synthesis route is often crucial for the correct three-dimensional folding of the expressed protein. 
     SUMMARY OF THE INVENTION 
     Thus, it is an object of the invention to provide a method for the production of functional antibodies and antibody fragments in  E. coli  in which the antigen-binding polypeptides can be isolated from the cytoplasm without the need for further processing such as protein folding and disulfide bond formation. 
     It is also an object of the invention to provide a method for the production of fusion polypeptides in  E. coli  comprising antibody or antibody fragment and an enzyme, in which the antibody or antibody fragment and the enzyme retain functionality and in which the functional fusion polypeptide can be isolated from the cytoplasm without the need for further processing such as protein folding and disulfide bond formation. 
     The invention relates, therefore, to processes for the recombinant expression of antibodies, antibody fragments or antibody fragment fusion molecules containing cytoplasmic mammalian or  E. coli  enzymes as fusion partners using thioredoxin reductase-deficient  E. coli  strains and subsequent isolation of the expression products from the cytoplasm. 
     The invention relates to the cytoplasmic expression of antibodies, antibody fragments and antibody fragment fusion molecules in  E. coli.  In particular, antibody fragment fusion molecules having an antibody moiety which is directed against tumors and an enzyme moiety which cleaves a nontoxic prodrug to give the toxic drug can be advantageously prepared in this way while retaining their respective functional properties. 
     Accordingly in one embodiment, the invention provides a method for producing an antibody or antibody fragment comprising: 
     a) transforming a thioredoxin reductase-deficient  E. coli  strain with a nucleotide molecule encoding said antibody or antibody fragment; 
     b) culturing said transformed  E.coli  strain to allow for expression of said antibody or antibody fragment; and 
     c) isolating said antibody or antibody fragment from the cytoplasm of said transformed  E.coli.    
     In a further embodiment, the invention also provides a method for producing a fusion protein comprising an antibody or antibody fragment and an enzyme, said method comprising: 
     a) transforming a thioredoxin reductase-deficient  E. coli  strain with a nucleotide molecule encoding said fusion protein; 
     b) culturing said transformed  E.coli  strain to allow for expression of said fusion polypeptide; and 
     c) isolating said fusion polypeptide from the cytoplasm of said transformed  E.coli.    
     In another embodiment, the antibody fragment used in the methods of the invention is selected from the group consisting of an Fab fragment, an Fv fragment, an sFv fragment and an F(ab′) 2  fragment. The antibody used in the methods of the invention can be a humanized antibody. The invention further provides an embodiment wherein the antibodies used in the method of the invention can be antibodies or antibody fragment binds specifically to tumor cells. 
     In another embodiment, an enzyme used in the method for making a fusion protein is capable of cleaving a nontoxic prodrug to produce a toxic drug and may be a human cytoplasmic enzyme. 
     In a further embodiment, the fusion protein produced according to the invention comprises an antibody or antibody fragments that is capable of specifically binding to tumor cells and an enzyme capable of cleaving a nontoxic prodrug to produce a toxic drug. 
     In a further embodiment, the fusion protein produced according to the invention comprises a humanized antibody and a human cytoplasmic enzyme. In another embodiment, the invention comprises a fusion protein comprising  E. coli  β-glucuronidase. 
     In another embodiment, the invention provides fusion proteins comprising an antibody or antibody fragment and an enzyme. The invention further provides fusion proteins produced according to the methods of the invention. In a further embodiment, a fusion polypeptide comprising  E. coli  β-glucuronidase and an antibody or antibody fragment is provided. 
     In a further embodiment, a nucleotide sequence encoding a fusion protein comprising an antibody or antibody fragment and an enzyme is provided. In still a further embodiment, the invention provides a nucleotide sequence encoding a fusion polypeptide comprising  E. coli  β-glucuronidase and an antibody or antibody fragment. In yet another embodiment, a nucleotide sequence encoding the amino acid sequence in FIG. 5 which begins at nucleotide number 666 and ends at nucleotide number 3162 is provided. In a final embodiment, a nucleotide sequence is provided wherein said sequence is the nucleotide sequence in FIG. 5 which beings at nucleotide number 666 and ends at nucleotide number 3165. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 illustrates the insertion of the DNA sequence for  E. coli  β-glucuronidase into the vector p bluescript II KS (KS) to produce vector KS/A.c.-β-Gluc. The sequences of the primers used to amplify the β-glucuronidase gene are also shown. 
     FIG. 2 shows the sequences of the primers used to amplify the antibody variable domain VH and the CH1 constant domain from the cDNA construct HC-hum-β-glucuronidase. This Figure also shows the cloning of the VH/CH1 region into vector KS by cleavage of the HC-hum-β-glucuronidase using XbaI and EcoRI restriction sites to produce vector KS/Fab HC. 
     FIG. 3 shows the ligation of the XbaI/NcoI fragment from vector KS/Fab HC into vector KS/A.c.-β-Gluc to produce vector KS/Fab HC/E.C. β-Gluc. 
     FIG. 4 shows the ligation of the XbaI/HindIII fragment of vector KS/Fab HC/E.C. β-Gluc into vector pTrc 99/FabLC to produce vector pTrc99/dicistr. Fab/E.c.β-Gluc. 
     FIG. 5 a  shows the organization of vector pTrc99/dicistr. Fab/E.c.β-Gluc. 
     FIG. 5 a  shows the nucleotide and corresponding amino acid sequences for the VK and CK domain coding sequences inserted into the vector pTrc99/dicistr. Fab/E.c.β-Gluc. 
     FIGS. 5 a - 5   i  shows the nucleotide and corresponding amino acid sequences for the VH, CHI and  E. coli  β-glucuronidase coding sequences inserted into the vector pTrc99/dicistr. Fab/E.c.β-Gluc. 
     FIG. 6 shows TSK-3000 gel chromatography of a purified fusion protein to determine the molecular weight of the fusion protein. The fusion protein eluted at 13.4 minutes, as evidenced in the peak absorbance at 280 nm. The positions of other molecular weight markers are shown. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     It has now been found that a antibody fragment, such as Mab BW 431/26 (an Fab molecule), Güssow &amp; Seemann,  Methods in Enzymology,  203:99-121 (1991), or a complete antibody molecule, can be expressed cytoplasmically in a functionally active state, i.e., while retaining its antigen-binding properties in an  E. coli  strain which is deficient in thioredoxin reductase. 
     Surprisingly, it has furthermore been possible to express an antibody fragment-enzyme fusion molecule composed of, for example, the cytoplasmic, non-disulfide-bridged  E. coli  enzyme β-glucuronidase and, for example, the Fab BW 431/26 which requires intramolecular cystine bridges for correct folding (Fab BW 431/26- E. coli  β-glucuronidase) in the cytoplasm and to isolate it therefrom in functional form. At the same time, the expressed molecule was soluble and no refolding was necessary to produce a functionally active polypeptide. This opens up novel opportunities for economically producing antibodies, antibody fragments and antibody fragment-enzyme fusion molecule for therapeutic and diagnostic use. 
     Antibodies and Antibody Fragments 
     Examples of antibody fragments of the invention include (A) a “half antibody” molecule, i.e., a single heavy:light chain pair, and (B) an antibody fragment, such as the univalent fragments Fab and (ab), the divalent fragment F(ab′) 2 , and a single or double chain Fv fragment. Antibody fragments according to the invention are preferably Fab fragments or antigen-binding regions such as sFv. See Plückthun and Skerra, Meth. Enzymol. 178, pp. 497-515, 1991. Many antibodes are known in the art. Antibodies according to the invention include human antibodies, humanized antibodies, and other antibodies known in the art. 
     Humanized antibodies are chimeric antibodies comprising non-human and human regions, and have reduced immunoreactivity when used therapeutically in humans. Typically, the variable domains or are of non-human origin and the constant domains are of human origin. Humanized antibodies can also be produced by inserting non-human complimentarity-determining-regions (CDRs) into the framework of a human antibody. An antigen binding site in an antibody is made up of CDRs in the light chain and CDRs in the heavy chain. Humanized antibodies can be produced using recombinant DNA technology well-known in the art. Briefly, oligonucleotides encoding CDRs with desired antigen-recognition properties are used to replace the CDR regions in a human antibody gene. In certain instances, a mouse monoclonal antibody will have the desired antigen-recognition characteristics. These CDR-encoding regions are sequenced and oligonucleotides encoding these regions are inserted into the human antibody gene. See Güssow,  Methods in Enzymology  203:99-121 (1991), which describes techniques well known in the art for humanization of antibodies and cloning antibody (immunoglobulin) genes. 
     The antibodies and antibody fragments according to the invention preferably bind specifically to malignant, cancerous, or tumorigenic cells. It is well known in the art that cancer cells often express specific antigens on their surface, and it is to these antigens that the antibodies and antibody fragments according to the invention specifically bind. 
     Also illustrative of an antibody fragment within the present invention is a non-peptide “mimetic,” i.e., a compound that mimics an epitope binding site of an antibody but that is water-soluble, resistant to proteolysis, and non-immunogenic. Conformationally restricted, cyclic organic peptides which mimic any of these antibodies can be produced in accordance with known methods described, for example, by Saragovi, et al.,  Science  253: 792 (1991). 
     In accordance with the present invention, antibody fragments within the invention can be obtained from a antibody by methods that include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer such as those supplied commercially by Applied Biosystems, Multiple Peptide Systems and others, or they may be produced manually, using techniques well known in the art. See Geysen et al.,  J. Immunol. Methods  102: 259 (1978). Direct determination of the amino acid sequences of the variable regions of the heavy and light chains of the antibodies according to the invention can be carried out using conventional techniques. 
     As noted, a fragment according to the present invention can be an Fv fragment. An Fv fragment of an antibody is made up of the variable region of the heavy chain (Vh) of an antibody and the variable region of the light chain of an antibody (Vl). Proteolytic cleavage of an antibody can produce double chain Fv fragments in which the Vh and Vl regions remain non-covalently associated and retain antigen binding capacity. 
     Double chain Fv fragments also can be produced by recombinant expression methods well known in the art. See Plückthun and Skerra, Meth. Enzymol. 178, pp. 497-515 (1991), Skerra et al.,  Science  240: 1038 (1988), and King et al.,  Biochemical J.  290: 723 (1991). Briefly, the amino acid sequence of the variable regions of the heavy and light chains of antibodies known in the art can be obtained by direct amino acid sequencing using methods well known to those in the art. From this amino acid sequence, synthetic genes can be designed which code for these variable regions and they can both be inserted into an expression vector. Alternatively, nucleotide sequences known in the art which encode antibodies can be employed. Two polypeptides can be expressed simultaneously from a mammalian or bacterial host, resulting in formation of an active Fv fragment. 
     An antibody fragment of the present invention also can be a single-chain molecule or so-called “single chain antigen binding polypeptide,” a phrase used in this description to denote a linear polypeptide that binds antigen with specificity and that comprises variable or hypervariable regions from the heavy and light chain chains of an antibody. Single chain antigen binding polypeptides that retain an antigen-binding capacity that is characteristic of the present invention can be produced by conventional methodology. The Vh and Vl regions of the Fv fragment can be covalently joined and stabilized by the insertion of a disulfide bond. See Glockshuber, et al.,  Biochemistry  1362 (1990). Alternatively, the Vh and Vl regions can be joined by the insertion of a peptide linker. A gene encoding the Vh, Vl and peptide linker sequences can be constructed and expressed using a recombinant expression vector. See Colcher, et al.,  J. Nat&#39;l Cancer Inst.  82: 1191 (1990). Amino acid sequences comprising hypervariable regions from the Vh and Vl antibody chains can also be constructed using disulfide bonds or peptide linkers, as described herein. 
     Fusion Proteins 
     Fusion proteins can be made in  E. coli  using recombinant DNA techniques that are well-known in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY pp. 16.0.5-16.7.7 (Ausubel, et al., eds. John Wiley &amp; Sons (1996)). Briefly, a fusion protein vector is constructed for insertion into  E. coli.  This vector will generally contain a selectable marker gene sequence, a controllable transcriptional promoter (such as lac, trp or tac), other translational control sequences such as appropriately positioned ribosome-binding site and initiator ATG and one or more polylinker sequences to facilitate insertion of the fusion protein gene in the correct orientation within the vector. This vector will also contain a “carrier sequence” that encodes a carrier protein; this carrier sequence is inserted into the expression vector 5′ of the gene to be expressed. The carrier sequence generally encodes a protein that is strongly expressed in  E. coli.  The carrier sequence will provide the necessary signals for proper expression and the expressed protein will contain an N-terminal region encoded by the carrier sequence. The carrier sequence can encode an entire protein, such as β-glucuronidase and β-galactosidase. 
     A fusion protein according to the invention generally comprises an antibody or antibody fragment as described above and an enzyme. The production of antibodies and antibody fragments according to the invention are described above, as are the methods for isolating nucleic acid sequences that encode such antibodies and antibody fragments. The enzymes of the fusion proteins according to the invention include cytoplasmic enzymes, including  E. coli  cytoplasmic enzymes such as  E. coli  β-glucuronidase. The  E.coli  β-glucuronidase gene (uid A) has been cloned, sequenced, and expressed as a fusion with the  E. coli  lacZ promoter and coding region by Jefferson et al. (PNAS vol. 83, pp. 8447-8451, 1986). The enzyme β-glucuronidase is capable of cleaving β-glucuronides to toxic counterparts. When fused to an antigen-binding polypeptide, an enzyme in a fusion protein according to the invention can cleave a non-toxic compound (known also as a prodrug) into its active, and toxic, form. 
     A recombinant expression vector encoding a fusion protein according to the invention can be expressed in  E. coli  using transformation and culturing techniques that are well known in the art, using techniques such as those described by Ausubel, supra, pp. 1.8.1-1.8.8 and 16.0.5-16.4.2. Briefly, calcium chloride can be used to transform  E. coli  with foreign DNA. Those of skill in the art will recognize that the following factors can influence the success of the transformation: harvesting bacterial cells during logarithmic growth phase, keeping cells on ice during transformation and avoiding prolonged exposure of cells to calcium chloride. Electroporation is also well-known in the art as an acceptable transformation method for  E. coli.  Those of skill in the art will recognize the need to vary the electric pulse strength and length to optimize transformation and to add sufficient DNA for transformation. 
     Thioredoxin Reductase-deficient  E.coli    
     An  E. coli  strain which is deficient in thioredoxin reductase, for example the strain AD 494, can form disulfide bridges in the cytoplasm and thus enzymes which are naturally secretory, for example alkaline phosphatase, can be expressed intracellularly (Derman et al., Science, vol. 262. 1744-1747, 1993). As used in this specification, a thioredoxin reductase-deficient  E. coli  strain is one which (1) has TRR activity that is eliminated or greatly reduced compared to the wild type strain and (2) is capable of producing cytoplasmic proteins with disulfide bonds.  E. coli  strains with (1) mutations in the trxB gene (which codes for TRR) and (2) that are capable of producing cytoplasmic proteins with disulfide bonds are included in the present invention. 
     A TRR-deficient  E.coli  strain can be isolated by selection methods known in the art. For example, it is known that alkaline phosphatase (AP) requires two intrachain disulfide bonds that are required for AP to retain its enzymatic activity. If AP is expressed in  E. coli  without its signal sequence, it remains in the cytoplasm and its disulfide bonds are not formed. Any mutant  E. coli  which produces enzymatically active, cytoplasmic AP is able to create disulfide bonds in the cytoplasm and is hence TRR-deficient. Thus, one assay to select for TRR-deficient strains is as follows. AP can function as a phosphomonoesterase. In  E. coli,  it is known that fructose-1,6-bisphosphatase (fbp) is a cytoplasmic phosphomonoesterase that is required for the growth of  E. coli  on gluconeogenic carbon sources such as glycerol. If an AP gene (without its signal sequence) that is expressed in an fbp− mutant will cause such a mutant to grow on glycerol, then the AP gene has enzymatic activity and the mutant is capable of creating disulfide bonds. An  E. coli  mutant selected in this way should be TRR-deficient. Jefferson, supra, describes the use of an fbp− mutant to select for mutant  E. coli  that allow for cytoplasmic disulfide bond formation. These fbp− mutants would only grow on glycerol if expression of a signal-sequenceless AP gene was induced with IPTG (isopropyl thio-β-D-galactopyranoside). 
     A second selection step can also be employed. AP can also perform the phosphoserine dephosphorylation function of the enzyme encoded by the  E. coil  serB gene. This is the final step in serine biosynthesis. Thus, if a deletion is introduced into the serB gene and induction of the signal-sequenceless AP gene restores the  E. coli  to Ser+, this indicates the presence of active cytoplasmic AP and hence the formation of disulfide bonds in the cytoplasm. 
     Purification of Fusion Proteins 
     The fusion proteins of the invention can be purified by various techniques well-known in the art. Following culturing of the transformed  E. coli  harboring the fusion protein gene, the cells are typically disrupted, suspended and pelletted to remove cell debris. Fusion protein can be purified from the supernatant. Numerous methods for ion-exchange chromatography and purification according to size are well characterized in the prior art and one of skill in the art could readily select appropriate purification techniques based on the properties of the fusion protein produced according to the invention. See Ausubel, supra, pp. 16.6.1-16.8.14. Affinity chromatography is also a technique well-known in the art. Anti-idiotype affinity chromatography is useful for purification of fusion proteins with an antibody or antibody fragment component. Briefly, the antiidiotypic antibody is ligated to CnBr-activated Sepharose to create the affinity matrix. The fusion-protein-containing supernatant is exposed to the affinity matrix and the fusion protein is eluted, typically using a pH gradient. 
     The following examples illustrate but do not limit the invention. 
     EXAMPLE 1 
     Construction of a dicistronic expression vector without signal sequences 
     A) Cloning the  E. coli  β-glucuronidase from  E. coli  RR1: 
     The DNA sequence encoding  E. coli  β-glucuronidase was amplified by PCR from the  E. coli  strain RR1 using the primers E.c.β Gluc. for (AAG CTT TCA TTG TTT GCC TCC CTG CTG CGG) and E.c.βGluc. back (TCT AGA CCA TGG TAC GTC CTG TAC AAA CCC CA), and cloned into the vector P bluescript II KS (Stratagene, La Jolla, Calif.) by way of the Xba I and Hind III sites (FIG.  1 ). 
     B) Cloning of VH/CH1 and linker Mab BW 431/26 in front of  E. coli  β-glucuronidase: 
     The antibody variable domain VH and the constant domain CH1 were amplified by PCR from an HC-human-β-glucuronidase cDNA construct, using the primers Fab HC for (GAA TTC CAT GGA ACC AGA ACC AGA ACC GAG CTC AAC TCT) and Fab HC back (TCT AGA TAA CGA GGG CAA AAA ATG GAG GTC CAA CTG CAG), and cloned into vector p bluescript II KS by way of the Xba I and Eco RI sites (FIG.  2 ). See Bosslet et al., Br. J, Cancer, 65, 234-238, 1992 and Güssow &amp; Seemann,  Methods in Enzymology,  203:99-121 (1991). Bosslet describes the construction of the vector comprising a cDNA sequence encoding humanized VH (derived from the VH gene of Mab BW431) and CH1, fused to the gene for human β-glucuronidase that was used in this step. Briefly, the fusion gene comprises the human IgG promoter region and signal peptide exon, the humanized version of the VH gene of Mab BW431 (described in Güssow, supra), and the CH1 exon of human IgG3. 
     After the DNA sequence had been verified, a DNA fragment which was isolated by digesting with Xbal and NcoI was ligated into the vector from cloning step A which had been cut with XbaI and NcoI (FIG.  3 ). 
     C) Cloning the Fab BW 431/26— E. coli  β-glucuronidase into pTrc 99: 
     An Xba I/Hind III fragment containing the human heavy chain construct fused to  E. coil  β-glucuronidase was ligated to the light chain of Mab BW 431/26, which was present in the expression vector pTrc99. See Amann et al., Gene 69, 301, 1988 (FIG.  4 ). The resulting construct has the structure: promotor (trc)—Shine Dalgarno sequence (SD)—VK/CK Mab BW 431/26—SD—VH/CH1 Mab 431/26— E. coli -β-gluc.-transcription termination signal (FIG.  5 ). This construct was used to transform  E. coli  AD 494 using standard transformation techniques known in the art. See, e.g., Ausubel, supra pp. 1.8.1-1.8.8. 
     EXAMPLE 2 
     Expression in AD 494 
     Overnight cultures of AD 494, harboring pTrc dicistr. Fab- E. coli -β-gluc., described in Example 1, were diluted 1:10 and incubated at 25° C. until they reached an OD 600  of 0.7. After inducing with 1 mM IPTG for 19-22 hours, the cells were incubated on ice for 1-1.5 hours. After the cells had been pelletted and resuspended in 10 ml of PBS, pH 7.2, per liter of culture volume, they were disrupted in a French press at 1000-1500 Psi. The disrupted cell suspension was clarified at 20,000 rpm in an SS-34 rotor and the supernatant was employed for the subsequent investigations. 
     EXAMPLE 3 
     Purification of the fusion molecule by affinity chromatography. 
     The disrupted cell suspension supernatant, which had been clarified by passing it through a 2 μm filter, was purified by affinity chromatography. The fusion protein was allowed to bind to an anti-idiotypic monoclonal antibody (BW 2064(34) (6 mg of Mab/ml of CnBr-activated Sepharose 4B) as described in Bosslet,  British J. Cancer  65:234 (1992) and Bosslett,  British J. Cancer  63:681 (1991). The fusion protein bound to the affinity column was eluted by pH shifting (pH 7.2-pH 5.0). The peak of antibody was eluted with pH 5.0. (Table 1). The eluate was then concentrated by means of ultrafiltration (Filtron Macrosep. Omega NMWL:30 KD). The cell disruption suspension supernatant, the void volume, the concentrated eluate and the filtrate from the ultrafiltration were analyzed by SDS-PAGE. The band appearing at about 97 KD corresponds, as regards its molecular weight, to the expected fusion protein comprising the heavy chain moiety of the antibody and the  E. coli  glucuronidase. The band appearing at about 70 KD represents endogenous β-glucuronidase which has been purified concomitantly, during the affinity chromatography, due to the formation of heterotetramers (see below) between expressed heavy chain/β-glucuronidase and endogenous β-glucuronidase. 
     EXAMPLE 4 
     TSK 3000 gel chromatography 
     The native molecule was examined by TSK 3000 gel chromatography and its molecular weight was found to be 450 KD. Since the glucuronidase forms a tetramer in the native state, the observed molecular weight corresponds to that which is to be expected theoretically. This step is described in detail below. 
     400 ng of a fusion protein, which had been purified by anti-idiotype affinity, in 25 μl were chromatographed on a TSK gel G 3000 SW XL column (TOSO HAAS, Cat. No. 3.5W×N3211, 7.8 mm×300 mm) in an appropriate mobile phase (PBS, pH 7.2, 5 g/l maltose, 4.2 g/l arginine) at a flow rate of 0.5 ml/min. The Merck Hitachi HPLC unit (L-400 UV detector, L-6210 intelligent pump, D-2500 chromato-integrator) was operated at about 20 bar, the optical density of the eluate was determined at 280 nm, and 0.5 ml fractions were collected, using an LKB 2111 Multisac fraction collector, and subsequently analyzed in an enzyme activity specificity test (Example 5). The experiment is depicted in FIG.  6 . Based on elution pattern of the the molecular weight markers (indicated by arrows), the functionally active Fab- E. coli  β-glucuronidase fusion protein was determined to have a molecular weight of about 450 KD. 
     EXAMPLE 5 
     Demonstration of the antigen-binding properties and the enzymatic activity 
     The ability of the Fab- E. coli  β-glucuronidase fusion protein to bind specifically to the Mab 431/26-defined epitope on CEA (carcino-embryonic antigen) and simultaneously to exert the enzymic activity of the β-glucuronidase was demonstrated in an enzyme activity specificity test. This assay is carried out as described below: 
     Polystyrene (96-well) microtiter plates (U shape, Type B, supplied by Nunc, Order No. 4-60445) are incubated with purified CEA (1-5 μg of CEA/ml, 75μ of this per well) or with GIT mucin (same amount as CEA) at R.T. overnight. 
     The non-adsorbed antigen is removed by aspiration and washed 3× with 0.05 M tris/citrate buffer, pH 7.4. 
     The microliter plates are left to stand at R.T. with the opening facing downwards on cellulose overnight. 
     The microtiter plates are incubated with 250 μl of 1% strength casein solution in PBS, pH 7.2, per well (blocking solution) at 20° C. for 30 minutes. 
     During the blocking, the substrate is made up. The amount of substrate depends on the number of supernatants to be assayed. The substrate is made up fresh for each assay. 
     Substrate: 4-methylumbelliferyl β-D-glucuronide (Order No.: M-9130 from Sigma), 2.5 mM in 200 mM sodium acetate buffer, pH 5.0, with 0.01% BSA. 
     The blocking solution is removed by aspiration, and in each case 50 μl of BHK call supernatant which contains the fusion protein are loaded onto the microtiter plate coated with CEA or GIT mucin (that is to say the sample volume required is at least 120 μl). 
     Incubation at R.T. is then carried out for 30 minutes. 
     The plates are washed 3× with ELISA washing buffer (Behring, OSEW 96). 
     The substrate is loaded in (50 μl/wall) and incubated at 37° C. for 2 hours. The plate is covered because of the possibility of evaporation. 
     After 2 hours, 150 μl of stop solution are pipetted into each well (stop solution=0.2 M glycine+0.2% SDS, pH 11.7). 
     Evaluation can now be carried out under a UV lamp (excitation energy 380 nm) or in a fluorescence measuring instrument (Fluorosean 11, ICN Biomedicals Cat. No.: 78-611-00). 
     See also EP-A-O 501 215 A2. The test determines the liberation of 4-methylumbelliferone from 4-methylumbelliferyl-β-glucuronide by the β-glucuronidase moiety of the fusion protein after the fusion protein has bound to the antigen by its Fab moiety. The fluorescence values which were determined are given as relative fluorescence units (FU) (Table 1). This assay demonstrates that the fusion protein elicits significant liberation of methylumbelliferone in the CEA-coated plates. 
     PEM (polymorphic epithelial mucin)-coated plates served as the control. In these plates, the fluorescence signal was always less than 30 FU. 
     
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 1:2 
                 1:4 
                 1:8 
                 1:16 
                 1:32 
                 1:64 
                 1:128 
                 1:256 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 FU, Disrupted cell 
                 8183 
                 8149 
                 7531 
                 6631 
                 4560 
                 2509 
                 1019 
                 421.9 
               
               
                 suspension 1:3 
               
               
                 FU, void volume 1:1 
                 6548 
                 5231 
                 3222 
                 1477 
                 525.2 
                 214 
                 86.19 
                 46.29 
               
               
                 FU, pH 5.0 eluate 1:3 
                 7782 
                 7571 
                 6360 
                 4239 
                 1983 
                 815.7 
                 302 
                 113.9 
               
               
                 FU, pH 5.0 eluate 
                 7904 
                 8106 
                 8036 
                 7153 
                 5802 
                 3618 
                 1651 
                 665.7 
               
               
                 Ultraconcent. 1:10 
               
               
                 FU pH 5.0 eluate 
                 74.65 
                 172.7 
                 90.23 
                 52.30 
                 38.84 
                 25.79 
                 23.51 
                 19.39 
               
               
                 Ultrafiltrate 1:1 
               
               
                   
               
             
          
         
       
     
     
       
         
           
             7 
           
           
             
               30 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Enterobacteriaceae Escherichia coli 
             
             
               KS + E.c.-Beta-Gluc 
             
              1
AAGCTTTCAT TGTTTGCCTC CCTGCTGCGG                                      30
 
           
           
             
               32 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Enterobacteriaceae Escherichia coli 
             
             
               KS + E.c.-Beta-Gluc 
             
              2
TCTAGACCAT GGTACGTCCT GTACAAACCC CA                                   32
 
           
           
             
               39 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Homo sapiens 
             
             
               pAB Stop c-DNA HC/hu-Beta-Gluc 
             
              3
GAATTCCATG GAACCAGAAC CAGAACCGAG CTCAACTCT                            39
 
           
           
             
               45 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Homo sapiens 
             
             
               pAB Stop c-DNA HC/hu-Beta-Gluc 
             
              4
TCTAGATAAC GAGGGCAAAA AATGGAGGTC CAACTGCAGG AGAGC                     45
 
           
           
             
               3169 base pairs 
               nucleic acid 
               double 
               circular 
             
             
               cDNA 
             
             NO 
             NO 
             
               Enterobacteriaceae Escherichia coli 
               pRAJ210 
             
             
               pTrc99 dicistr. Fab/E.c.-Beta-Gluc 
             
             
               CDS 
               3..641
 
             
             
               CDS 
               666..3162
 
             
              5
CC ATG GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGC        47
   Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
     1               5                  10                  15
GTG GGT GAC AGA GTG ACC ATC ACC TGT AGT ACC AGC TCG AGT GTA AGT       95
Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser
                 20                  25                  30
TAC ATG CAC TGG TAC CAG CAG AAG CCA GGT AAG GCT CCA AAG CTG CTG      143
Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
             35                  40                  45
ATC TAC AGC ACA TCC AAC CTG GCT TCT GGT GTG CCA AGC AGA TTC AGC      191
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
         50                  55                  60
GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC CTC CAG      239
Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln
     65                  70                  75
CCA GAG GAC ATC GCC ACC TAC TAC TGC CAT CAG TGG AGT AGT TAT CCC      287
Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro
 80                  85                  90                  95
ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGT ACT GTG GCT GCA      335
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
                100                 105                 110
CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA      383
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
            115                 120                 125
ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC      431
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
        130                 135                 140
AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC CAA TCG GGT AAC TCC CAG      479
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
    145                 150                 155
GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC CTC AGC      527
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
160                 165                 170                 175
AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC      575
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                180                 185                 190
GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC ACA AAG AGC      623
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
            195                 200                 205
TTC AAC AGG GGA GAG TGT TAGTCTAGAT AACGAGGGCA AAAA ATG GAG GTC       674
Phe Asn Arg Gly Glu Cys                            Met Glu Val
        210                                          1
CAA CTG CAG GAG AGC GGT CCA GGT CTT GTG AGA CCT AGC CAG ACC CTG      722
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu
      5                  10                  15
AGC CTG ACC TGC ACC GTG TCT GGC TTC ACC ATC AGC AGT GGT TAT AGC      770
Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser
 20                  25                  30                  35
TGG CAC TGG GTG AGA CAG CCA CCT GGA CGA GGT CTT GAG TGG ATT GGA      818
Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly
                 40                  45                  50
TAC ATA CAG TAC AGT GGT ATC ACT AAC TAC AAC CCC TCT CTC AAA AGT      866
Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser
             55                  60                  65
AGA GTG ACA ATG CTG GTA GAC ACC AGC AAG AAC CAG TTC AGC CTG AGA      914
Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg
         70                  75                  80
CTC AGC AGC GTG ACA GCC GCC GAC ACC GCG GTC TAT TAT TGT GCA AGA      962
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
     85                  90                  95
GAA GAC TAT GAT TAC CAC TGG TAC TTC GAT GTC TGG GGT CAA GGC AGC     1010
Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser
100                 105                 110                 115
CTC GTC ACA GTC ACA GTC TCC TCA GCT TCC ACC AAG GGC CCA TCG GTC     1058
Leu Val Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                120                 125                 130
TTC CCC CTG GCG CCC TGC TCC AGG AGC ACC TCT GGG GGC ACA GCG GCC     1106
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly Thr Ala Ala
            135                 140                 145
CTG GGC TGC CTG GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG     1154
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
        150                 155                 160
TGG AAC TCA GGC GCC CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC     1202
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
    165                 170                 175
CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC     1250
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180                 185                 190                 195
TCC AGC AGC TTG GGC ACC CAG ACC TAC ACC TGC AAC GTG AAT CAC AAG     1298
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asn His Lys
                200                 205                 210
CCC AGC AAC ACC AAG GTG GAC AAG AGA GTT GAG CTC GGT TCT GGT TCT     1346
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Leu Gly Ser Gly Ser
            215                 220                 225
GGT TCC ATG GTA CGT CCT GTA GAA ACC CCA ACC CGT GAA ATC AAA AAA     1394
Gly Ser Met Val Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys
        230                 235                 240
CTC GAC GGC CTG TGG GCA TTC AGT CTG GAT CGC GAA AAC TGT GGA ATT     1442
Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile
    245                 250                 255
GAT CAG CGT TGG TGG GAA AGC GCG TTA CAA GAA AGC CGG GCA ATT GCT     1490
Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala
260                 265                 270                 275
GTG CCA GGC AGT TTT AAC GAT CAG TTC GCC GAT GCA GAT ATT CGT AAT     1538
Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn
                280                 285                 290
TAT GCG GGC AAC GTC TGG TAT CAG CGC GAA GTC TTT ATA CCG AAA GGT     1586
Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly
            295                 300                 305
TGG GCA GGC CAG CGT ATC GTG CTG CGT TTC GAT GCG GTC ACT CAT TAC     1634
Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr
        310                 315                 320
GGC AAA GTG TGG GTC AAT AAT CAG GAA GTG ATG GAG CAT CAG GGC GGC     1682
Gly Lys Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly
    325                 330                 335
TAT ACG CCA TTT GAA GCC GAT GTC ACG CCG TAT GTT ATT GCC GGG AAA     1730
Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys
340                 345                 350                 355
AGT GTA CGT ATC ACC GTT TGT GTG AAC AAC GAA CTG AAC TGG CAG ACT     1778
Ser Val Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr
                360                 365                 370
ATC CCG CCG GGA ATG GTG ATT ACC GAC GAA AAC GGC AAG AAA AAG CAG     1826
Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln
            375                 380                 385
TCT TAC TTC CAT AAT TTC TTT AAC TAT GCC GGG ATC CAT CGC AGC GTA     1874
Ser Tyr Phe His Asn Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val
        390                 395                 400
ATG CTC TAC ACC ACG CCG AAC ACC TGG GTG GAC GAT ATC ACC GTG GTG     1922
Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val
    405                 410                 415
ACG CAT GTC GCG CAA GAC TGT AAC CAC GCG TCT GTT GAC TGG CAG GTG     1970
Thr His Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val
420                 425                 430                 435
GTG GCC AAT GGT GAT GTC AGC GTT GAA CTG CGT GAT GCG GAT CAA CAG     2018
Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln
                440                 445                 450
GTG GTT GCA ACT GGA CAA GGC ACT AGC GGG ACT TTG CAA GTG GTG AAT     2066
Val Val Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn
            455                 460                 465
CCG CAC CTC TGG CAA CCG GGT GAA GGT TAT CTC TAT GAA CTG TGC GTC     2114
Pro His Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val
        470                 475                 480
ACA GCC AAA AGC CAG ACA GAG TGT GAT ATC TAC CCG CTT CGC GTC GGC     2162
Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly
    485                 490                 495
ATC CGG TCA GTG GCA GTG AAG GGC GAA CAG TTC CTG ATT AAC CAC AAA     2210
Ile Arg Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys
500                 505                 510                 515
CCG TTC TAC TTT ACT GGC TTT GGT CGT CAT GAA GAT GCG GAC TTA CGT     2258
Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg
                520                 525                 530
GGC AAA GGA TTC GAT AAC GTG CTG ATG GTG CAC GAC CAC GCA TTA ATG     2306
Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met
            535                 540                 545
GAC TGG ATT GGG GCC AAC TCC TAC CGT ACC TCG CAT TAC CCT TAC GCT     2354
Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala
        550                 555                 560
GAA GAG ATG CTC GAC TGG GCA GAT GAA CAT GGC ATC GTG GTG ATT GAT     2402
Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp
    565                 570                 575
GAA ACT GCT GCT GTC GGC TTT AAC CTC TCT TTA GGC ATT GGT TTC GAA     2450
Glu Thr Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu
580                 585                 590                 595
GCG GGC AAC AAG CCG AAA GAA CTG TAC AGC GAA GAG GCA GTC AAC GGG     2498
Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly
                600                 605                 610
GAA ACT CAG CAA GCG CAC TTA CAG GCG ATT AAA GAG CTG ATA GCG CGT     2546
Glu Thr Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg
            615                 620                 625
GAC AAA AAC CAC CCA AGC GTG GTG ATG TGG AGT ATT GCC AAC GAA CCG     2594
Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro
        630                 635                 640
GAT ACC CGT CCG CAA GGT GCA CGG GAA TAT TTC GCG CCA CTG GCG GAA     2642
Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu
    645                 650                 655
GCA ACG CGT AAA CTC GAC CCG ACG CGT CCG ATC ACC TGC GTC AAT GTA     2690
Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val
660                 665                 670                 675
ATG TTC TGC GAC GCT CAC ACC GAT ACC ATC AGC GAT CTC TTT GAT GTG     2738
Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val
                680                 685                 690
CTG TGC CTG AAC CGT TAT TAC GGA TGG TAT GTC CAA AGC GGC GAT TTG     2786
Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu
            695                 700                 705
GAA ACG GCA GAG AAG GTA CTG GAA AAA GAA CTT CTG GCC TGG CAG GAG     2834
Glu Thr Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu
        710                 715                 720
AAA CTG CAT CAG CCG ATT ATC ATC ACC GAA TAC GGC GTG GAT ACG TTA     2882
Lys Leu His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu
    725                 730                 735
GCC GGG CTG CAC TCA ATG TAC ACC GAC ATG TGG AGT GAA GAG TAT CAG     2930
Ala Gly Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln
740                 745                 750                 755
TGT GCA TGG CTG GAT ATG TAT CAC CGC GTC TTT GAT CGC GTC AGC GCC     2978
Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala
                760                 765                 770
GTC GTC GGT GAA CAG GTA TGG AAT TTC GCC GAT TTT GCG ACC TCG CAA     3026
Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln
            775                 780                 785
GGC ATA TTG CGC GTT GGC GGT AAC AAG AAA GGG ATC TTC ACT CGC GAC     3074
Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp
        790                 795                 800
CGC AAA CCG AAG TCG GCG GCT TTT CTG CTG CAA AAA CGC TGG ACT GGC     3122
Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly
    805                 810                 815
ATG AAC TTC GGT GAA AAA CCG CAG CAG GGA GGC AAA CAA TGAAGCTT        3169
Met Asn Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln
820                 825                 830
 
           
           
             
               213 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              6
Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
  1               5                  10                  15
Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr
             20                  25                  30
Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
         35                  40                  45
Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
 65                  70                  75                  80
Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr
                 85                  90                  95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
            100                 105                 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
        115                 120                 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
    130                 135                 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145                 150                 155                 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
                165                 170                 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
            180                 185                 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
        195                 200                 205
Asn Arg Gly Glu Cys
    210
 
           
           
             
               832 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              7
Met Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser
  1               5                  10                  15
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser
             20                  25                  30
Gly Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu
         35                  40                  45
Trp Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser
     50                  55                  60
Leu Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe
 65                  70                  75                  80
Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
                 85                  90                  95
Cys Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly
            100                 105                 110
Gln Gly Ser Leu Val Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
        115                 120                 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly
    130                 135                 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145                 150                 155                 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
                165                 170                 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
            180                 185                 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
        195                 200                 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Leu Gly
    210                 215                 220
Ser Gly Ser Gly Ser Met Val Arg Pro Val Glu Thr Pro Thr Arg Glu
225                 230                 235                 240
Ile Lys Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn
                245                 250                 255
Cys Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg
            260                 265                 270
Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp
        275                 280                 285
Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile
    290                 295                 300
Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val
305                 310                 315                 320
Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val Met Glu His
                325                 330                 335
Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile
            340                 345                 350
Ala Gly Lys Ser Val Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn
        355                 360                 365
Trp Gln Thr Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys
    370                 375                 380
Lys Lys Gln Ser Tyr Phe His Asn Phe Phe Asn Tyr Ala Gly Ile His
385                 390                 395                 400
Arg Ser Val Met Leu Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile
                405                 410                 415
Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala Ser Val Asp
            420                 425                 430
Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala
        435                 440                 445
Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln
    450                 455                 460
Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu
465                 470                 475                 480
Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu
                485                 490                 495
Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile
            500                 505                 510
Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala
        515                 520                 525
Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Met Val His Asp His
    530                 535                 540
Ala Leu Met Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr
545                 550                 555                 560
Pro Tyr Ala Glu Glu Met Leu Asp Trp Ala Asp Glu His Gly Ile Val
                565                 570                 575
Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile
            580                 585                 590
Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala
        595                 600                 605
Val Asn Gly Glu Thr Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu
    610                 615                 620
Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Met Trp Ser Ile Ala
625                 630                 635                 640
Asn Glu Pro Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro
                645                 650                 655
Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys
            660                 665                 670
Val Asn Val Met Phe Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu
        675                 680                 685
Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser
    690                 695                 700
Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala
705                 710                 715                 720
Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val
                725                 730                 735
Asp Thr Leu Ala Gly Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu
            740                 745                 750
Glu Tyr Gln Cys Ala Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg
        755                 760                 765
Val Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala
    770                 775                 780
Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe
785                 790                 795                 800
Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg
                805                 810                 815
Trp Thr Gly Met Asn Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln
            820                 825                 830