DE4237244A1 - Process for the production of high-purity human GAD-1 and GAD-2 proteins - Google Patents

Abstract

The invention pertains to a process for producing high-purity human GAD-1 and GAD-2 proteins using the baculovirus/Sf9 expression system; the process comprises the following steps: a) preparation of cDNA sequences from human cDNA libraries which code GAD-1 and GAD-2 proteins at full length or in modifications thereof; b) insertion of the GAD-1 or GAD-2 cDNA sequence into a baculovirus transfer vector; c) co-transfection of Sf9 cells with baculovirus transfer vectors containing GAD-1 or GAD-2 cDNA and with baculovirus DNA to produce recombinant baculoviruses; d) identification, selection and enrichment of recombinant baculoviruses; e) extraction of GAD-1 or GAD-2 protein after infection of Sf9 cells with recombinant baculoviruses, and f) purification of the recombinant GAD-1 or GAD-2 protein. The process as per the invention provides high-purity, nearly homogeneous GAD-1 or GAD-2 proteins with a high rate of expression and a high degree of purity. The isolated GAD proteins are used in immunoassays for early detection of type I diabetes mellitus (IDDM).

Description

The present invention relates to a method for manufacturing high purity human GAD-1 and GAD-2 proteins according to the characteristics of the main claim.

The result is insulin-dependent diabetes mellitus type I (IDDM)  from a selective destruction of the insulin-producing kör Pere's own pancreatic β cells. The progressive Destruction process of the β cells leads a few years before Outbreak of autoimmune disease due to a certain pro Release of the complexion to form specific autoantibodies.

From the prior art it is known that the formation of these Autoantibodies make an early diagnosis of autoimmune diseases made possible by suitable immunoassays. Consequently was searched for specific target antigens, with their help the antibodies can be detected.

Baekkeskov et al (S. Baekkeskov, J.H. Nielsen, B. Marner, T. Bilde, J. Ludvigsson, A. Lernmark, Nature 298 (1982), pages 167 to 169) found that a specific protein of Langerhans islet cells, namely a 64 kDa protein, the one you are looking for Represents target antigen.

In a later investigation, the 64 kDa protein was identified as Glutamine decarboxylase (GAD) identified (S. Baekkeskov, H.-J. Aanstoot, S. Christgau et al, Nature 347 (1990), pp 151 to 156).

The GAD proteins are two isoforms with different molecular weights, GAD ₆₅ with 65 kDa (GAD-1) and GAD ₆₇ with 67 kDa (GAD-2).

The natural GAD proteins are found in the Langerhans Inselzel len formed the pancreas. The GAD proteins form the autoantibodies formed in type I diabetes mellitus an antibody-antigen complex. So far, some Immunoassays based on the antibody-antigen complex  that wraps the early detection of IDDM before the actual one Allow outbreak of the disease. The antigen substrates that are in the generally used in these immunoassays so far directly from the pancreas or the Langerhans islet cells won.

WO-90/07117 describes a method for early detection and Treatment of IDDM, the antigen substrate being a 64 kDa protein alone or together with other antibodies holds, is obtained directly from human pancreas.

The disadvantages of these immunoassays lie in the time it takes is needed to get the antigen substrate out of the pancreas or out win the Langerhans islet cells, as well as in quality of the sera obtained in this way, since both human and animal Tissues contain variable amounts of antigens that add purity and the quality of the isolated and included in the immunoassays set antigen substrates significantly impair. They use antigen substrates, which often contain only small amounts of GAD-Pro contain stones, because of their molecular size are more difficult to express as a result of existing ones Contamination from other substances also with non-specific ones Components react. As a result, both the significance as the reliability of the immunoassay is also significantly impaired does. Furthermore, it is not possible to use the two GAD forms in Distinguish antigen substrate since they are only limited Amounts are present and those present in the antigen substrate Impurities do not allow differentiation.

To address the disadvantages of immunoassay systems that are natural GAD proteins obtained from sources as an antigen substrate bacterial expressions have been used to avoid  systems proposed to make recombinant GAD-1 and GAD-2 proteins to express.

The use of bacterial expression systems, especially from Escherichia coli cells, has proven to be unsuccessful because most of the expressed recombinant GAD Proteins neither the correct modification nor the natural one Has molecular conformation. The specific reaction of the Antibody with the GAD proteins depends on the antigens Epitopes of the GAD proteins recognized by the antibodies Need to become. A change in the molecular conformation of the GAD Proteins prevent the antibodies from targeting the specific epitopes can recognize. As a result, the use results in bacterial expressed GAD proteins for immunoassays, their results are not reliably meaningful because the serums used of the IDDM patients on the bacterially expressed GAD proteins address poorly.

In addition, the expressed GAD proteins are in the bacterial cell often in the form of insoluble inclusion bodies in front. This is due to an intracellular accumulation of expri mated proteins, which are characterized by an increased expression rate is called. These insoluble inclusion bodies turn out to be problematic since it is often not possible is recombinant GAD from these insoluble inclusion bodies To obtain proteins with the correct conformation.

Baekkeskov et al (Journal of Cell Biology, Volume 118, No. 2, July 1992, pages 309 to 320) published a baculovirus Sf9 system in which recombinant GAD proteins starting from Rat GAD cDNA can be expressed. The isolated GAD proteins are in impure form because a cleaning step is not  is provided. In addition, the article deals specifically fish with the binding of the GAD-1 autoantigen to the cell membrane. It was previously unknown to have purified human GAD antigen sub strate manufacture and the disadvantages of the previously used to overcome unpurified antigen substrates and these in Use immunoassays.

Furthermore, the purification of the recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen is used as the expression system pH6EX3. The cleaning step is carried out by metal chelate affinity chromatography (H. Berthold, M. Scanarini, C. C. Abney, B. Frorath, W. Northemann, Prot Exp Purif 3, pages 50 to 56 (1992)). Of the Article contains no clue regarding the expression of GAD- Proteins and their purification.

It is therefore an object of the invention to produce a method human GAD-1 and GAD-2 proteins using the Baculovirus / Sf9 expression system indicate the disadvantages the state of the art, in particular the impurities of the antigen substrates as well as the small amounts of the isolated Affect GAD proteins, overcomes.

The features of the Hauptan are used to solve this task saying. Advantageous configurations are in the Unteran sayings defined.

The present invention thus relates to a method for Production of high-purity human GAD-1 and GAD-2 proteins under Use of the baculovirus / Sf9 expression system, the Procedure includes the following steps:

• a) Preparation of cDNA sequences from human cDNA library ken, each for GAD-1 and GAD-2 proteins in full Encode length;
• b) insertion of the two GAD-1 and GAD-2 cDNA sequences into one baculovirus transfer vector each to form recombinant baculovirus transfer vectors;
• c) Transfection of Sf9 cells, with the recombinant Baculo virus transfer vectors that encode the GAD-1 and GAD-2 cDNA Se quenz included;
• d) selecting the positively infected Sf9 cells;
• e) Obtaining the GAD-1 and GAD-2 proteins by cultivation the positively infected Sf9 cells and
• f) Purification of the recombinant GAD-1 and GAD-2 proteins.

The invention is explained in more detail by the figures. Show it:

. Figure 1 shows the analysis of the expression of GAD-1 and GAD-2 cDNA in Sf9 cells and their value analysis;

Fig. 2, the measurement of GAD-1 and GAD-2 enzyme activity in infected Sf9 cells;

Fig. 3, the immunoprecipitation of recombinant GAD-1 proteins with sera from IDDM patients and

Fig. 4 shows the analysis of the expression of GAD-1 and GAD-2 cDNA in E. coli and their analysis value.

The method according to the invention preferably uses the Bacu lovirus-Spodoptera frugiperda (Sf9 cell) expression system, that provides a large amount of biologically active proteins. Furthermore, it is possible to recome with this expression system  binante GAD-1 and GAD-2 proteins in ultra pure form put. Here the baculovirus switches the vector to viroal Basis available, while in the Sf9 insect cell the Ex pression takes place.

The baculovirus / Sf9 system has the advantage that the expression cell system under serum-free conditions as a suspension culture can be bred and propagated.

According to the present invention, the DNA to be expressed, which codes for the human GAD proteins, is preferably produced from cDNA libraries derived from a pancreatic carcinoma cell line or a hippocampal cell line. In a screening process, the specific cDNAs coding for the GAD-1 or GAD-2 proteins are hybridized with oligonucleotide probes from a human pancreatic carcinoma cDNA library or a human hippocampus cDNA library and PCR amplifi cation produced. The synthetic oligonucleotides used in this screening method are based on the homologous sequence that has been published for rat brain GAD ₆₇ cDNA (JF Julien, P. Samama, J. Mallet, J. Neurochem 57 (1990), pages 703 to 705). The isolated cDNA fragments were characterized by cDNA analysis and linked together to produce 2.0 and 1.8 kb full-length cDNAs that are required for the proteins of the 585 amino acids of GAD-1 and the 594 amino acids of GAD- 2 code.

In the next step, the specific cDNAs preferably in the baculovirus transfer vector pVL1393 Formation of clones pAc GAD-1 and pAc GAD-2 inserted. In addition a DNA is inserted into the vector which is suitable for a histidine Encoded hexapeptide. The expressed GAD fusion proteins are  via its N-terminus to the human GAD-1 and GAD-2 proteins and bound to a histidine hexapeptide via its C-terminus. The histidine hexapeptide is for the method according to the invention particularly preferred for the production of the GAD proteins.

Then the transfection into the Sf9 (insect) cells takes place together with the linearized wild type Baculovirus Autographa california. This is followed by the expression of rekom binant GAD fusion proteins.

In order to be able to compare the expression rate of the baculovirus / Sf9 system, Sf9 cell cultures are advantageously created which are provided with the wild-type baculovirus and a non-recombinant vector (MOCK cells). No expression takes place in this control cell system (MOCK) ( FIG. 1, column 1, 2). In a further comparison method, the expression of the GAD proteins can be carried out in prokaryotic E. coli cells. Expression takes place in the bacterial cells, which has considerable disadvantages, which will be discussed in more detail later.

After transfection of the Sf9 cells and selection of the positive infected Sf9 cells are cultivated cultures as suspension cultures, preferably in an SF900- Medium containing fetal calf serum. The recombinant GAD 1- and GAD-2 proteins are obtained by lysis and centrifugation Sf9 cells were obtained, one insoluble and one soluble Cell fraction arises. The GAD-1 or GAD-2 protein is located in the supernatant, d. H. in the soluble cell fraction.

The solution is then purified, particularly preferably by means of metal chelate affinity chromatography. For this purpose, the cytosolic solution containing the GAD-1 or the GAD-2 fusion proteins is placed on a column which contains a matrix with metal ions, in particular Ni ²⁺ ions, as the stationary phase. The GAD proteins form a metal chelate complex with the Ni ²⁺ ions. An eluent, in particular imidazole, is then added to the column in order to elute the GAD proteins from the column.

This particularly preferred embodiment of the invention provides by the one-step affinity chromatography over a metal ion matrix an almost homogeneous GAD antigen substrate for ver addition. The chromatography is carried out at physiological pH and carried out without additional detergents to the natural to obtain the conformation of both GAD forms. this will based on the enzyme activity and the immunoprecipitation ver drive clearly occupied.

With the aid of SDS-polyacrylamide gel electrophoresis and the Western blotting method ( FIG. 1), it can be demonstrated that both recombinant GAD forms are mainly contained in the soluble cell fraction ( FIG. 1, columns 3, 4 and 5, 6). The supernatant liquid of the centrifugate, which contains the soluble cell fraction, is treated with a specific solution (40 mM Tris / HCl, pH 7.6, 0.5 mol NaCl).

The antigenic function of the GAD-1 and GAD-2 proteins produced according to the invention is demonstrated by Western blotting methods. Figure 1A shows expression and Western blotting analysis of the GAD-1 and GAD-2 proteins expressed in the Sf9 cells. A GAD-1-specific IDDM autoimmune serum, which is designated as anti-GAD924, ( FIG. 1B) and a GAD-2-specific mouse monoclonal antibody ( FIG. 1C) are used as test serum. From FIGS. 1A and 1B is obtained that specific antigen-antibody complexes form. Furthermore, they show that the GAD-1 proteins have an excellent antigen function, which can be used in immunoassays. - It should be noted that the numbering in FIGS . 1B and 1C corresponds to that in FIG. 1A.

The GAD proteins produced according to the invention were related to tested for their enzyme activity and with natural GAD proteins, which are derived from pig islet cells and brain tissue, compared.

. The Figure 2 it can be seen that the present invention manufactured in, human GAD-1 (GAD ₆₄ -Sf9) - and GAD-2 (GAD ₆₇ -Sf9) proteins have a much higher enzyme activity than the GAD-Pro proteins from the Pig islets and brains. The enzyme activity of the GAD-1 and GAD-2 proteins produced according to the invention is 400 or 45 times for GAD-1 and 600 or 75 times for GAD-2 compared to GAD from pancreatic islet cells or from Brain cells. Naturally, no GAD enzyme activity is detected in the control Sf9 cells which contain baculovirus wild-type (MOCK).

Approx. 2 to 3 mg of each GAD-1 and GAD-2 protein are made 1000 ml of culture suspension of the infected cells were obtained.

Fig. 3 shows the immunoprecipitation of the invention manufactured in, GAD-1 protein with ten IDDM patient sera and two normal sera. As can be seen from the immunoprecipitations of columns 1 to 10, the GAD-1 proteins produced according to the invention are outstandingly suitable as reagents and thus as antigen substrates in immunoprecipitations with sera from patients with GAD-1-specific IDDM. The recombinant GAD-1 proteins are not immunoprecipitated with normal sera (columns 11 and 12).

The comparison process in which the production of GAD-1 and GAD-2 proteins by expression in prokaryotic E. coli System takes place, shows different results.

The SDS-polyacrylamide gel electrophoresis and the Western blotting method from FIG. 4 show that in addition to the GAD-1 protein expressed in the E. coli cell, a smaller GAD ₆₅ protein with a molecular weight of 41 kDa is also expressed (column 1). In Fig. 4, the band sequence of GAD-1 is numbered 1, while the band sequence of GAD-2 is number 2. This smaller GAD form has a different conformation than that of the specific GAD-1 and is only found in prokaryotic expression systems. The recombinant GAD proteins of the bacterial system are analyzed by means of the Western blotting method, also using ten IDDM test sera. All test sera had to be pretreated with bacterial extracts to minimize side reactions with other bacterial proteins.

The immunoprecipitation procedure showed that only five IDDM sera (50%) contain autoantibodies that react with the linear epitope of GAD-1, while four IDDM sera (20%) recognize the linear epitope of GAD-2. Only one of the IDDM sera shows a specifically strong interaction with the recombinant GAD-1 ( Fig. 4e, column 1).

This result shows that the GAD-1 specific autoantibodies mainly with the antigen epitopes caused by the Conformation can be reached, react. As a result, they are  Approaches using anti-GAD autoantibodies in IDDM patient sera prokaryotic expressed recombinant GAD-1 proteins measure, not representative, since the GAD autoantibodies with the prokaryotic expressed GAD-1 proteins not specifically react.

The method according to the invention represents highly pure recombinant GAD-1 and GAD-2 proteins expressed in baculovirus / Sf9 system are available. The manufactured recombinant GAD-1 and GAD-2 proteins have an outstanding de enzyme activity and a highly specific antigenicity. The The inventive method enables a quick and effec tive isolation of the recombinant GAD-1 and GAD-2 proteins have a high degree of cleaning.

In addition, the high purity GAD-1 and GAD-2 proteins as an antigen substrate in immunoassays, e.g. B. Solid phase immunoassay and Elisa.

The following example illustrates the invention without it however restrict.

example 1. Preparation of the GAD-1 and GAD-2 cDNA sequences required for Encode full-length GAD-1 or GAD-2 fusion proteins

With the help of the screening process, a human Pancreatic carcinoma cDNA library or a human hippo campus cDNA library the specific cDNA sequences that code for human GAD-1 or GAD-2 proteins by Hybri mix with oligonucleotide samples and PCR amplification  produced. The synthetic oligonucleotides that use are based on the known sequence of rat rat brain GAD-2 cDNA (J.F. Julien, P. Samama, J. Mallet, Neurochem 54 (1990), pages 703 to 705).

The recombinant DNA was isolated for PCR (polymerase chain reaction) amplification from 2 × 10 ⁶ plaques from the lambda cDNA library by known methods. 1 µg of the combined cDNA is amplified with a "1 unit" Tac polymerase in 10 mM Tris / HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl ₂ , 4 pmol of each primer and 100 µM dNTPS . The amplification reaction was carried out in 30 cycles with the following cycle times: denaturation, 1 minute at 95 ° C, heating, 2 minutes at 60 ° C and primer extension, 2 minutes at 72 ° C. The synthesized GAD cDNA fragments were analyzed by DNA sequence analysis using T7DNA polymerase. Suitable cDNA fragments are combined according to known methods to form 2.0 kb and 1.8 kb cDNAs which code for full-length GAD-1 and GAD-2 proteins. An additional cDNA sequence coding for a histidine hexapeptide was inserted into the GAD-1 cDNA and the GAD-2 cDNA by side-specific mutagenesis in the baculovirus transfer vector pVL1393.

2. Expression of the GAD-1 and GAD-2 cDNAs in Baculovirus / Sf9- Expression system

The 2.0 kb and 1.8 kb cDNAs produced under 1., which code for both human GAD-1 and GAD-2 proteins, were inserted into the baculovirus transfer vector pVL1393 to convert the cDNA- Prepare clones pAc GAD-2 and pAc GAD-1 encoding the recombinant GAD-1 and GAD-2 fusion proteins. The recombinant fusion proteins have the GAD-1 or GAD-2 protein at the N-terminus and the histidine hexapeptide at the C-terminus. Spodoptera frugiperda (Sf9) cells were isolated with the recombinant transfer vector and the linearized wild-type baculovirus auto grapha california by lipofection at a density of 1.6 × 10 ⁶ cells / ml with an infection multiplicity (MOI) of 5 according to conventional transfection methods with Baculo virus cotransfected. The positive recombinant Baculovi rus clones were selected according to the standard methods of expression according to the plague cleaning process. The positively infected Sf9 cells were cultured as suspension cultures in the SF900 medium containing 0.04% fetal calf serum for 48 hours. Sf9 cells which were infected only with the wild-type Baculo virus and a non-recombinant vector pLV1393 were used as control cells.

3. Purification of the recombinant human GAD-1 and GAD-2-Pro teine

Before lysis in a lysis buffer containing 20 mM Tris / HCl pH 7.4, 1mM PMSF (phenylmethylsulfonyl fluoride), 1mM AET (2-aminoethylisothiuronium bromide), 0.02 mM PLP (pyridoxal-5- Phosphate) and 2 µg / ml of the proteinase inhibitors leupeptin, Containing aprotinin, bestatin and pepstatin, were the cult fourth cells sedimented. Approx. 8 ml of the sedimented Cells were resuspended and in 30 ml lysis buffer at 0 ° C homogenized and by centrifugation (100,000 × g and 4 ° C) separated into soluble and insoluble cell fractions.

The supernatant liquid, which contains the GAD-1 or GAD-2 proteins, was treated with 40 mM Tris / HCl, pH 7.6 and 0.5 mol NaCl and applied directly to a chelated Sepharose FF column, which Ni ²⁺ ions was prepared, placed. The loaded column was washed with 10mM, 30mM and 500mM imidazole lysis buffer containing 0.5M NaCl. The recombinant GAD proteins were then eluted from the column with 500 mM imidazole, which additionally contains 40% sucrose. The isolated GAD proteins can be used directly in immunoassays.

4. Western blotting analysis of human GAD-1 and GAD-2-Pro teine

The GAD-1 or GAD-2- cleaned as described under 3. Protein was determined by SDS-polyacrylamide gel electrophoresis under reducing and denaturing conditions separates and transferred to nitrocellulose filters, under Use of a "transblot" semi-dry electrophore table transfer cell (BioRad). The unoccupied protein bin The stains on the filter were degreased by 5% Dry milk in TBST buffer (10 mM Tris / HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) blocked. The immobilized pro stones were diluted 500-fold for 90 minutes of a patient's autoimmune serum that contains 0.1 mg / ml E. coli Extracts was pre-absorbed, incubated. The bound anti body was made with anti-human immunoglobin, which with alkaline phosphatase was conjugated, visualized.

5. Immunoprecipitation of the metabolically labeled GAD-1 and GAD-2 proteins

Approximately 5 × 10 ⁶ infected Sf9 cells per petri dish with a diameter of 100 mm were cultivated in Grace's medium, which contains 10% fetal calf serum, at 27 ° C. for 36 hours. The fetal calf serum was then replaced by a serum- and methionine-free medium for 60 minutes and then the infected Sf9 cells were labeled with 10 ml of a serum-free medium containing 200 μCi ³⁵ S-methionine for a further six hours at 27 ° C.

The cells were washed with 0.5 ml hypotonic buffer (20 mM Sodium phosphate, pH 7.0, 2 mM EDTA, 2 mM PMSF (polymethyl sulfonyl fluoride), 1 mM AET (2-aminoethylisothiuronium bro mid), 2 µg / ml aprotinin, 0.2 mM PLP (pyridoxal-5-phosphate) lysed and centrifuged at 10,000 × g for ten minutes, to remove the cell shells.

A 100 µl aliquot of the supernatant was mixed with 25 µl antiserum mixed and incubated overnight at 4 ° C. Of the Immune complex was closely linked to 10 Sepharose protein at room temperature bound two hours, four times with 1 ml of TNET buffer (20 mM Tris / HCl, pH 7.5, 140 mM MaCl, 5 mM EDTA, 1% Triton X-100) and eluted before fluorography and by SDS-polyacrylamide gel electrophoresis separated.

6. Immunoprecipitation of the metabolically labeled high purity GAD-1 and GAD-2 proteins

The immunoprecipitation was described under 5 guided. The GAD-1 and GAD-2 proteins show a high specific antigenicity.

7. Measurement of the enzyme activities of the recombinant GAD-1 and GAD-2 proteins

GAD activity is measured according to standard Krieger and Heller methods (O'Reilly, Miller, Lucow, Baculovirus expression vectors, Freeman and Company, New York, (1992)). The formation of the ¹⁴ CO ₂ by decarboxylation of 0.1 μCi L- [1- ¹⁴ C] glutamate was determined in 200 μl of a tissue or cell homogenate in cell lysis buffer (50 mM KH ₂ PO ₄ , pH 7.0, 1 mM EDTA, 1mM AET, 0.2mM PLP, 1% Triton X-100). The GAD activity was calibrated for the incubation time and the protein concentration and identified as mU / mg of the total cell proteins (1 unit = 1 μM ¹⁴ CO ₂ / min).

Comparison test Expression of human GAD-1 and GAD-2 in E. coli cells

The GAD-1 and GAD-2 cDNAs were found in the prokaryotic express ion vector pH6EX3 inserted. The cDNA clones produced pGAD-E22 and pGAD-E11 synthesize the recombinant GAD- Proteins with a histidine hexapeptide fragment at the N-terminus under the control function of a Tac promoter. After Transforma E. coli strand K5254 expression of the Fu ion genes by 1 mM IPTG (isopropylthiogalactoside) for eight hours long induced.

To analyze the bacterial recombinant human GAD proteins the transformed E. coli strands were cultivated K5254 fourth, induced with 1 mM IPTG and with lysozyme and before lysis Triton X-100 sedimented. The insoluble inclusion bodies were dissolved in 8 moles of urea and by SDS-polyacrylamide Gel electrophoresis and Western blotting analysis separately.  

Anti-GAD antibody

The autoimmune sera used in the present invention have been obtained from IDDM patients with a new onset of the disease and are positive for anti-GAD-1 autoantibodies, as demonstrated by immunoprecipitation. Isolated pig pancreatic islet cells were used after metabolic labeling with ³⁵ S-methionine. The patient serum, which is designated as anti-GAD924, reacts with linear autoantigenic epitopes of the human GAD-1 proteins. As a result, this serum was selected for Western blotting analysis of the expressed recombinant GAD-1 proteins.

The mouse anti-GAD monoclonal antibody that specifically only the recognizing linear epitopes of the GAD-2 protein became friendlier wise of Dr. B. Ziegler and Dr. M. Ziegler (Diabetes Institute, University in Greifswald, Karlsburg, Germany) posed. The liquid culture of a corresponding mouse hybri doma cell line containing the anti-GAD monoclonal antibodies was diluted 200 times for Western blotting.

The example of the invention is illustrated in the drawings which are in The following are described in more detail.

Fig. 1 shows the expression and Western blotting analysis of the GAD-1 and GAD-2 proteins expressed in the Sf9 insect cells. The proteins separated by SDS-polyacrylamide gel electrophoresis are stained (A) with the dye Coomassie Brilliant Blue or (B) by the Western blotting method which uses the anti-GAD924-IDDM patient serum, which is specific to GAD- 1 is used or (C) analyzed by mouse anti-GAD monoclonal antibodies specific for GAD-2. Column 1 shows the soluble, column 2 the insoluble fractions of the MOCK cells; column 3 lists the soluble fractions and column 4 the insoluble fractions of the GAD-1 protein expressed in the Sf9 cell; Columns 5 and 6 show the soluble and insoluble fractions of the GAD-2 proteins expressed in the Sf9 insect cell; Column 7 contains 2.5 mg of purified GAD-1 and column 8 2.5 mg of purified GAD-2.

Figure 2 shows the measurement of GAD enzyme activity in infected Sf9 insect cells. Sf9 cells infected with the wild-type baculovirus (MOCK), Sf9 cells expressing GAD-1 (GAD ₆₅ -Sf9) and GAD-2 (GAD ₆₇ -Sf9), and isolated pancreatic islet cells and porcine brain tissue that were homogenized and were used to measure GAD activity.

Figure 3 shows the immunoprecipitation of the recombined human GAD-1 with GAD-1 specific IDDM patient sera. In columns 1 to 10 the sera of newly ill IDDM patients and in columns 11 and 12 two normal sera are used. While an antibody-antigen complex is formed in columns 1 to 10, there is no reaction between the GAD-1 protein and the normal sera. Column 13 shows the dissolved cell pellet of a metabolically labeled Sf9 control insect cell. The precipitated proteins were separated by 10% polyacrylamide gel electrophoresis and made visible by fluorography.

FIG. 4 shows the Western blotting analysis of the GAD-1 and GAD-2 proteins expressed in E. coli with specific IDDM patient sera. The inclusion bodies of E. coli, which are transformed with the recombinant expression vectors pGAD-E22 and pGAD-E11, which are transformed for GAD-1 (number 1 in FIG. 4) and GAD-2 (number 2 in FIG. 4) were isolated and dissolved in 8 moles of urea. Aliquots containing 10 ul of the bacterial cultures were analyzed by 10% polyacrylamide SDS gel electrophoresis. The GAD proteins isolated from the inclusion bodies are stained with Coomassie brilliant blue (A) or with the Western blotting method (BK), in which ten sera from newly ill IDDM patients were used, or with anti-GAD924-IDDM serum (L) analyzed. Mouse anti-GAD monoclonal antibodies (M) served as control serum.

Claims (17)

1. A method of producing high purity human GAD-1 and GAD-2 proteins using the baculovirus / Sf9 expression system, the method comprising the following steps

• a) Preparation of cDNA sequences from human cDNA libraries, each coding for full-length GAD-1 and GAD-2 proteins;
• b) insertion of the two GAD-1 and GAD-2 cDNA sequences into a baculovirus transfer vector in each case to form recombinant baculovirus transfer vectors;
• c) transfection of Sf9 cells with the recombinant Baculo virus transfer vectors containing the GAD-1 and GAD-2 cDNA sequences;
• d) selecting the positively infected Sf9 cells;
• e) obtaining the GAD-1 and GAD-2 proteins by culturing the positively infected Sf9 cells;
• f) Purification of the recombinant GAD-1 and GAD-2 proteins.

2. The method according to claim 1, characterized in that the cDNA sequences from a human Pancreatic carcinoma cDNA library or from a hippocampus cDNA library. 3. The method according to claim 1 or 2, characterized in that the recombinant baculovirus trans fervectors additionally contain a DNA sequence which is suitable for encodes a histidine hexapeptide. 4. The method according to any one of claims 1 to 3,  characterized in that the human cDNA sequences for Encode GAD-1 and GAD-2 into the baculovirus transfer vector pVL1393 can be inserted. 5. The method according to claim 4, characterized in that the recombinant baculovirus trans Form vector cDNA clones pAc GAD-1 and pAc GAD-2. 6. The method according to claim 5, characterized in that the clones pAc GAD-1 and pAc GAD-2 encode for recombinant fusion proteins that contain the GAD-1 protein or the GAD-2 protein at its N-terminus and a histidine Have hexapeptide at their C-terminus. 7. The method according to claim 1, characterized in that the transfection by means of Spodoptera frugiperda (Sf9 insect cells), preferably with the recom binant baculovirus transfer vectors together with the lineari wild-type Baculovirus Autographa california becomes. 8. The method according to any one of claims 1 to 7, characterized in that the positively infected Sf9 cells in a medium containing a specific serum , especially in the SF900 medium, the 0.04% fetal calf contains serum. 9. The method according to claim 8, characterized in that the Sf9 cells are cultivated for 48 hours become. 10. The method according to any one of claims 1 to 9,  characterized in that after culturing the Sf9 cells be lysed and by centrifugation in soluble and insoluble cell fractions are separated. 11. The method according to claim 10, characterized in that the soluble cell fraction the GAD-1- and contains GAD-2 fusion proteins. 12. The method according to claim 1, characterized in that the soluble cell fraction by a single-stage metal chelate affinity chromatography purified becomes. 13. The method according to claim 1, characterized in that the soluble cell fraction is passed through a chromatographic column which is equipped with a metal ion matrix, in particular Ni ²⁺ ion matrix. 14. The method according to claim 1, characterized in that the GAD proteins with an eluent, especially imidazole. 15. immunoassay, characterized in that the immunoassay by the Ver start up produced according to one of claims 1 to 14 pure GAD-1 and GAD-2 proteins. 16. Immunoassay according to claim 15, characterized in that the immunoassay for early detection diagnosis of type I diabetes mellitus. 17. Immunoassay according to one of claims 15 and 16,  characterized in that the immunoassay Elisa, solid phase Immunoassay, etc.