Hybrid plasmid for 38 kDa antigen of M. tuberculosis

The invention concerns a hybrid plasmid for the expression of unfused 38 kDa antigen of M. tuberculosis in E. coli, E. coli with the plasmid, an 38 kDa antigen and a protein of about 33 kDa useful for diagnoses.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to polypeptides and more particularly to the 38 kDa 
antigen of M. tuberculosis. 
2. Brief Description of Related Art 
Tuberculosis is a highly contagious human disease with over 3 million 
deaths and 8 million new cases occurring annually. The advent of AIDS is 
expected to worsen the situation because of reactivation of the dormant M. 
tuberculosis in immunocompromised individuals. The infectious dose in 
tuberculosis is exceedingly low, e. g. one to three tubercle bacilli are 
sufficient to initiate a primary lesion in the lung. The diagnosis of the 
infected individuals plays a vital role in the epidemiology and prevention 
and spread of the disease. Currently, diagnosis rests on the cultivation 
of M. tuberculosis from the sputum which takes about 6 weeks because of 
the slow growth rate of the organism. Another important part of the 
diagnosis is the `Tuberculin-test`. Tuberculin or the purified protein 
derivative (PPD) is a mixture of proteins from heat killed M. tuberculosis 
culture filtrate. This test suffers from high non-specificity due to 
cross-reactions in individuals infected with or vaccinated with other 
mycobacteria. Thus, there is an obvious need for developing defined and 
specific sero-diagnostic and skin-testing reagents for tuberculosis. 
Serological studies have shown that the 38 kDa antigen of M. tuberculosis 
contains immunodominant epitopes specific to the virulent strains of M. 
tuberculosis. This antigen is produced in minor quantities in the vaccine 
strain BCG which is an avirulent derivative of the bovine-tubercle 
bacillus M. bovis. Thus, on the basis of serology, this antigen can be 
used to distinguish between organisms of M. tuberculosis complex and other 
mycobacteria. Purification of the native 38 kDa antigen directly from M. 
tuberculosis is not practicable because of the low yields, slow growth 
rates and the virulent nature of the organism. 
As already stated, Mycobacterium tuberculosis is the causative agent of 
tuberculosis, a widespread human disease claiming about 3 million lives 
each year. Important goals of mycobacterial research are the provision of 
protective immunity against tuberculosis through more effective vaccines, 
and the development of specific skin-test/serodiagnostic reagents. A 
pre-requisite of such goals is the characterization and detailed 
evaluation of the immunological role of individual mycobacterial antigens. 
For this purpose, substantial amounts of such antigens are required. 
Purification of antigens directly from M. tuberculosis is difficult 
because of low cell yields, slow growth rates and the virulent nature of 
the organism (Kadival, G. V., S. D. Chaparas, and D. Hussong. 1987. 
Characterization of serologic and cell-mediated reactivity of a 38 kDa 
antigen isolated from Mycobacterium tuberculosis. J. Immunol. 139: 
2447-2451. Young, D., L. Kent, A. Rees, J. Lamb, and J. Ivanyi. 1986. 
Immunological activity of a 38 kDa-kilodalton protein purified from 
Mycobacterium tuberculosis. Infect. Immun. 54: 177-183). A potential 
solution to this problem is the production of recombinant antigens in 
biotechnologically emanable organisms such as E. coli. 
The immunological and diagnostic relevance of the 38 kDa protein antigen of 
M. tuberculosis has been shown previously (Andersen, A. B., Z.-L. Yuan, K. 
Haslov, B, Vergmann, and J. Bennedsen. 1986. Interspecies reactivity of 
five monoclonal antibodies to Mycobacterium tuberculosis as examined by 
immunoblotting and enzyme-linked immunosorbent assay. J. Clin. Microbiol. 
23: 446-451; Young, D. et al. 1986. supra.). The protein contains species 
specific B-cell epitopes (Anderson, A. B. et al., supra.), and T-cells 
isolated from immunized mice, guinea pigs or humans proliferate when 
cultivated in its presence (Kadival, G. V. et al. 1987. supra.; Worsaae, 
A., L. Ljungqvist, K. Haslov, I. Heron, and J. Bennedsen. 1987. Allergenic 
and blastogenic reactivity of three antigens from Mycobacterium 
tuberculosis in sensitized guinea pigs. Infect. Immmun. 55: 2922-2927; 
Young, D. et al. 1986. supra.). The majority of humans (especially of the 
HLA type DR2), suffering from active tuberculosis develop antibodies 
against the 38 kDa antigen (Bothamley, G. H., J. S. Beck, G. M. T. 
Schreuder, J. D'Amaro, R. R. P. de Vries, T. Kardijito, and J. Ivanyi. 
1989. Association of tuberculosis and Mycobacterium tuberculosis--specific 
antibody level with HLA. J. Infect. Dis. 19: 549-555). 
The 38 kDa protein of the Gram positive bacterium Mycobacterium 
tuberculosis H37Rv is an immunodominant antigen of potential utility for 
diagnosis and vaccine development. Assessment of this potential requires 
large amounts of the purified protein that would be difficult if not 
impossible to obtain from M. tuberculosis itself. 
SUMMARY OF THE INVENTION 
According to one embodiment of the invention, a hybrid plasmid for the 
expression of an unfused 38 kDa antigen of M. tuberculosis in E. coli is 
provided, the plasmid comprising 
the signal sequence of the 38 kDa antigen (pre-protein) and 
a restriction site comprising within its recognition sequence the base 
triplet ATG which codes the first amino acid M in frame. 
According to one specific embodiment of the invention the hybrid plasmid is 
characterized in that the 38 kDa antigen is a protein of M. tuberculosis 
of a wild type or of a variant of M. tuberculosis which likewise causes 
tuberculosis. 
The signal sequence comprises for example 17, 18, 19, 20, 21, 22, 23 or 24 
codons. 
According to a specific embodiment the hybrid plasmid is characterized in 
that the DNA sequence coding the 38 kDa antigen (pre-protein) is inserted 
N-terminally in a NcoI-, NdeI- or SphI restriction sequence of the 
starting vector for example pJLA 603. 
Examples of DNA sequences coding the 38 kDa antigen (pre-protein) are given 
in FIGS. 1A and 1C. 
According to another embodiment the invention is directed to E. coli 
comprising a hybrid plasmid according to the invention. 
According to another embodiment the invention is directed to a 38 kDa 
antigen (pre-protein) of M. tuberculosis which can be produced by means of 
E. coli comprising a hybrid plasmid according to the invention, the signal 
sequence comprising 17 to 24 codons, a signal sequence of 23 codons being 
excluded. 
The 38 kDa antigen (pre-protein) according to the invention can be 
characterized by 22 to 17 amino acids of the following signal sequence 
(SEQ ID NO.1): 
MKIRLHTLLAVLTAAPLLLAAAG 
for example amino acid 1 plus amino acids 8 to 23. 
According to another embodiment the invention is directed to a protein of 
about 33 kDa which can be obtained by means of E. coli as host of pJLA 603 
as expression plasmid 
a DNA sequence coding the 38 kDa antigen of M. tuberculosis (pre-protein) 
being inserted into a NcoI, NdeI or SphI restriction site of pJLA 603, 
the inserted DNA sequence comprising the signal sequence of the antigen, 
the recognition sequence of the restriction site of the base triplett 
comprising ATG, coding the first codon M in frame and 
if wanted, the protein of about 33 kDa being separated from the 
simultaneously expressed 38 kDa antigen. 
According to a specific embodiment of the invention a protein of about 33 
kD is characterized in that it is deleted N-terminally by the signal 
sequence and additional 24 amino acids compared with the 38 kDa antigen of 
M. tuberculosis (pre-protein). 
According to another embodiment the invention concerns a 38 kDa antigen of 
M. tuberculosis and/or a protein of about 33 kDa produced by means of E. 
coli comprising a hybrid plasmid according to the invention and being 
renaturated as follows: 
solubilization of the antigen and/or protein, obtained as inclusion body, 
in guanidine-HCl (if wanted in the presence of a reducing agent) and 
subsequent renaturation by means of cross-linked dextran; SEPHADEX 
chromatography. 
According to a specific embodiment of the invention 
the solubilization is carried out with about 6 M guanidine-HCl and/or in 
the presence of dithiothreithol (DTT) and/or 
the renaturation is carried out by means of sephadex G-25. 
According to the invention recombinant plasmids have been constructed which 
produce high levels of the 38 kDa antigen of M. tuberculosis in 
Escherichia coli. With the inventive recombinant constructions, large 
quantities of the unfused (unique) 38 kDa protein can be produced in E. 
coli mostly in the form of inclusion bodies. According to the invention a 
method for the isolation and purification of the recombinant antigen and 
inclusion bodies has also been developed. The purified 38 kDa antigen 
thereby prepared is immunologically indistinguishable from the native 38 
kDa antigen of M. tuberculosis. Due to its high specificity to M. 
tuberculosis, this recombinant antigen may be useful for the serological 
diagnosis of tuberculosis worldwide. The antigen is also a potential 
candidate for vaccines against tuberculosis because it contains 
immunodominant T-cell epitopes. 
The gene coding for the 38 kDa antigen had been previously cloned and in 
the present study was expressed as an unfused protein in Escherichia coli 
under the control of strong transcriptional (bacteriophage lambda P.sub.R 
P.sub.L) and translational (atpE) signals. Fermentation of the recombinant 
E. coli K-12 strain CAG629 (pMS9-2), which is deficient in on protease and 
the heat shock response, produced the recombinant antigen at high levels 
(about 10% of total cellular protein). The recombinant antigen, which 
accumulated as inclusion bodies, was completely solubilized in 6 M 
guanidine-HCl, refolded and purified to apparent homogeneity. The product 
showed the expected amino acid composition and molecular weight, and as 
strong reactivities with three different monoclonal antibodies as the 
native protein. Polyclonal antibodies raised against the recombinant 
antigen reacted strongly with the native antigen in enzyme-linked 
immunosorbent assay. These results demonstrated that recombinant 38 kDa 
antigen which cannot be distinguished antigenically from the native 
protein of M. tuberculosis can be prepared in quantity from E. coli.

A DETAILED DESCRIPTION OF THE INVENTION FOLLOWS 
MATERIAL AND METHODS 
Bacterial Strains, Phage, Plasmids and Growth Conditions 
The E. coli strains used in this study were TG-1 (lac-pro, supE, thi, 
hsdD5/F'traD36, proA.sup.+ B.sup.+, lacI.sup.q, lacZ M15) DH5alpha(endA1, 
recA1, hsdR17, supE44, thi-1, gyrA96, relA1, (lacZYA-argF), 80d/lacZ M15), 
EC 538 and CAG629 (lon, htpR165-Tn10; C. Gross). The recombinant lambda 
gt11 bacteriophage clone AA59 was isolated in a previous study (Andersen, 
A. B., A. Worsaae, and S. D. Chaparas. 1988. Isolation and 
characterization of recombinant lambda gt11 bacteriophages expressing 
eight different mycobacterial antigens of potential immunological 
relevance. Infect. Immun. 56: 1344-1351) from an M. tuberculosis genomic 
DNA library constructed by R. A. Young (Young, R. A., B. R. Bloom, C. M. 
Grosskinsky, J. Ivanyi, D. D. Thomas, and R. W. Davis. 1985. Dissection of 
Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. 
Acad. Sci. USA 82: 2583-2587). 
Unless otherwise stated, the strains were grown in Luria-Bertani broth 
(Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a 
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y.) at 37.degree. C. Liquid cultures were aerated by shaking at 160 
r.p.m. in a Pilot-Shake shaker (Kuhne, Switzerland). 
DNA Manipulations 
Preparation and handling of DNA was according to standard protocols 
(Maniates, T., supra.). Transformation was performed as described by 
Hanahan (Hanahan, D. 1983. Studies on transformation of Escherichia coli 
with plasmids. J. Mol. Biol. 166: 557-580). DNA sequencing was done by the 
dideoxynucleotide chain termination method (Sanger, F., S. Nicklen, and A. 
R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. 
Natl. Acad. Sci. USA 74: 5463-5467). Oligonucleotides were synthesized 
using an Applied Biosystems model 380B DNA synthesizer and purified with 
OPC columns (Applied Biosystems Inc.). 
Oligonucleotide Mutagenesis 
The 2.0 kb EcoRI fragment from the genomic clone lambda AA59 (Anderson, A. 
B. et al., 1988, supra) was transferred into M13mp19. Preparation of 
single stranded DNA and oligonucleotide mutagenesis was carried out using 
the Amersham kit (RPN 1523). The DNA sequence of the template before and 
after mutagenesis was confirmed by DNA sequencing (Sanger, F., supra.). 
Small-Scale Preparation of Crude Protein Extracts 
Strains were grown in LB medium containing ampicillin (100 .mu.g/ml) at 
30.degree. C. to an absorbance at 580 nm of 0.6. Cultures were induced by 
shifting to 42.degree. C. for 3 h in a shaking water bath. Bacteria were 
harvested from 1 ml culture, suspended in 100 .mu.l of sample buffer (62 
mM Tris-HCL, pH 6.8; 2% sodium dodecyl sulphate; 0.7 M 2.mercaptoethanol; 
10% glycerol; 0.002% bromophenol blue) and broken by sonication in ice 
(3.times.30 sec; 50 W) using a Braun Labsonic 2000. Samples were heated at 
95.degree. C. for 10 min and 10 .mu.l were analysed by polyacrylamide gel 
electrophoresis. 
Cultivation in a Bioreactor 
30 l of a modified concentrated LB medium (tryptone, 40 g/l; yeast extract, 
20 g/l; NaCl, 5 g/l) were sterilized in a 50 l bioreacter (Biostat U30D, 
Braun Melsungen, FRG) in the presence of 3.5 ml Ucolub 
N38(polyalkylene-glycol) antifoam (Brenntag, Mulheim, FRG). Cultivation 
was initiated by inoculation with a 0.5 l overnight culture of the 
organism in the same medium to give an initial A.sub.546 =0.04. Stirrer 
speed was maintained constant at 300 rpm, which ensured a dissolved oxygen 
concentration close to saturation, and pH was held at 6.9. After 4 hours 
of fermentation (A.sub.546 =0.4) the temperature was shifted from 
30.degree. C. to 42.degree. C., and maintained at this temperature for a 
further 4 hours. At the end of the induction period the broth was 
concentrated in a closed system by crossflow microfiltration (Enka module 
type A7 ABA 3A) with 0.23 m.sup.2 Accural membrane (0.2 .mu.m pore 
diameter; Enka, Wuppertal, FRG) until a final volume of 10 l was obtained. 
Protein Purification 
Cells obtained from the bioreactor were disrupted by single pass through a 
high pressure homogenizer LAB 60/500/2 (A. P. V. -Schroder, Lubeck, FRG) 
at 500 bar with a flow rate of 60 l/h. Inclusion bodies were crudely 
separated using a centrifugal separator SA 1-01-175 (Westfalia, Oelde, 
FRG). This device allows isolation of inclusion bodies from the broth at a 
flow rate of 15-20 l/h. The inclusion bodies were washed twice with 200 ml 
of buffer L (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) containing 2% TRITON 
X-100, supra. Washed pellets were resuspended in 3 l of buffer L 
containing 6M guanidine-HCl, 20 mM DTT for 16 hrs at 4.degree. C. with 
slow stirring. After centrifugation at 7000 g, the supernatant fluid was 
passed through a Sephadex G-25 gel filteration column (10.times.90 cm) 
equilibrated with 10 mM Tris-HCl buffer, pH 7.0, containing 100 mM sodium 
chloride in order to remove the guanidine-HCl and effect renaturing of the 
recombinant antigen. The solution was applied in 2 l aliquots with a flow 
rate of 7 l/h and the eluate was monitored for O.D.sub.260 and 
conductivity. The desalted and renatured antigen solution was recovered in 
starting buffer with a conductivity of 8 mS cm.sup.-1, well separated from 
the salt peak containing mainly guanidine-HCl. The antigen peaks were 
combined and diluted with distilled water to obtain a conductivity of 5 mS 
cm.sup.-1 and the pH adjusted to 8.5 with 1M Tris. The solution was 
divided into two parts for the following purification step. 
One part of the solution was applied to an FPLC-column (QAE-SEPHAROSE, 
supra; 5.times.18 cm) equilibrated with 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 the starting buffer, elution of the antigen 
was performed by application of a step gradient consisting of 50 mM, 100 
mM, 250 mM, 500 mM and 1M sodium chloride in starting buffer. Afterwards 
the column was re-equilibrated with starting buffer, the second portion 
from the gel filtration column was applied and eluted as described before. 
The antigen-containing fractions from the two QAE-SEPHAROSE, supra runs 
were pooled, concentrated and diafiltered (2 mS cm.sup.-1) by 
ultrafiltration using an Amicon Hollowfiber Cartridge (type H1P10; cutoff 
10.000). 
Around 20 mg of the protein obtained was applied to a Mono Q HR (5/5) 
FPLC-column equilibrated with 20 mM Tris-HCl, pH 8.0. The column was 
washed with starting buffer at a flow rate of 2 ml/min and at a pressure 
of 25 bar. Thereafter, the antigen was eluted by gradually increasing the 
sodium chloride concentration to 0.5 M in starting buffer. Altogether 
three Mono Q HR (5/5) runs were carried out under the conditions described 
above. 
Polyacrylamide Gel Electrophoresis and Immunoblotting 
The crude and purified proteins were analyzed by sodium lauryl 
sulfate/polyacrylamide (12%) gel electrophoresis (Laemmli, U. K. 1970. 
Cleavage of structural proteins during the assembly of the head of 
bacteriophage T4. Nature (London) 227: 680-683). Protein samples were 
mixed 1:1 with 2.times. sample buffer and heated at 95.degree. C. for 10 
min before loading on the gels. After electrophoresis the polypeptides 
were visualized by silver staining (Damerval, C., M. le Guilloux, J. 
Blaisonneau, and D. de Vienne. 1987. A simplification of Heukeshoven and 
Dernick's silver staining of proteins. Electrophoresis. 8: 158-159). 
Protein concentrations were determined by the method of Lowry et al. 
(Lowry, O. H., A. L. Farr, N. J. Rosebrough, and R. Randall. 1951. Protein 
measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275). 
Proteins were transferred to a nitrocellulose membrane (BioRad) with a 
home 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 in TBS (50 mM Tris-HCl; 200 mM NaCl, pH 7.5) containing a 10% 
solution of milk (0.3% fat). The primary antibodies (mouse monoclonal 
antibodies) were diluted 1000-fold in TBS and incubated with the filters. 
overnight at 4.degree. C. The filters were washed 3 times with TBS and 
immunodetection was carried out with a biotinylated anti-mouse IgG and 
streptavidin-alkaline phosphatase conjugate (BRL, Gaithersburg, Md., USA). 
For the immunodot-blot assay, protein samples were filtered through a 
nitrocellulose membrane using a BioRad bio-dot apparatus and processed 
further as described above. Monoclonal antibodies HAT2, HBT12, HYT28 have 
been described earlier (Andersen, A. B., et al. 1986. supra; Ljungqvist, 
L., A. Worsaae, and I. Heron. 1988. Antibody responses against 
Mycobacterium tuberculosis in 11 strains of inbred mice: noval monoclonal 
antibody specifities generated by fusions, using spleens from BALB.B10 and 
CBA/J mice. Infect. Immun. 56: 1994-1998; Schou, C., Z.-L. Yuan, A. B. 
Andersen, and J. Bennedsen. 1985. Production and partial characterization 
of monoclonal hybridoma antibodies to Mycobacterium tuberculosis. Acta 
Pathol. Microbiol. Immunol. Scand. Sect. C 93: 265-272). Densitometric 
measurements of silver stained gels and western blots were done on a laser 
densitometer (LKB). 
Amino Acid Analyses 
Amino acid analyses were performed on a Biotronik LC-5001 amino acid 
analyzer (Maintal, Germany) after hydrolysis of the protein sample in 6N 
HCl containing 0.1 % phenol for 24 h at 105.degree. C. 
Production of Polyclonal Anti-38 kDa Protein Sera 
Rabbits were immunized subcutaneously with either affinity purified 38 kDa 
protein (Worsaae, A., L. Ljungqvist, and I. Heron. 1988. Monoclonal 
antibodies produced in BALB.B10 mice define new antigenic determinants in 
culture filtrate preparations of Mycobacterium tuberculosis. Infect. 
Immun. 56: 2608-2614) or with recombinant 38 kDa protein (Preperation I). 
The antigens (10 .mu.g per dose) were adsorbed to aluminium hydroxide (2.4 
mg) and subsequently mixed with 1 ml of Freund's incomplete adjuvant. The 
rabbits were immunized three times with intervals of two weeks. The blood 
was drawn ten days after the last immunization and the IgG fraction was 
purified as described by Harboe and Inglid (Harboe, N., and A. Inglid. 
1983. Immunization, isolation of immunoglobulins and antibody titre 
determination. Scand. J. Immunol. 17S10: 345-351). 
Electron Microscopy 
The cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde in PBS 
(50 mM K-phosphate, 0.9% NaCl, pH 6.9) for 1 h on ice. After several 
washing steps in PBS, cells were embedded following the progressive 
lowering of temperature (PLT) method (17). Cells were dehydrated with 10%, 
then 30% ethanol for 30 min on ice, then with 50% ethanol for 30 min at 
-20.degree. C., and 70%, 90%, 100% ethanol for 30 min each at -35.degree. 
C., and with 100% ethanol for 1 h at -35.degree. C. Infiltration with the 
Lowicryl resin K4M was done as follows: 1 part ethanol/1 part K4M resin 
for overnight at -35.degree. C., 1 part ethanol/2 parts K4M resin for 12 h 
and pure K4M resin for 2 days at -35.degree. C. with several changes of 
the resin mixture. Polymerisation of the resin was achieved with UV-light 
(366 nm) for 1 day at -35.degree. C., and at room temperature for another 
2 days. Ultrathin sections were poststained with uranyl acetate and lead 
citrate before they were examined with a Zeiss EM 10B transmission 
electron microscope at an acceleration voltage of 80 kV. 
RESULTS 
Construction of expression plasmids. The DNA sequence of the 38 kDa gene 
revealed an open reading frame encoding a polypeptide of 374 amino acids 
and containing GTG as the initiation codon (Andersen, A. B., and E. B. 
Hansen. 1989. Structure and mapping of antigenic domains of protein 
antigen b, a 38,000-molecular-weight protein of Mycobacterium 
tuberculosis. Infect. Immun. 57: 2481-2488). There is also a 24 amino acid 
long signal sequence (FIG. 1A) showing similarity to those of bacterial 
lipoproteins. In the present study, the gene was manipulated so that high 
level expression of the unfused 38 kDa antigen could be achieved in the 
pJLA603 expression vector (8; FIG. 2). For cloning and expression in these 
vectors, it is required that the foreign gene contains a restriction site, 
e.g., NcoI, NdeI, SphI, which includes an in-frame ATG within its 
recognition sequence. Since there is no such site around the initiation 
codon of the 38 kDa gene, oligonucleotide mutagenesis in M13mp19 was 
carried out to create an NdeI site at the N-terminus and to change the 
initiation codon from GTG to ATG (FIG. 1 and 2). The 1.2 kb NdeI-SphI 
fragment that could be excised from the M13 derivative after mutagenesis 
was then cloned between the NdeI-SphI sites of pJLA603. The recombinant 
plasmid, which was designed to express the 38 kDa protein with its 
original signal peptide intact, was designated pMS9-2. In the same way, 
another recombinant plasmid pMS10-4 containing a deletion of the first 6 
amino acids in the signal sequence was constructed (FIG. 1C). Computer 
analysis of the translation initiation region of pMS9-2-specified mRNA 
predicted a loose secondary structure (FIG. 1B) which should be highly 
suitable for high level expression in E. coli (McCarthy, J. E. G., and C. 
Bokelmann. 1988. Determinants of translational initiation efficiency in 
the atp operon of Escherichia coli. Molec. Microbiol. 2: 455-465). On the 
other hand pMS10-4 showed a more stable secondary structure (FIG. 1D) 
indicating that the expression from this plasmid might not be as good as 
from pMS9-2. Both recombinant plasmids were used for expression studies. 
Expression of the Recombinant Antigen in Small-Scale Culture 
Several E. coli strains (DH5.zeta.; EC538 and CAG629) were tested for 
expression of the 38 kDa recombinant antigen encoded by pMS9-2 and 
pMS10-4. The lon, htpR strain CAG629 showed the strongest expression. 
Extracts of CAG629(pMS9-2) cells induced at 42.degree. C. contained 
substantial amounts of the recombinant protein (FIG. 3, lane 3, and 4) 
whereas it was absent in extracts from uninduced cells (FIG. 3, lane 1). 
The recombinant strain exhibited no obvious perturbations after induction 
and continued to grow exponentially (data not shown). Immunoblotting with 
MAbs HBT12 (FIG. 3A, lanes 10, and 11), HAT2 and HYT28 (data not shown) 
yielded positive reactions with the recombinant 38 kDa protein. Most of 
the recombinant protein was present in the cell pellet fraction of 
disrupted cells and only a small fraction was detected in the supernatant 
fluid (FIG. 3A, lanes 10, 11). The recombinant clone CAG629(pMS10-4), as 
expected from the secondary structure prediction, produced considerably 
less protein than pMS9-2 (FIG. 3A, lanes 13, 14). The faint bands seen on 
the immunoblots which run slower than the 38 kDa protein correspond to 
SDS-insoluble, aggregated forms of the recombinant protein. 
The recombinant 38 kDa protein was produced at high levels (about 10% of 
the total cellular protein) as measured by laser densitometry of the 
silver stained SDS-PAGE gels. As is often the case with recombinant 
proteins produced at high levels in E. coli (Halenbeck, R., E. Kawasaki, 
J. Wrin, and K. Koths. 1989. Renaturation and purification of biologically 
active recombinant human macrophage colony-stimulating factor expressed in 
Escherichia coli. Biotechnology 7: 710-715; Sarmientos, P., M. Duchesne, 
P. Denefle, J. Boiziau, N. Fromage, N. Delaporte, F. Parker, Y. Lelievre, 
J-F. Mayaux, and T. Cartwright. 1989. Synthesis and purification of active 
human tissue plasminogen activator from Escherichia coli. Biotechnology 7: 
495-501), the 38 kDa antigen was mostly contained in cytoplasmic 
aggregates or inclusion bodies (FIG. 4). 
Fermentation of Recombinant E. coli and Purification of the Recombinant 
Antigen 
Recombinant clone CAG629(pMS9-2) was chosen for a 30 L fermentation because 
it produced and tolerated the antigen at high levels in batch cultures, 
and coded for the 38 kDa protein with an intact signal sequence. The time 
course of production of the recombinant antigen in the bioreactor was 
monitored (FIG. 3B). SDS-PAGE of the whole cell extracts showed that 
within 30 min after increasing the temperature from 30.degree. C. to 
42.degree. C., a double band of 38 kDa size was clearly visible on silver 
stained gels. 
Washing of the inclusion bodies obtained from the fermentor culture with 
buffer containing TRITON X-100 resulted in the removal of some of the 
contaminating proteins (FIG. 5, lane 3) without any apparent loss of the 
recombinant antigen (FIG. 5, lane 2). Using a SEPHADEX G-25, supra column 
the antigen was desalted and renatured (FIG. 5, lanes 6); we did not 
observe any reaggregation of the antigen at this stage. Further 
purification of the antigen was obtained by multiple rounds of 
FPLC-anion-exchange chromatograpy (FIG. 6) where the antigen was found to 
elute at 100 mM NaCl (Preparation I) and between 130-200 mM NaCl 
(Preparation III). Preparation II shown in FIG. 6 represents the antigen 
purified on FPLC-anion-exchange column in presence of TRITON X-100 from 
the aggregated and contaminated fractions obtained from earlier 
anion-exchange chromatographic steps. 
Structure, immunological reaction and immunogenicity of purified 
recombinant protein. The immunological reactions of the purified antigen 
preparations were tested with monoclonal antibodies HAT2, HBT12 and HYT28. 
All the three antibodies reacted with recombinant antigen preparations 
(FIG. 6). When the affinity purified native antigen was compared with the 
recombinant antigen (Preparation I) by SDS-PAGE and immunoblotting, no 
difference was observed (FIG. 7A). The silver stained gel and the 
immunoblot shown in FIG. 7A were also traced with a laser densitometer 
and, after normalizing for differences in the amounts of protein present, 
the native and the recombinant antigen were seen to have given identical 
reactions with the monoclonal antibodies HBT12 (FIG. 7B), HAT2 and HYT28 
(data not shown). 
The immunogenicity of the recombinant antigen was also tested by raising 
polyclonal antisera in rabbits against the native and the recombinant 
antigen under similar conditions. The two sera were then assayed by enzyme 
linked immunosorbent assay (ELISA) against both recombinant (FIG. 8A) and 
native antigens (FIG. 8B). The slopes of the curves are identical showing 
that the two sera bind native and the recombinant 38 kDa protein equally 
well. 
The amino acid composition of the 38 kDa recombinant protein closely 
resembles the amino acid composition derived from the nucleotide sequence 
(Table. 1) 
About 33 kDa Protein 
The deletion in comparison to the 38 kDa antigen has completely removed the 
signal sequence. Still, the truncated protein reacts strongly to the 
monoclonal antibodies HAT2, HBT12 and HYT28 showing that the three 
epitopes are intact. The truncated protein also shows strong reaction in 
ELISA and western blotting to sera from mice infected with Mycobacterium 
tuberculosis. We have found that about 85% of the sera from Tuberculosis 
patients reacted positively with the truncated protein and antigen, 
respectively, showing that the truncated antigen can be effectively used 
for the diagnosis. 
DISCUSSION 
A strategy was developed and implemented to clone a Mycobacterium 
tuberculosis DNA fragment in expression vector such that unfused 38 kDa 
protein would be produced at high levels. The vector contained the lambda 
P.sub.R P.sub.L promoter and the efficient translation initiation region 
of the atpE gene which resulted in expression of the heterologous 38 kDa 
antigen gene in E. coli to a level representing 10% of the total cellular 
protein. About 15 mg recombinant protein/liter was produced under the 
conditions given. It should be emphasized, however, that the objective of 
the present work was to determine whether hyperproduced recombinant 
antigen could be recovered in an antigenic form immunologically 
indistinguishable from native antigen obtained from Mycobacterium 
tuberculosis, and not to optimize fermentation yields. 
Most of the recombinant 38 kDa protein accumulated as inclusion bodies. 6M 
guanidine-HCl in the presence of a reducing agent (DTT) at high 
concentration was found to be suitable for the solubilization of the 
inclusion bodies. Renaturation of inclusion body proteins, specially 
hydrophobic, membrane proteins, is often difficult and needs careful 
optimization of experimental conditions. In our case, the usual procedure 
of dialysis or step-dialysis resulted in the precipitation of the 
recombinant 38 kDa protein even at very low protein concentrations (0.05 
mg/ml). Renaturation of the recombinant protein on SEPHADEX G-25, supra 
proved on the other hand to be effective and no significant reaggregation 
of the protein was observed. Starting with inclusion bodies containing 
about 200 mg total protein, we were able to purify 19 mg of the 38 kDa 
antigen with more than 95% purity as judged by silver staining of 
SDS-polyacrylamide gels. 
The 38 kDa antigen of M. tuberculosis is most probably a lipoprotein (D. B. 
Young and T. R. Garbe, Res. Microbiol. 142: -, 1991, in press). 
Lipoproteins show aberrant, diffuse bands on SDS-PAGE gels (Pugsley, A. 
P., C. Chapon, and M. Schwartz. 1986. Extracellular pullulanase of 
Klebsiella pneumoniae is a lipoprotein. J. Bacteriol. 166: 1083-1088; 
Schouls, L. M., R. Mount, J. Dekkert, and J. D. A. van Embden. 1989. 
Characterization of lipid-modified immunogenic proteins of Treponema 
palidum expressed in Escherichia coli. Microbial. Pathogen. 7: 175-188). 
As is true of other lipoproteins, we observed a strong tendency of the 
recombinant 38 kDa antigen to aggregate during anion-exchange 
chromatography and during concentration by ultrafiltration. Part of the 
antigen which aggregated during ultrafiltration could be recovered by 
solubilization in 2% TRITON X-100, supra followed by FPLC on MonoQ. 
The native 38 kDa protein runs as a doublet on SDS-polyacrylamide gels. The 
reason for this is not known but it might be due to acylation and 
processing of the pre-protein. We did not remove the signal sequence 
before producing the antigen in large amounts because the presence of the 
lipoyl moities on the N-terminal cystein may play important role in the 
immunogenicity of protein antigens (Deres, K., H. Schild, K-H-Wiesmuller, 
G. Jung, and H-G. Ramensee. 1989. In vivo priming of virus-specific 
cytotoxic lymphocytes with synthetic lipopeptide vaccine. Nature. 342: 
561-564). The recombinant protein purified in this study also showed the 
doublet characteristic. Preparation I contains mostly the upper band and 
only minor amounts of the lower band. Preparation II contains both the 
upper and the lower bands in almost equal amounts. Preparation III 
represents a proteolytically truncated derivative (34 kDa) of the 38 kDa 
antigen. 
The native 38 kDa protein isolated from the culture supernatant of M. 
tuberculosis and the purified recombinant proteins present in preparations 
I and II are of the same size and show the doublet character on SDS-PAGE. 
Furthermore, the purified proteins and the native antigen showed identical 
reaction on immunoblots with monoclonal antibodies HAT2, HBT12, and HBT28. 
Polyclonal serum raised against the recombinant protein (Preparation I) 
recognized the native antigen and showed the same reaction in ELISA as the 
serum raised against the native antigen. Similar results were obtained 
when the two sera were tested against the recombinant antigen by ELISA 
demonstrating that the recombinant protein posseses similar epitopes and 
is as immunogenic as the native 38 kDa protein purified from M. 
tuberculosis. 
In conclusion, we have developed an expression system and production and 
purification procedure for the 38 kDa protein of M. tuberculosis in E. 
coli that enable the ready isolation of significant quantities of the 
antigen. The recombinant antigen is immunologically indistinguishable from 
the native antigen. These results should significantly accelerate 
assessment of the utility of the antigen for diagnosis and vaccine 
development. 
______________________________________ 
Living Material 
______________________________________ 
Lambda-AA59: 
genomic clone for 2.0 kb EcoRI fragment 
Supply: Literature (3); DSM 6524 
M13mp19: Phagen for transferring a 2.0 kb EcoRI fragment 
Supply: Pharmacia 
pJLA603: Vector for inserting a NdeI-SphI fragment of 
M13mp19 after transferring the 2.0 kb EcoRI 
fragment and mutagenization 
Supply: Literature (20); Medac (Hamburg) 
______________________________________ 
TABLE 1 
______________________________________ 
Amino acid composition of purified 38kDa antigen 
no. of residues 
Deduced from 
Amino acid 
Amino acid DNA sequence 
analysis 
______________________________________ 
Ala 55 56.8 
Arg 5 6.9 
Asn 18 39.3 
Asp 16 
Cys 3 ND.sup.a 
Gln 19 32.8 
Glu 10 
Gly 43 41.2 
His 7 7.6 
Ile 19 17.8 
Leu 36 32.4 
Lys 12 12.5 
Met 6 0.7 
Phe 13 13.0 
Pro 26 23.4 
Ser 26 22.3 
Thr 27 23.8 
Trp 4 ND 
Tyr 10 10.8 
Val 19 19.0 
______________________________________ 
.sup.a ND Not determined 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 1 
- (2) INFORMATION FOR SEQ ID NO: 1: 
- (i) SEQUENCE CHARACTERISTICS: 
# 78 base pairsH: 
# nucleic acid 
# single (C) STRANDEDNESS: 
# linearD) TOPOLOGY: 
#antigeni) MOLECULE TYPE: 
- (iii) HYPOTHETICAL: 
# no (iv) ANTI-SENSE: 
- (v) FRAGMENT TYPE: 
- (vi) ORIGINAL SOURCE: 
# M. TuberculosisM: 
(B) STRAIN: 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: 
(G) CELL TYPE: 
(H) CELL LINE: 
(I) ORGANELLE: 
- (vii) IMMEDIATE SOURCE: 
(B) CLONE: 
#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
# 50TTTGCA TACGCTGTTG GCCGTGTTGA CCGCTGCGCC 
# 73GCGG GCT 
__________________________________________________________________________