Hybrid-plasmid for the 38 kDa antigen from M. tuberculosis, E. coli as host, the 38 kDa antigen and a 33 kDa protein.

Background of the Invention

Tuberculosis is a highly contagious human disease with over 3 million deaths and 8 million new cases each year. The appearance of AIDS will likely make the situation dormant because of the reactivation<i>M. tuberculosis</i> worse in immunocompromised patients. The infection dose for tuberculosis is extremely low, which means that one to three tubercle bacilli are sufficient to initiate a primary effect in the lungs. The diagnosis of sick people plays an essential role in epidemiology, prevention and spread of the disease. The diagnosis is currently based on the cultivation of<i>M. tuberculosis</i> from the sputum, which takes about 6 weeks due to the slow growth of the organism. Another important part of the diagnosis is the "tuberculin test". Tuberculin or the purified protein derivative thereof (PPD) is a mixture of proteins from the culture medium filtrate of those killed by heat<i>M. tuberculosis</i>. This test is highly non-specific due to cross-reactivities in individuals who have been infected with or have been vaccinated against other mycobacteria. There was therefore an urgent need to develop defined and specific serodiagnostic reagents suitable for skin testing for tuberculosis. Serological studies have shown that the 38 kDa antigen of <i>M. tuberculosis</i> includes immunodominant epitopes relevant to the virulent strains of <i>M. tuberculosis</i> are specific. This antigen is produced in small quantities in the BCG vaccine strain, which is an avirulent derivative of the bovine tubercle bacillus<i>M. bovis</i> represents. So, based on serological methods, this antigen can be used to move between organisms of the<i>M. tuberculosis</i>-Distinguish complex and other mycobacteria. Purification of the native 38 kDa antigen directly<i>M. tuberculosis</i> is not practical due to the low yields, slow growth and the virulent nature of the organism.

As already mentioned <i>Mycobacterium tuberculosis</i> the causal agent of tuberculosis, a widespread human disease that kills around 3 million people each year. Significant goals of mycobacteria research are to provide protective immunity to tuberculosis through more effective vaccines and to develop specific skin test / serodiagnostic reagents. A precondition for such goals is the characterization and detailed determination of the immunological role of the individual mycobacterial antigens. Sufficient amounts of these antigens are required for this purpose. The purification of antigens directly<i>M. tuberculosis</i> is difficult because of the low cell yields, slow washing and the virulent nature of the organism (10, 26). One possible solution to this problem is the production of recombinant antigens in biotechnologically accessible organisms such as, for example<i>E. coli</i>.

The immunological and diagnostic relevance of the 38 kDa antigen protein <i>M. tuberculosis</i> has already been shown (2, 26). The protein includes species-specific B cell epitopes (2), and T cells isolated from immunized mice, guinea pigs or humans proliferate when cultured in the presence of the antigen (10, 24, 26). The majority of people (especially those of the HLA type DR2) who suffer from active tuberculosis develop antibodies against the 38 kDa antigen (4).

The 38 kDa protein of the gram-positive bacterium <i>Mycobacterium tuberculosis</i> H37Rv is an immunodominant antigen of potential utility for the diagnosis and development of a vaccine. Estimating this potential requires large amounts of the purified protein, which would be difficult if not impossible if it were to be eliminated<i>M. tuberculosis</i> win yourself.

According to one embodiment of the invention, a hybrid plasmid for the expression of an unfused 38 kDa antigen of <i>M. tuberculosis</i> in <i>E. coli</i> provided the plasmid<ul id="ul0001" list-style="dash"><li>the signal sequence of the 38 kDa antigen (pre-protein) and</li><li>comprises a restriction interface which, within its recognition sequence, comprises the base triplet ATG, which codes the first codon M in the reading direction.</li></ul>

This hybrid plasmid can be characterized in that the 38 kDa antigen is a protein of <i>M. tuberculosis</i> is about the wild type or about one <i>M. tuberculosis</i>-Variant that can also cause tuberculosis.

A hybrid plasmid according to the invention can further be characterized in that the signal sequence comprises 17, 18, 19, 20, 21, 22, 23 or 24 codons.

A hybrid plasmid according to the invention can further be characterized in that the DNA sequence encoding the 38 kDa antigen (pre-protein) has been inserted N-terminally into an NcoI, NdeI or SphI site of the starting vector, for example pJLA603 .

A hybrid plasmid according to the invention can also be characterized by a DNA sequence coding for the 38 kDa antigen (pre-protein) as shown in FIG. 1A or 1C.

According to a further embodiment, the invention relates <i>E. coli</i> with a hybrid plasmid according to the invention.

According to a further embodiment, the invention relates to a 38 kDa antigen (pre-protein) from <i>M. tuberculosis</i>, producible with the help of <i>E. coli</i> according to the invention, the signal sequence comprising 17 to 24 codons, with the exception of a signal sequence with 23 codons.

This 38 kDa antigen (pre-protein) can contain 22 to 17 amino acids of the following signal sequence: MKIRLHTLLAVLTAAPLLLAAAG, for example, amino acids 1 can be labeled 8 to 23.

According to a further embodiment, the invention relates to an approximately 33 kDa protein, obtainable with the aid of <i>E. coli</i> as the host of pJLA 603 as an expression plasmid, where<ul id="ul0002" list-style="dash"><li>a the 38 kDa antigen of <i>M. tuberculosis</i> (Pre-protein) coding DNA sequence has been inserted N-terminally into an NcoI, NdeI or SphI site of pJLA 603,</li><li>this DNA sequence used comprises the signal sequence of the antigen,</li><li>the recognition sequence of the interface comprises the base triplet ATG, which encodes the first codon M in the reading direction and</li><li>the approximately 33 kDa protein is optionally separated from the likewise expressed 38 kDa antigen.</li></ul>

According to a further embodiment, the invention relates to an approximately 33 kDa protein which is characterized in that it is opposite the 38 kDa antigen of <i>M. tuberculosis</i> (Pre-protein) N-terminal around the signal sequence and another 24 amino acids is deleted.

According to a further embodiment, the invention relates to a 38 kDa antigen from <i>M. tuberculosis</i> and / or about 33 kDa protein made using <i>E. coli</i> renatured according to the invention and as follows:<ul id="ul0003" list-style="dash"><li>Solubilize the antigen and / or protein obtained as an inclusion body in guanidinium HCl (optionally in the presence of a reducing agent) and</li><li>subsequent renaturation using Sephadex chromatography.</li></ul>

This 38 kDa antigen and / or approximately 33 kDa protein can be characterized in that<ul id="ul0004" list-style="dash"><li>has worked with about 6 M guanidinium HCl in the solubilization and / or in the presence of dithiothreitol (DTT) and / or</li><li>carried out the renaturation with the help of Sephadex G-25.</li></ul>

According to the invention, recombinant plasmids were constructed which <i>Escherichia coli</i> the 38 kDa antigen of <i>M. tuberculosis</i> to produce. With the recombinant constructs according to the invention, large amounts of unfused (unique) 38 kDa protein in<i>E. coli</i>, mainly in the form of inclusion bodies. In addition, a method for isolating and purifying the recombinant antigen from the inclusion bodies was developed within the scope of the present invention. The purified 38 kDa antigen prepared in this way is immunologically indistinguishable from the native 38 kDa antigen of<i>M. tuberculosis</i>. Because of its high specificity for<i>M. tuberculosis</i> this recombinant antigen can be used worldwide for the serological diagnosis of tuberculosis. The antigen is also a potential candidate for tuberculosis vaccines because it includes immunodominant T cell epitopes.

The gene encoding the 38 kDa antigen has already been cloned and is used as an unfused protein in the present invention <i>Escherichia coli</i> under the control of strong transcriptional (bacteriophage lambda P_RP_L) and translational (<i>atpE</i>) Signals expressed. The fermentation of an ion protease and the heat shock response are absent, provided the recombinant antigen in high levels (approximately 10% of the total cell protein). The recombinant antigen, which accumulated as inclusion bodies, was completely solubilized in 6 M guanidinium hydrochloride, refolded and appeared homogeneous after purification. The product showed the expected amino acid composition and the expected molecular weight, and also reactivities of the same strength with three different monoclonal antibodies as the native protein. Polyclonal antibodies developed against the recombinant antigen reacted strongly with the native antigen in an enzyme-linked immunosorbent assay. These results show that the recombinant 38 kDa antigen, which is not immunologically derived from the native protein<i>M. tuberculosis</i> can be distinguished in sufficient quantities <i>E. coli</i> can be produced.

Description of the pictures

<dl id="dl0001"><dt>Illustration 1:</dt><dd>Nucleotide sequences of the 5 'ends of the engineered genes encoding the pre-form (A) and the truncated form (C) of the 38 kDa protein. The<i>Nde</i>I (CATATG) interface was by oligonucleotide mutagenesis using the oligonucleotides 5'-AGCACAGAAAGGTATCATATGAAAATTCGTTTGCATA-3 '(for A) and 5'-ATTCGTTTGCATATGCTGTTGGCCGTGT-3' (for C). The derived amino acid sequence is shown in the one-letter code and the arrow above the sequence shows the suspected processing site. The RNA stability in the region of the ATG start codon of the pre-form (B) and the shortened form (D) was calculated using the method of Zucker and Stiegler (28). The ribosome binding site on the<i>atp</i>The E gene and the initiation codon of the 38 kDa antigen gene are marked with an asterisk and a triangle, respectively.</dd><dt>Figure 2:</dt><dd>Construction of an expression plasmid. A 2.0 kb<i>Eco</i>RI fragment comprising the gene for the 38 kDa protein was cloned from Lambda AA59 (3) in M13mp19 and mutagenized to a <i>Nde</i>I (CATATG) interface in such a way that it contains the initiation codon of the gene. A 1.2 kb<i>Nde</i>I-<i>Sph</i>I fragment was then inserted into the <i>Nde</i>I-<i>Sph</i>I interface of the expression vector, pJLA603, subcloned, which promoter P_R and P_L of the bacteriophage lambda in a tandem arrangement, the <i>atp</i>E-translation initiation region (white box) containing the transcriptional terminator fd and translational stop codons in all three reading frames (20). The plasmids are not drawn to scale.</dd><dt>Figure 3:</dt><dd>Expression of the recombinant 38 kDa protein in <i>E. coli</i> CAG629 in batch cultures (A) and in a bioreactor (B). A: Silver-stained gels (lanes 1-7) and the corresponding immunoblots with MAb HBT12 (lanes 8-14) showing the proteins derived from CAG629 (pMS9-2) after growing at 30 ° C (lanes 1/8 and 2 / 9) and at 42 ° C (tracks 3/10 and 4/11). The protein patterns of CAG629 (pMS10-4) after induction at 42 ° C are shown in lanes 6/13 and 7/14. Lanes 1, 3 and 6 represent the soluble fractions and lanes 2, 4 and 7 the inclusion bodies. Lane 5: molecular weight markers (the values ​​are given on the left in kilodaltons), 12: previously colored standard. B: Accumulation of the recombinant 38 kDa protein in the bioreactor at 30 ° C (lane 3) and after induction at 42 ° C (lanes 4-9). Samples were taken every 30 minutes after induction. Lane 1 shows the molecular weight marker and Lane 2 shows small amounts of the 38 kDa protein, which from inclusion bodies from one batch points to the double band corresponding to the recombinant product.</dd><dt>Figure 4:</dt><dd>Electron microscopic image of sections of CAG629 (pMS9-2 showing inclusion bodies (arrows).</dd><dt>Figure 5:</dt><dd>SDS-12.5% ​​-PAGE- (silver staining) analysis of various cleaning steps. Lanes 1 and 4 show the molecular weight standards. Lane 2: solubilized inclusion bodies; Lane 3: proteins released during the washing of the inclusion bodies before solubilization; Lane 5: proteins renatured on Sephadex G25; Lanes 6-7: QAE-Sepharose eluates. The arrow shows the recombinant 38 kDa protein.</dd><dt>Figure 6:</dt><dd>SDS-PAGE and immunoblot analyzes of the FPLC-purified 38 kDa protein preparations. Samples containing about 1 µg protein were separated by SDS-12.5% ​​-PAGE and either silver stained (lanes 1-3) or immunoblotted using anti-38 kDa MAbs: HAT2 (lanes 4-6), HBT12 (lanes 7-9) and HYT28 (lanes 10-12). The recombinant protein that reacted with the three MAbs was found in 2 major peaks: one at 100 mM NaCl (lanes 1, 4, 7 and 10) and the other at 130-200 mM NaCl (lanes 3, 6, 9 and 12). Lanes 2, 5, 8 and 11 show the protein that was purified in the presence of Triton X-100 (eluted at 170 mM NaCl). The molecular weight standard was plotted in lane S (sizes are given in kilodaltons on the left) and lane P represents the previously stained marker used during immunoblotting.</dd><dt>Figure 7:</dt><dd>Comparison of the native and the recombinant 38 kDa antigen. A: About 1 µg of the native protein purified by affinity chromatography (lanes 1 and 3) and the recombinant protein, preparation-I (lanes 2 and 4) were separated by SDS-12.5% ​​PAGE and either silver-stained (lanes 1 and 2) or below Use of the MAb's HBT12 immunoblotted (lanes 3 and 4). Lane P shows the previously stained molecular weight standard. B: Laser densitometric comparison of the immunoreactivity of the native and the recombinant 38 kDa protein. The silver-colored gel and the immunoblot from part (A) were scanned with a laser densitometer. The peak areas of the silver-colored native (Nat. Silver) and the recombinant protein (Rec. Silver) as well as those of the immunoblotted native (Nat. Immuno) and the recombinant protein (Rec. Immuno) are shown.</dd><dt>Figure 8:</dt><dd>A: Titration curves of polyclonal sera with the 38 kDa antigen. Sera from rabbits immunized with the native (dot) or recombinant 38 kDa protein (solid box). Serial two-fold dilutions, starting with a 1: 100 dilution, were titrated in microtiter plates which had been coated with the preparation I of the recombinant 38 kDa protein (0.1 μg / well). The bound immunoglobulins were detected using horseradish peroxidase coupled to pork anti-rabbit immunoglobulins in a 1: 1000-fold dilution (P 217, Dakopatts, Glostrup, DK). B: As (A), except that the microtiter plates were coated with the native 38 kDa antigen (0.1 µg / well).</dd></dl>

The invention is explained in detail below.

material and methods

Bacterial strains, phages, plasmids and growth conditions.

Those used in this invention <i>E. coli</i>Strains were TG-1 (Δlac-pro, <i>sup</i>E, <i>thi</i>, <i>hsd</i>D5 / F '<i>tra</i>D36, proA⁺ B⁺, <i>lac</i>I.^q, <i>lac</i>Z) ΔM15), DH5alpha (<i>end</i>A1, <i>rec</i>A1, <i>hsd</i>R17, <i>sup</i>E44, <i>thi</i>-1, <i>gyr</i>A96, <i>rel</i>A1, (<i>lac</i>ZYA-argF), φ 80d /<i>lac</i>Z ΔM15), EC 538 and CAG629 (<i>lon</i>, <i>htp</i>R165-Tn<i>10</i>; C. Gross). The recombinant Lambda gt11 bacteriophage, clone AA59, was obtained in a previous study (3) from a genomic DNA library from<i>M. tuberculosis</i> (constructed by RA Young (27)) isolated.

Unless otherwise stated, the strains were cultured in Luria Bertani medium (15) at 37 ° C. The liquid cultures were supplied with oxygen by circulating them at 160 rpm in a pilot shake shaker (Kuehne, Switzerland).

DNA manipulation.

Preparation and treatment of the DNA was carried out according to standardized protocols (15). The transformation was accomplished as described in Hanahan (8). DNA sequencing was carried out using the dideoxynucleotide chain termination method (18). Oligonucleotides were made on an Applied Biosystems Model 380B DNA synthesizer and purified on OPC columns (Applied Biosystems Inc.).

Oligonucleotide mutagenesis.

The 2.0 kb <i>Eco</i>RI fragment from the genomic clone Lambda-AA59 (3) was transferred to M13mp19. Single-stranded DNA preparation and oligonucleotide mutagenesis were performed using the Amersham kit (RPN 1523). The DNA sequence of the oligonucleotide before and after mutagenization was confirmed by DNA sequencing (18).

Preparation of a coarse protein extract on a small scale.

The strains were cultured in LB medium supplemented with ampicillin (100 μg / ml) at 30 ° C. to an absorption of 0.6 at 580 nm. The cultures were induced by raising the temperature to 42 ° C. in a shaking water bath for three hours. Bacteria from 1 ml culture medium were harvested, suspended in 100 µl sample buffer (62 mM Tris-HCl, pH 6.8; 2% sodium lauryl sulfate; 0.7 M 2-mercaptoethanol; 10% glycerol; 0.002% bromophenol blue) and treated with ultrasound on ice (3rd x 30 seconds; 50 W) using a Braun Labsonic 2000. The samples were heated to 95 ° C for 10 minutes and 10 ul was analyzed by polyacrylamide gel electrophoresis.

Cultivation in the bioreactor.

30th 1 of a modified concentrated LB medium (trypton 40 g / l; yeast extract 20 g / l; NaCl 5 g / l) were in the presence of 3.5 ml Ucolub N38 Antifoam (Brenntag, Mülheim, FRG) in a 50 l bioreactor (Biostat U30D , Braun Melsungen, FRG) sterilized. The cultivation was started by inoculation with 0.5 l of a culture of the organisms that had grown overnight in the same medium, the initial absorption being A₅₄₆ = 0.04. The stirring speed was kept constant at 300 rpm, which kept the concentration of dissolved oxygen near saturation, and the pH was adjusted at 6.9. After 4 hours fermentation time (A₅₄₆ = 0.4) the temperature was increased from 30 ° C to 42 ° C and left at this temperature for a further 4 hours. At the end of the induction period, the suspension was concentrated in a closed system by means of shear current microfiltration (Enka module type A7 ABA 3A) with a 0.23 m² Accural membrane (0.2 µm pore diameter; Enka, Wuppertal, FRG) until a final volume of 10 l was obtained.

Protein purification.

The cells obtained from the bioreactor were broken up by passing them once at 500 bar at a flow rate of 60 l / h through a high-pressure homogenizer LAB 60/500/2 (APV-Schröder, Lübeck, FRG). The inclusion bodies were roughly separated using a centrifugal separator SA 1-01-175 (Westfalia, Oelde, FRG). This device allows the isolation of inclusion bodies from the medium with a flow rate of 15-20 l / h. The inclusion bodies were washed twice with 200 ml buffer L (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) containing 2% Triton X-100. The washed precipitates were resuspended in 3 l of buffer L, which contained 6 M guanidinium hydrochloride and 20 mM DTT, with slow stirring, at 4 ° C. for 16 hours. After centrifugation at 7000 g, the liquid supernatant was applied to a Sephadex G-25 gel filtration column (10 x 90 cm) which had been equilibrated with 10 mM Tris-HCl buffer, pH 7.0, which contained 100 mM NaCl to do this To remove guanidinium HCl and to accomplish the renaturation of the recombinant antigen. The solution was applied in 2 l aliquots at a flow rate of 7 l / h and in the eluate OD₂₆₀ and conductivity were measured. The desalted and renatured antigen solution was found in the start buffer with conductivity of 8 mS / cm, well separated from the salt peak, which mainly contained guanidinium HCl. The antigen peaks were pooled and diluted with distilled water to achieve a conductivity of 5 mS / cm and the pH was adjusted to 8.5 with 1 M Tris. This solution was divided into two parts for the following cleaning steps. A portion of the solution was applied to an FPLC column (QAE-Sepharose FF; 5 x 18 cm), which had been equilibrated with a 20 mM Tris-HCl buffer, pH 8.0. The flow rate was 1.72 l / h, corresponding to a linear flow rate of 86.6 cm / h. After extensive washing with start buffer, the antigen was eluted by applying a graded gradient consisting of 50 mM, 100 mM, 250 mM, 500 mM and 1 M sodium chloride in start buffer. The column was then re-equilibrated with starting buffer, the second part was applied from the gel filtration column and eluted as described above. The antigen-containing fractions from the two QAE-Sepharose runs were combined and concentrated by ultrafiltration using an Amicon hollow fiber device (type H1P10; cut-off limit 10,000) and diafiltered (2 mS / cm). About 20 mg of the protein thus obtained was applied to a Mono Q HR (5/5) FPLC column which had been equilibrated with 20 mM Tris-HCl, pH 8.0. The column was washed with start buffer at a flow rate of 2 ml / min and a pressure of 25 bar. The antigen was then eluted by gradually increasing the NaCl concentration to 0.5 M in start buffer. A total of three Mono Q HR (5/5) runs were performed under the conditions described above.

Polyacrylamide gel electrophoresis and immunoblotting.

The coarsely purified and highly purified proteins were analyzed using sodium lauryl sulfate / polyacrylamide (12%) gel electrophoresis (11). The protein samples were mixed with a 2 x 1: 1 sample buffer and heated to 95 ° C for 10 minutes before being applied to the gel. Following electrophoresis, the polypeptides were visualized by silver staining (5). The protein concentrations were determined by the method of Lowry et al. measured (13). The proteins were transferred to a nitrocellulose membrane (BioRad) using a self-made semi-dry blotting apparatus using 25 mM Tris, 192 mM glycine, 20% methanol, pH 7.4. Non-specific binding was blocked by incubating the filter with TBS (50 mM Tris-HCl; 200 mM NaCl, pH 7.5), which contained a 10% milk solution (0.3% fat). The primary antibodies (monoclonal antibodies from mice) were diluted 1000-fold in TBS and the filters were incubated with them at 4 ° C. overnight. The filters were washed 3 times with TBS and immunodetection was performed with a biotinylated anti-mouse IgG and a conjugate of streptavidin and alkaline phosphatase (BRL, Gaithersburg, MD, USA). For an immunodot blot assay, the protein samples were filtered through a nitrocellulose membrane using a BioRad Bio-Dot apparatus and then treated as described above. The monoclonal antibodies HAT2, HBT12 and HYT28 have been described earlier (2, 12, 21). Densitometric measurements of the silver-colored gels and the Western blots were carried out with a laser densitometer (LKB).

Amino acid analysis

.

The amino acid analyzes were carried out with a Biotronik LC-5001 amino acid analyzer (Maintal, FRG) after the protein sample had been hydrolyzed in 6 N HCl, which contained 0.1% phenol, at 105 ° C. for 24 hours.

Preparation of the polyclonal anti-38 kDa protein sera.

Rabbits were immunized subcutaneously either with affinity-purified 38 kDa protein (25) or with recombinant 38 kDa protein (preparation I). The antigen (10 µg / dose) was adsorbed on aluminum hydroxide (2.4 mg) and subsequently mixed with 1 ml Freund's incomplete adjuvant. The rabbits were immunized three times at two week intervals. Blood was obtained 10 days after the last immunization and the IgG fraction was purified using the Harboe and Inglid (9) method.

Electron microscopy.

The cells were fixed on ice with 1% formaldehyde and 0.2% glutaraldehyde in PBS (50 mM K-phosphate, 0.9% NaCl, pH 6.9) for one hour. After washing several times with PBS, the cells were embedded according to the progressive temperature reduction method (PLT), (17). The cells were first on ice with 10%, then with 30% ethanol for 30 minutes, then with 50% ethanol for 30 minutes at -20 ° C, and finally with 70%, 90% and 100% ethanol at -35 ° C each for 30 minutes and dehydrated with 100% ethanol at -35 ° C for 1 hour. The infiltration with the Lowicryl resin K4M was carried out as follows: 1 part ethanol / 1 part K4M resin overnight at -35 ° C, 1 part ethanol / 2 parts K4M resin for 12 hours and pure K4M resin for 2 days at - 35 ° C with repeated changes of the resin mixture. The polymerization of the resin was achieved by UV light (366 nm) for 1 day at -35 ° C and at room temperature for a further 2 days. Ultrathin sections were stained with uranyl acetate and lead citrate before being examined with a Zeiss EM 10B transmission electron microscope at an acceleration voltage of 80 kV.

Results

Construction of the expression plasmids.

DNA sequencing of the 38 kDa protein gene revealed an open reading frame encoding a 374 amino acid polypeptide and containing GTG as the start codon (1). There was also a 24 amino acid signal sequence (Figure 1A) that was similar to that of bacterial lipoproteins. In the present invention, the gene was manipulated so that the unfused 38 kDa antigen could be expressed to a high degree in the expression vector pJLA603 (Figure 2). For cloning and expression in such vectors, it is necessary that the foreign gene have a restriction site, for example <i>Nco</i>I, <i>Nde</i>I, <i>Sph</i>I, which includes ATG in reading frame within its recognition sequence. Because there is no such interface in the region around the initiation codon of the 38 kDa gene, an oligonucleotide mutagenesis was carried out in M13mp19 in order to exchange the start codon from GTG to ATG and one<i>Nde</i>I interface to be provided at the N terminus (Figures 1 and 2). The 1.2 kb<i>Nde</i>I-<i>Sph</i>I fragment that could be excised from the M13 derivative after mutagenization was then inserted between the <i>Nde</i>I-<i>Sph</i>I interfaces cloned from pJLA603. The recombinant plasmid, which was constructed so that the 38 kDa protein could be expressed with its original, intact signal peptide, was named pMS9-2. Another recombinant plasmid, pMS10-4, which had a deletion of the first 6 amino acids in the signal sequence, was prepared in the same way (Figure 1C). Computer analysis of the translation initiation region of the m-RNA specified by pMS9-2 predicted a loose secondary structure (Figure 1B), which is essential for high-level expression in <i>E. coli</i> should be very beneficial (14). On the other hand, pMS10-4 showed a more stable secondary structure (Figure 1D), suggesting that the expression of this plasmid would not be as good as that of pMS9-2. Both recombinant plasmids were used for the expression studies.

Expression of the recombinant antigen in a culture on a small scale

.

Several <i>E. coli</i>Strains (DH5α; EC538 and CAG629) were examined for expression of the 38 kDa recombinant antigen encoded by pMS9-2 and pMS10-4. Of the<i>lon</i>, <i>htp</i>R strain CAG629 showed the strongest expression. Extracts from CAG629 (pMS9-2) cells, which had been induced at 42 ° C, contained sufficient amounts of the recombinant protein (Figure 3, lane 3 and 4), whereas it was not found in extracts from uninduced cells (Figure 3, Lane 1). The recombinant strain showed no apparent interference after induction and continued to grow and exponentially (data not shown). Immunoblotting with MAb's HBT12 (Figure 3A, lanes 10 and 11), HAT2 and HYT28 (data not shown) gave positive reactions with the recombinant 38 kDa protein. Most of the recombinant protein was found in the cell pellet fraction of the disrupted cells, and only a small fraction was detectable in the liquid supernatant (Figure 3A, lanes 10 and 11). The recombinant clone CAG629 (pMS10-4) produced, as expected from the prediction of the secondary structure, considerably less protein than pMS9-2 (Figure 3A, lanes 13 and 14). The weak bands recognizable on the immunoblot, which migrated more slowly than the 38 kDa protein, correspond to SDS-insoluble, aggregated forms of the recombinant protein. The recombinant 38 kDa protein was produced to a large extent (approximately 10% of the total cell protein), as could be measured by means of measurements with a laser densitometer on silver-colored SDS-PAGE gels. As is often the case with recombinant proteins that are present in high levels in<i>E. coli</i> are produced (7, 19), the 38 kDa protein is mainly present in cytoplasmic aggregates or inclusion bodies (Figure 4).

Fermentation of the recombinant

E. coli

and purification of the recombinant antigen.

The recombinant clone CAG629 (pMS9-2) was selected for a 30 l fermentation because it produced and tolerated the highest antigen levels in batch cultures and because it encoded a 38 kDa protein with an intact signal sequence. The time course of the production of recombinant antigen in the bioreactor was observed (Figure 3B). SDS-PAGE of the entire cell extract showed that within 30 minutes after increasing the temperature from 30 ° C to 42 ° C a double band of 38 kDa size was clearly visible on silver-colored gels.

Washing the inclusion bodies obtained from the fermenter culture with buffer containing Triton X-100 caused some contaminating proteins (Figure 5, lane 3) to be removed without an apparent loss of recombinant antigen (Figure 5; lane 2). The antigen was desalted and renatured using a Sephadex G-25 column (Figure 5, lane 6); we observed no reagent aggregation of the antigen at this stage. Further purification of the antigen was achieved by several cycles of FPLC anion exchange chromatography (Figure 6), in which it was found that the antigen eluted at 100 mM NaCl (preparation I) and between 130-200 mM NaCl (preparation III). Preparation II shown in Figure 6 represents the antigen which was purified on an FPLC anion exchange column in the presence of Triton X-100 from the aggregated and contaminated fractions from previous anion exchange chromatography steps.

Structure, immunological response and immunogenicity of the purified, recombinant protein.

The immunological reaction of the purified antigen preparations was tested with the monoclonal antibodies HAT2, HBT12 and HYT28. All three antibodies reacted with the recombinant antigen preparations (Figure 6). When the affinity-purified native antigen was compared with the recombinant antigen (preparation I) using SDS-PAGE and immunoblot, there were no differences (Figure 7A). The silver stained gel and immunoblot from Figure 7A were also analyzed with a laser densitometer, and after normalization for differences in the amounts of protein applied, we observed that the native and recombinant antigen reacted identically with the HBT12 monoclonal antibodies (Figure 7B), HAT2 and HYT28 (data not shown) resulted.

The immunogenicity of the recombinant antigen was further tested by obtaining polyclonal antisera from rabbits against the native and the recombinant antigen under similar conditions. The two sera were subsequently tested in the enzyme-linked immunosorbent assay (ELISA) against both the recombinant (Figure 8A) and the native antigen (Figure 8B). The slope of the curves is identical, which shows that the two sera bind the native and the recombinant 38 kDa protein equally well.

The amino acid composition of the recombinant 38 kDa protein pretty well reflects the amino acid composition that can be derived from the nucleotide sequence (Table 1).

<b>About 33 kDa protein.</b> The reduction compared to the 38 kDa antigen completely removed the signal sequence. However, the truncated protein reacts strongly with the HAT2, HBT12 and HYT28 monoclonal antibodies, indicating that the three epitopes are intact. The truncated protein also shows a strong response in the ELISA and Western blotting test to sera from mice with<i>Mycobacterium tuberculosis</i> had been infected. In the context of the present invention, it was found that approximately 85% of the sera from tuberculosis patients reacted positively with the truncated protein or the trimmed antigen, which shows that the trimmed antigen can be used effectively for diagnosis.

discussion

A strategy was developed and executed, a DNA fragment from <i>Mycobacterium tuberculosis</i> clone into an expression vector so that unfused 38 kDa protein would be produced to a high degree. The vector included the Lambda-P_RP_LPromoter and the efficient translation initiation region of the <i>atp</i>E gene, which is an expression of the heterologous 38 kDa antigen in <i>E. coli</i> to an extent that corresponded to 10% of the total cell protein. About 15 mg recombinant protein / liter was produced under these given conditions. However, it should be emphasized that the aim of the present invention was to find out whether the overexpressed recombinant antigen could be found in an antigenic form which is immunologically indistinguishable from the native antigen which is made from<i>Mycobacterium tuberculosis</i> wins, and not to optimize the fermentation yields.

The majority of the recombinant 38 kDa protein accumulated in the form of inclusion bodies. 6 M guanidinium HCl in the presence of a reducing agent (DTT) in high concentration turned out to be the most suitable for the solubilization of the inclusion bodies. The renaturation of the proteins of the inclusion bodies, in particular the hydrophobic membrane proteins, is often difficult and the experimental conditions have to be carefully optimized. In our case, the usual method of dialysis or step dialysis caused the recombinant 38 kDa protein to precipitate, even at very low protein concentrations (0.05 mg / ml). Renaturation of the recombinant protein on Sephadex G-25, on the other hand, was found to be effective and no significant reaggregation of the protein was observed. Starting with the inclusion bodies, which contain about 200 mg of total protein, we were able to prepare 19 mg of the 38 kDa protein with a purity of greater than 95%, as was shown on silver-colored SDS-PAGE gels.

The 38 kDa antigen <i>M. tuberculosis</i> is most likely a lipoprotein (DB Young and TR Garbe, Res. Microbiol. 142: - (1991), in press). Lipoproteins show aberrant, diffuse bands on SDS-PAGE gels (16, 22). As is true for other lipoproteins, we observed a clear tendency for the 38 kDa recombinant antigen to aggregate during anion exchange chromatography and during concentration by ultrafiltration. Part of the antigen that aggregated during ultrafiltration could be recovered by solubilization in 2% Triton X-100, followed by FPLC on Mono Q.

The native 38 kDa protein appears as a double band on SDS polyacrylamide gels. The reason for this is not known, but it could result from acylation and processing of the pre-protein. We did not remove the signal sequence until we produced the antigen in large quantities because the presence of lipoylan moieties on the N-terminal cysteine ​​could play an important role in the immunogenicity of protein antigens (6). The recombinant protein which was purified in this invention also showed a double band characteristic. Preparation I mainly contains the upper band and only small amounts of the lower band. Preparation II contains both the upper and lower bands in almost equal amounts. Preparation III represents a proteolytically truncated derivative (about 33 kDa) of the 38 kDa antigen.

The native 38 kDa protein, which is obtained from the culture supernatant of <i>M. tuberculosis</i> is isolated, and the purified recombinant protein, which can be found in preparations I and II, were of the same size and showed a double-band character on SDS-PAGE gels. In addition, the purified proteins and the native antigen on immunoblots showed an identical reaction with the monoclonal antibodies HAT2, HBT12 and HYT28. A polyclonal serum, which was produced against the recombinant protein (preparation I), recognized the native antigen and showed the same reaction in ELISA as a serum which was produced against the native antigen. Similar results were obtained when the two sera were tested in ELISA against the recombinant antigen, which shows that the recombinant protein has similar epitopes and is as immunogenic as the native 38 kDa protein made from<i>M. tuberculosis</i> can be cleaned.

In summary, we have an expression system and a manufacturing and purification process for the 38 kDa protein <i>M. tuberculosis</i> in <i>E. coli</i> that allows easy isolation of significant amounts of the antigen. The recombinant antigen is immunologically indistinguishable from the native antigen. These results should significantly speed up the assessment of the value of the antigen for the diagnosis and development of vaccines.

bibliography

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Dis. 159: 549-555.</li><li>5. Damerval, C., M. le Guilloux, J. Blaissonneau, and D. de Vienne. 1987. A simplification of Heukeshoven and Bernick's silver staining of proteins. Electrophoresis 8: 158-169.</li><li>6. Deres, K., H. Schild, K.-H. Wiesmüller, G. Jung, and HG Ramensee. 1989.<i>In vivo</i> priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342: 561-564 Halenbeck, R., E. Kawasaki, J. Wrin, and K. Koths. 1989. Renaturation and purification of biologically active recombinant human macrophage colony-stimulating factor expressed in<i>Escherichia coli</i>. Biotechnology 7: 710-715.</li><li>8th. Hanahan, D. 1983. Studies on transformation of <i>Escherichia coli</i> with plasmids. J. Mol. Biol. 166: 557-580.</li><li>9. Harboe, N., and A. Inglid. 1983. Immunization, isolation of immunoglobulins and antibody titre determination. Scand. J. Immunol. 17S10: 345-351.</li><li>10th Kadival, GV, SD Chaparas, and D. Hussong. 1987. 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Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY</li><li>16. Pugsley, AP, C. Chapon, and M. Schwartz. 1986. Extracellular pullulanase of<i>Klebsiella pneumoniae</i> is a lipoprotein. J. Bacteriol. 166: 1083-1088.</li><li>17th Roth, J., M. Bendayan, E. Carlemalm, W. Villiger, and M. Garavito. 1981. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J. Histochem. Cytochem. 29: 663-669.</li><li>18th Sanger, F., S. Nicklen, and AR Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.</li><li>19th Sarmientos, P., M. Duchesne, P. Denefle; J. Boiziau, N, Fromage, N. Delaporte, F. Parker, Y. Lelievre, JF. Mayaux, and T. Cartwright. 1989. Synthesis and purification of active human tissue plasminogen activator from<i>Escherichia coli</i>. Biotechnology 7: 495-501</li><li>20th Schauder, B., H. Blöcker, R. Frank, and JEG Mccarthy. 1987. Inducible expression vectors incorporating the<i>Escherichia coli atp</i>E translational initiation region. Genes. 52: 279-283.</li><li>21. Schou, C., Z.-L. Yuan, AB Andersen, and J. Bennedsen. 1985. Production and partial characterization of monoclonal hybridoma antibodies to<i>Mycobacterium tuberculosis</i>. Acta Pathol. Microbiol. Immunol. Scand. Sect. C 93: 265-272</li><li>22. Schouls, LM, R. Mount, J. Dekkert, and JDA van Embden. 1989. Characterization of lipid-modified immunogenic proteins of<i>Treponema palidum</i> expressed in <i>Escherichia coli</i>. Microbiol. Pathogenic. 7: 175-188.</li><li>23. Styblo, K. 1989. Overview and epidemiological assessment of the current global tuberculosis situation with an emphasis on control in developing countries. Rev. Infect. Dis. 11: 5339-5346.</li><li>24th Worsaae, A., L. Ljungqvist, K. Haslov, I. Heron, and J. Bennedsen. 1987. 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Living material

<dl id="dl0002"><dt>Lambda-AA59:</dt><dd>genomic clone for 2.0 kb EcoRI fragment Access: literature (3); DSM 6524</dd><dt>M13mp19:</dt><dd>Transfer phage for 2.0 kb EcoRI fragment Access: Pharmacia</dd><dt>pJLA603:</dt><dd>Vector for the uptake of a NdeI-SphI fragment from M13mp19 after transfer of the 2.0 kb EcoRI fragment and mutagenization Access: literature (20); Medac (Hamburg)</dd></dl>

<tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Amino acid composition of the purified 38 kDa antigen</entry></row><row><entry namest="col1" nameend="col1" align="center">amino acid</entry><entry namest="col2" nameend="col3" align="center">Number of leftovers</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">derived from the DNA sequence</entry><entry namest="col3" nameend="col3" align="center">Amino acid analysis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Ala</entry><entry namest="col2" nameend="col2" align="right">55</entry><entry namest="col3" nameend="col3" align="char" char=".">56.8</entry></row><row><entry namest="col1" nameend="col1" align="left">Arg</entry><entry namest="col2" nameend="col2" align="right">5</entry><entry namest="col3" nameend="col3" align="char" char=".">6.9</entry></row><row><entry namest="col1" nameend="col1" align="left">Asn</entry><entry namest="col2" nameend="col2" align="right">18</entry><entry namest="col3" nameend="col3" align="char" char=".">39.3</entry></row><row><entry namest="col1" nameend="col1" align="left">Asp</entry><entry namest="col2" nameend="col2" align="right">16</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">Cys</entry><entry namest="col2" nameend="col2" align="right">3</entry><entry namest="col3" nameend="col3" align="left">ND^a</entry></row><row><entry namest="col1" nameend="col1" align="left">Gln</entry><entry namest="col2" nameend="col2" align="right">19</entry><entry namest="col3" nameend="col3" align="char" char=".">32.8</entry></row><row><entry namest="col1" nameend="col1" align="left">Glu</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">Gly</entry><entry namest="col2" nameend="col2" align="right">43</entry><entry namest="col3" nameend="col3" align="char" char=".">41.2</entry></row><row><entry namest="col1" nameend="col1" align="left">His</entry><entry namest="col2" nameend="col2" align="right">7</entry><entry namest="col3" nameend="col3" align="char" char=".">7.6</entry></row><row><entry namest="col1" nameend="col1" align="left">Ile</entry><entry namest="col2" nameend="col2" align="right">19</entry><entry namest="col3" nameend="col3" align="char" char=".">17.8</entry></row><row><entry namest="col1" nameend="col1" align="left">Leu</entry><entry namest="col2" nameend="col2" align="right">36</entry><entry namest="col3" nameend="col3" align="char" char=".">32.4</entry></row><row><entry namest="col1" nameend="col1" align="left">Lys</entry><entry namest="col2" nameend="col2" align="right">12</entry><entry namest="col3" nameend="col3" align="char" char=".">12.5</entry></row><row><entry namest="col1" nameend="col1" align="left">Mead</entry><entry namest="col2" nameend="col2" align="right">6</entry><entry namest="col3" nameend="col3" align="char" char=".">0.7</entry></row><row><entry namest="col1" nameend="col1" align="left">Phe</entry><entry namest="col2" nameend="col2" align="right">13</entry><entry namest="col3" nameend="col3" align="char" char=".">13.0</entry></row><row><entry namest="col1" nameend="col1" align="left">Per</entry><entry namest="col2" nameend="col2" align="right">26</entry><entry namest="col3" nameend="col3" align="char" char=".">23.4</entry></row><row><entry namest="col1" nameend="col1" align="left">Ser</entry><entry namest="col2" nameend="col2" align="right">26</entry><entry namest="col3" nameend="col3" align="char" char=".">22.3</entry></row><row><entry namest="col1" nameend="col1" align="left">Thr</entry><entry namest="col2" nameend="col2" align="right">27</entry><entry namest="col3" nameend="col3" align="char" char=".">23.8</entry></row><row><entry namest="col1" nameend="col1" align="left">Trp</entry><entry namest="col2" nameend="col2" align="right">4</entry><entry namest="col3" nameend="col3" align="left">ND</entry></row><row><entry namest="col1" nameend="col1" align="left">Tyr</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">10.8</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Val</entry><entry namest="col2" nameend="col2" align="right">19</entry><entry namest="col3" nameend="col3" align="char" char=".">19.0</entry></row></tbody></tgroup><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col3" align="justify">^aND not determined</entry></row></tbody></tgroup></table></tables>