Patent Application
20070003569
1. Field of the Invention
The invention relates to recombinant polypeptides and peptides, which can be used for the diagnosis of tuberculosis. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against tuberculosis.
It also relates to nucleic acids coding for said polypeptides and peptides.
Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against tuberculosis.
By “recombinant polypeptides or peptides” it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression “recombinant polypeptides” such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
The term “recombinant” indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host.
Nevertheless, it must be understood that this expression does not exclude the possibility for the polypeptide to be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules.
The expression “biologically pure” or “biological purity” means on the one hand a grade of purity such that the recombinant polypeptide can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants.
2. Description of the Prior Art
Tuberculosis remains a major disease in developing countries. The situation is dramatic in some countries, particularly where high incidence of tuberculosis among AIDS patients represents a new source of dissemination of the disease.
Tuberculosis is a chronic infectious disease in which cell-mediated immune mechanisms play an essential role both for protection against and control of the disease.
Despite BCG vaccination, and some effective drugs, tuberculosis remains a major global problem. Skin testing with tuberculin PPD (protein-purified derivative) largely used for screening of the disease is poorly specific, due to cross reactivity with other pathogenic or environmental saprophytic mycobacteria.
Moreover, tuberculin PPD when used in serological tests (ELISA) does not allow to discriminate between patients who have been vaccinated by BCG, or those who have been primo-infected, from those who are developing evolutive tuberculosis and for whom an early and rapid diagnosis would be necessary.
A protein with a molecular weight of 32-kDa has been purified (9) from zinc deficient Mycobacterium bovis BCG culture filtrate (8). This 32-kDa protein of M. bovis BCG has been purified from Sauton zinc deficient culture filtrate of M. bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation has been found to be homogeneous as based on several analyses. This P32 protein is a constituent of BCG cells grown in normal conditions. It represents about 3% of the soluble fraction of a cellular extract, and appears as the major protein released in normal Sauton culture filtrate. This protein has been found to have a molecular weight of 32 000 by SDS-polyacrylamide gel electrophoresis and by molecular sieving.
The NH2-terminal amino acid sequence of the 32-kDa protein of M. bovis BCG (Phe-Ser-Arg-Pro-Gly-Leu (SEQ ID NO:49)) is identical to that reported for the MPB 59 protein purified from M. bovis BCG substrain Tokyo (34).
Purified P32 of M. bovis BCG has been tested by various cross immunoelectrophoresis techniques, and has been shown to belong to the antigen 85 complex in the reference system for BCG antigens. It has been more precisely identified as antigen 85A in the Closs [[closs]] reference system for BCG antigens (7).
Increased levels of immunoglobulin G antibodies towards the 32-kDa protein of M. bovis BCG could be detected in 70% of tuberculous patients (30).
Furthermore, the 32-kDa protein of M. bovis BCG induces specific lymphoproliferation and interferon-(IFN-Γ) production in peripheral blood leucocytes from patients with active tuberculosis (12) and PPD-positive healthy subjects. Recent findings indicate that the amount of 32-kDa protein of M. bovis BOG-induced IFN-Γ. in BCG-sensitized mouse spleen cells is under probable H-2 control (13). Finally, the high affinity of mycobacteria for fibronectin is related to proteins of the BCG 85 antigen complex (1).
Matsuo et al. (17) recently cloned the gene encoding the antigen α a major protein secreted by BCG (substrain Tokyo) and highly homologous to MPB 59 antigen in its NH2-terminal amino acid sequence, and even identical for its first 6 amino acids: Phe-Ser-Arg-Pro-Gly-Leu (SEQ ID NO:49).
This gene was cloned by using a nucleotide probe homologous to the N-terminal amino acid sequence of antigen α, purified from M. tuberculosis as described in Tasaka, H. et al., 1983. “Purification and antigenic specificity of alpha protein (Yoneda and Fukui) from Mycobacterium tuberculosis and Mycobacterium intracellulare. Hiroshima J. Med. Sci. 32, 1-8.
The presence of antigens of around 30-32-kDa, named antigen 85 complex, has been revealed from electrophoretic patterns of proteins originating from culture media of mycobacteria, such as Mycobacterium tuberculosis. By immunoblotting techniques, it has been shown that these antigens cross-react with rabbit sera raised against the 32-kDa protein of BCG (8).
A recent study reported on the preferential humoral response to a 30-kDa and 31-kDa antigen in lepromatous leprosy patients, and to a 32-kDa antigen in tuberculoid leprosy patients (24).
It has also been found that fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major (FN)-binding molecule. In 21-day-old supernatants, FN was bound to a double protein band of around 30 to 32-kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa) (1).
In other experiments, recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M. tuberculosis. Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) (“Expression of Proteins of Mycobacterium tuberculosis in Escherichia coli and Potential of Recombinant Genes and Proteins for Development of Diagnostic Reagents”, Mitchell L Cohen et al., Journal of Clinical Microbiology, July 1987, p. 1176-1180).
Concerning the various results known to date, the physico-chemical characteristics of the antigen P32 of Mycobacterium tuberculosis are not precise and, furthermore, insufficient to enable its unambiguous identifiability, as well as the characterization of its structural and functional elements.
Moreover, the pathogenicity and the potentially infectious property of M. tuberculosis has hampered research enabling to identify, purify and characterize the constituents as well as the secretion products of this bacteria.
An aspect of the invention is to provide recombinant polypeptides which can be used as purified antigens for the detection and control of tuberculosis.
Another aspect of the invention is to provide nucleic acids coding for the peptidic chains of biologically pure recombinant polypeptides which enable their preparation on a large scale.
Another aspect of the invention is to provide antigens which can be used in serological tests as an in vitro rapid diagnostic of tuberculosis.
Another aspect of the invention is to provide a rapid in vitro diagnostic means for tuberculosis, enabling it to discriminate between patients suffering from an evolutive tuberculosis from those who have been vaccinated against BCG or who have been primo-infected.
Another aspect of the invention is to provide nucleic probes which can be used as in vitro diagnostic reagent for tuberculosis, as well as in vitro diagnostic reagent for identifying M. tuberculosis from other strains of mycobacteria.
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
On
X represents G or GG,
Y represents C or CC,
Z represents C or G,
W represents C or G and is different from Z,
K represents C or CG,
L represents G or CC,
a1-b1 represents ALA-ARG or GLY-ALA-ALA,
a2 represents arg or gly,
a3-b3-c3-d3-e3-f3-represents his-trp-val-pro-arg-pro or ala-leu-gly-ala,
a4 represents pro or pro-asn-thr,
a5 represents pro or ala-pro.
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
The recombinant polypeptides of the invention contain in their polypeptidic chain one at least of the following amino acid sequences:
Advantageous polypeptides of the invention are characterized by the fact that they react with rabbit polyclonal antiserum raised against the protein of 32-kDa of M. bovis BCG culture filtrate, hereafter designated by “P32 protein of BCG”.
Advantageous polypeptides of the invention are characterized by the fact that they selectively react with human sera from tuberculous patients and particularly patients developing an evolutive tuberculosis at an early stage.
Hereafter is given, in a non limitative way a process for preparing rabbit polyclonal antiserum raised against the P32 protein of BCG and a test for giving evidence of the reaction between the polypeptides of the invention and said rabbit polyclonal antiserum raised against the P32 protein of BCG.
1) Process for Preparing Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG:
Purified P32 protein of BCG from culture filtrate is used.
a) Purification of Protein P32 of BCG:
P32 protein can be purified as follows:
The bacterial strains used are M. bovis BCG substrains 1173P2 (Pasteur Institute, Paris) and GL2 (Pasteur Institute, Brussels).
The culture of bacteria is obtained as follows:
Mycobacterium bovis BCG is grown as a pellicle on Sauton medium containing 4 g Aspargine, 57 ml 99% Glycerine (or 60 ml 87% Glycerine), 2 g Citric Acid, 0.5 g K2HPO4, 0.5 g MgSO4, 0.05 g Iron Citrate, 5×10−6 M Ammonium (17% Fe III) SO4Zn-7H2O and adjusted to 1 liter distilled water adjusted to pH 7.2 with NH4OH, at 37.5° C. for 14 days. As the medium is prepared with distilled water, zinc sulfate is added to the final concentration of 5 .μM (normal Sauton medium) (De Bruyn J., Weckx M., Beumer-Jochmans M.-P. Effect of zinc deficiency on Mycobacterium tuberculosis var. bovis (BCG). J. Gen. Microbiol. 1981; 124:353-7). When zinc deficient medium was needed, zinc sulfate is omitted.
The filtrates from zinc deficient cultures are obtained as follows:
The culture medium is clarified by decantation. The remaining bacteria are removed by filtration through Millipak 100 filter unit (Millipore Corp., Bedford, Mass.). When used for purification, the filtrate is adjusted to 20 mM in phosphate, 450 mM in NaCl, 1 mM in EDTA, and the pH is brought to 7.3 with 5 M HCl before sterile filtration.
The protein analysis is carried out by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done on 13% (w/v) acrylamide-containing gels as described by Laemmli UK. (Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5). The gels are stained with Coomassie Brilliant Blue R-250 and for quantitative analysis, scanned at 595 nm with a DU8 Beckman spectrophotometer. For control of purity the gel is revealed with silver stain (Biorad Laboratories, Richmond, Calif.).
The purification step of P32 is carried out as follows:
Except for hydrophobic chromatography on Phenyl-Sepharose, all buffers contain Tween 80 (0.005% final concentration). The pH is adjusted to 7.3 before sterilization. All purification steps are carried out at +4° C. Elutions are followed by recording the absorbance at 280 nm. The fractions containing proteins are analysed by SDS-PAGE.
(i) The treated filtrate from a 4 liters zinc-deficient culture, usually containing 125 to 150 mg protein per liter, is applied to a column (5.0 by 5.0 cm) of Phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden), which is previously equilibrated with 20 mM phosphate buffer (PB) containing 0.45 M NaCl and 1 mM EDTA, at a flow rate of 800 ml per hour. The gel is then washed with one column volume of the same buffer to remove unfixed material and successively with 300 ml of 20 mM and 4 mM PB and 10% ethanol (v/v). The P32 appears in the fraction eluted with 10% ethanol.
(ii) After the phosphate concentration of this fraction has been brought to 4 mM, it is applied to a column (2.6 by 10 cm) of DEAE-Sephacel (Pharmacia Fine Chemicals), which is equilibrated with 4 mM PB. After washing with the equilibrating buffer the sample is eluted with 25 mM phosphate at a flow rate of 50 ml per hour. The eluate is concentrated in a 202 Amicon stirred cell equipped with a PM 10 membrane (Amicon Corp., Lexington, Mass.).
(iii) The concentrated material is submitted to 4 mg of P32 protein of BCG (soluble extract) or molecular sieving on a Sephadex G-100 (Pharmacia) column (2.6 by 45 cm) equilibrated with 50 mM PB, at a flow rate of 12 ml per hour. The fractions of the peak giving one band in SDS-PAGE are pooled. The purity of the final preparation obtained is controlled by SDS-PAGE followed by silverstaining and by molecular sieving on a Superose 12 (Pharmacia) column (12.0 by 30 cm) equilibrated with 50 mM PB containing 0.005% Tween 80 at a flow rate of 0.2 ml/min. in the Fast Protein Liquid Chromatography system (Pharmacia). Elution is followed by recording the absorbance at 280 nm and 214 nm.
b) Preparation of Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG:
400 μg of purified P32 protein of BCG per ml physiological saline are mixed with one volume of incomplete Freund's adjuvant. The material is homogenized and injected intradermally in 50 μl doses delivered at 10 sites in the back of the rabbits, at 0, 4, 7 and 8 weeks (adjuvant is replaced by the diluent for the last injection). One week later, the rabbits are bled and the sera tested for antibody level before being distributed in aliquots and stored at −80° C.;
2) Test for Giving Evidence of the Reaction Between the Polypeptides of the Invention and Said Rabbit Polyclonal Antiserum Raised Against the P32 Protein of BCG:
the test used was an ELISA test; the ELISA for antibody determination is based on the method of Engvall and Perlmann (Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871-874)
Immulon Microelisa plates (Dynatech, Kloten, Switzerland) are coated by adding to each well 1 μg of one of the polypeptides of the invention in 100 μl Tris hydrochloride buffer 50 mM (pH 8.2). After incubation for 2 h at 27° C. in a moist chamber, the plates are kept overnight at 4° C. They are washed four times with 0.01 M phosphate-buffered saline (pH 7.2) containing 0.05% Tween 20 by using a Titertek microplate washer (Flow Laboratories. Brussels. Belgium). Blocking is done with 0.5% gelatin in 0.06 M carbonate buffer (pH 9.6) for 1 h. Wells are then washed as before, and 100 μl of above mentioned serum diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin is added. According to the results obtained in preliminary experiments, the working dilutions are set at 1:200 for IgG, 1:20 for IgA and 1:80 for IgM determinations. Each dilution is run in duplicate. After 2 h of incubation and after the wells are washed, they are filled with 100 μl of peroxidase-conjugated rabbit immunoglobulins directed against human IgG, IgA or IgM (Dakopatts, Copenhagen, Denmark), diluted 1:400, 1:400 and 1:1,200, respectively in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin and incubated for 90 min. After the wash, the amount of peroxidase bound to the wells is quantified by using a freshly prepared solution of o-phenylenediamine (10 mg/100 ml) and hydrogen peroxide (8 μl of 30% H2O2 per 100 ml) in 0.15 M citrate buffer (pH 5.0) as a substrate. The enzymatic reaction is stopped with 8 N H2SO4 after 15 min. of incubation. The optical density is read at 492 nm with a Titertek Multiskan photometer (Flow Laboratories).
Wells without sera are used as controls for the conjugates. Each experiment is done by including on each plate one negative and two positive reference sera with medium and low antibody levels to correct for plate-to-plate and day-to-day variations. The antibody concentrations are expressed as the optical density values obtained after correction of the readings according to the mean variations of the reference sera.
Hereafter is also given in a non limitative way, a test for giving evidence of the fact that polypeptides of the invention are recognized selectively by human sera from tuberculous patients.
This test is an immunoblotting (Western blotting) analysis, in the case where the polypeptides of the invention are obtained by recombinant techniques. This test can also be used for polypeptides of the invention obtained by a different preparation process. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polypeptides of the invention are blotted onto nitrocellulose membranes (Hybond C. (Amersham)) as described by Towbin et al. (29). The expression of polypeptides of the invention fused to β-galactosidase in E. coli Y1089, is visualized by the binding of a polyclonal rabbit anti-32-kDa BCG protein serum (1:1,000) or by using a monoclonal anti-β-galactosidase antibody (Promega). The secondary antibody (alkaline phosphatase anti-rabbit immunoglobulin G and anti-mouse alkaline phosphatase immunoglobulin G conjugates, respectively) is diluted as recommended by the supplier (Promega).
In order to identify selective recognition of polypeptides of the invention and of fusion proteins of the invention by human tuberculous sera, nitrocellulose sheets are incubated overnight with these sera (1:50) (after blocking a specific protein-binding sites). The human tuberculous sera are selected for their reactivity (high or low) against the purified 32-kDa antigen of BCG tested in a dot blot assay as described in document (31) of the bibliography hereafter. Reactive areas on the nitrocellulose sheets are revealed by incubation with peroxidase conjugated goat anti-human immunoglobulin G antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 h, and after repeated washings, color reaction is developed by adding peroxidase substrate
(α-chloronaphtol) (Bio-Rad Laboratories, Richmond, Calif.) in the presence of peroxidase and hydrogen peroxide.
It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the polypeptides of the invention, particularly the free carboxyl groups which are carried by the groups Glu or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acid inside the peptidic chain, for instance Lys, on the other hand, can be modified in so far as this modification does not alter the above mentioned properties of the polypeptide.
The molecules which are thus modified are naturally part of the invention. The above mentioned carboxyl groups can be acylated or esterified.
Other modifications are also part of the invention. Particularly, the amine or ester functions or both of terminal amino acids can be themselves involved in the bond with other amino acids. For instance, the N-terminal amino acid can be linked to a sequence comprising from 1 to several amino acids corresponding to a part of the C-terminal region of another peptide.
Furthermore, any peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids of the polypeptides according to the invention are part of the invention in so far as this modification does not alter the above mentioned properties of said polypeptides.
The polypeptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
Advantageous recombinant polypeptides of the invention contain in their polypeptidic chain, one at least of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (−30) to the extremity constituted by amino acid at position (295) represented on
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
Other advantageous recombinant polypeptides of the invention consist in one of the following amino acid sequences:
In eukaryotic cells, these polypeptides can be used as signal peptides, the role of which is to initiate the translocation of a protein from its site of synthesis, but which is excised during translocation.
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
Other advantageous peptides of the invention consist in one of the following amino acid sequence:
It is to be noted that the above mentioned polypeptides are derived from the expression products of a DNA derived from the nucleotide sequence coding for a protein of 32-kDa secreted by Mycobacterium tuberculosis as explained hereafter in the examples.
The invention also relates to the amino acid sequences constituted by the above mentioned polypeptides and a protein or an heterologous sequence with respect to said polypeptide, said protein or heterologous sequence comprising for instance from about 1 to about 1000 amino acids. These amino acid sequences will be called fusion proteins.
In an advantageous fusion protein of the invention, the heterologous protein is β-galactosidase.
Other advantageous fusion proteins of the invention are the ones containing an heterologous protein resulting from the expression of one of the following plasmids:
The invention also relates to any nucleotide sequence coding for a polypeptide of the invention.
The invention also relates to nucleic acids comprising nucleotide sequences which hybridize with the nucleotide sequences coding for any of the above mentioned polypeptides under the following hybridization conditions:
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by x-y: i.e. by the sequence extending from the extremity constituted by the nucleotide at position (x) to the extremity constituted by the nucleotide at position (y) represented on
The above mentioned temperatures are to be considered as approximately ±5° C.
The invention also relates to nucleic acids comprising nucleotide sequences which are complementary to the nucleotide sequences coding for any of the above mentioned polypeptides.
It is to be noted that in the above defined nucleic acids, as well as in the hereafter defined nucleic acids, the nucleotide sequences which are brought into play are such that T can be replaced by U.
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
A group of preferred nucleic acids of the invention comprises one at least of the following nucleotide sequences:
Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
Other preferred nucleic acids of the invention comprise one at least of the following nucleotide sequences:
Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
Another preferred group of nucleic acids of the invention comprises the following nucleotide sequences:
According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
According to another advantageous embodiment, nucleic acids of the invention comprises one of the following sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
These nucleotide sequence can be used as nucleotide signal sequences, coding for the corresponding signal peptide.
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
Preferred nucleic acids of the invention consist in one of the following nucleotide sequences:
The invention also relates to any recombinant nucleic acids containing at least a nucleic acid of the invention inserted in an heterologous nucleic acid.
The invention relates more particularly to recombinant nucleic acid such as defined, in which the nucleotide sequence of the invention is preceded by a promoter (particularly an inducible promoter) under the control of which the transcription of said sequence is liable to be processed and possibly followed by a sequence coding for transcription termination signals.
The invention also relates to the recombinant nucleic acids in which the nucleic acid sequences coding for the polypeptide of the invention and possibly the signal peptide, are recombined with control elements which are heterologous with respect to the ones to which they are normally associated within the bacteria gene and, more particularly, the regulation elements adapted to control their expression in the cellular host which has been chosen for their production.
The invention also relates to recombinant vectors, particularly for cloning and/or expression, comprising a vector sequence, notably of the type plasmid, cosmid or phage, and a recombinant nucleic acid of the invention, in one of the non essential sites for its replication.
Appropriate vectors for expression of the recombinant antigen are the following one:
The pEX1, pEX2 and pEX3 vectors are commercially available and can be obtained from Boehringer Mannheim.
The pUEX1, pUEX2 and pUEX3 vectors are also commercially available and can be obtained from Amersham.
According to an advantageous embodiment of the invention, the recombinant vector contains, in one of its non essential sites for its replication, necessary elements to promote the expression of polypeptides according to the invention in a cellular host and possibly a promoter recognized by the polymerase of the cellular host, particularly an inducible promoter and possibly a signal sequence and/or an anchor sequence.
According to another additional embodiment of the invention, the recombinant vector contains the elements enabling the expression by E. coli of a nucleic acid according to the invention inserted in the vector, and particularly the elements enabling the expression of the gene or part thereof of β-galactosidase.
The invention also relates to a cellular host which is transformed by a recombinant vector according to the invention, and comprising the regulation elements enabling the expression of the nucleotide sequence coding for the polypeptide according to the invention in this host.
The invention also relates to a cellular host chosen from among bacteria such as E. coli, transformed by a vector as above defined, and defined hereafter in the examples, or chosen from among eukaryotic organism, such as CHO cells, insect cells, Sf9 cells [Spodoptera frugiperda] infected by the virus Ac NPV (Autographa californica nuclear polyhydrosis virus) containing suitable vectors such as pAc 373 pYM1 or pVC3, BmN [Bombyx mori] infected by the virus BmNPV containing suitable vectors such as pBE520 or p89B310.
The invention relates to an expression product of a nucleic acid expressed by a transformed cellular host according to the invention.
The invention also relates to nucleotidic probes, hybridizing with anyone of the nucleic acids or with their complementary sequences, and particularly the probes chosen among the following nucleotidic sequences gathered in Table 1, and represented in
The hybridization conditions can be the following ones:
hybridization temperature (HT) and wash temperature (WT):
These probes might enable to differentiate M. tuberculosis from other bacterial strains and in particular from the following mycobacteria species:
The sequences which can be used as primers are given in Table 2 hereafter (sequences P1 to P6 or their complement) and illustrated in
compl. = complement
The sequences can be combined in twelve different primer-sets (given in Table 3) which allow enzymatical amplification by the polymerase chain reaction (PCR) technique of any of the nucleotide sequences of the invention, and more particularly the one extending from the extremity constituted by nucleotide at position 1 to the extremity constituted by nucleotide at position 1358, as well as the nucleotide sequence of antigen α of BCG (17).
The detection of the PCR amplified product can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides which is located between PCR primers which have been used to amplify the DNA.
The PCR products of the nucleotide sequences of the invention can be distinguished from the α-antigen gene of BCG or part thereof by hybridization techniques (dot-spot, Southern blotting, etc.) with the probes indicated in Table 3. The sequences of these probes can be found in Table 1 hereabove.
It is to be noted that enzymatic amplification can also be achieved with all oligonucleotides with sequences of about 15 consecutive bases of the primers given in Table 2. Primers with elongation at the 5′-end or with a small degree of mismatch may not considerably affect the outcome of the enzymatic amplification if the mismatches do not interfere with the base-pairing at the 3′-end of the primers.
Specific enzymatic amplification of the nucleotide sequences of the invention and not of the BCG gene can be achieved when the probes (given in Table 1) or their complements are used as amplification primers.
When the above mentioned probes of Table 1 are used as primers, the primer sets are constituted by any of the nucleotide sequences (A, B, C, D, E, F) of Table 1 in association with the complement of any other nucleotide sequence, chosen from A, B, C, D, E or F, it being understood that sequence A means any of the sequences A(i) , A(ii) , A(iii), A(iv) , A(v) and sequence F, any of the sequences F(i), F(ii), F(iii) and F(iv).
Advantageous primer sets for enzymatic amplification of the nucleotide sequence of the invention can be one of the following primer sets given in Table 3bis hereafter:
A(i), A(ii), A(iii), A(iv), A(v), B, C, D, E and F having the nucleotide sequence indicated in Table 1.
In the case of amplification of a nucleotide sequence of the invention with any of the above mentioned primer sets defined in Table 3bis hereabove, the detection of the amplified nucleotide sequence can be achieved by a hybridization reaction with an oligonucleotide sequence of at least 10 nucleotides, said sequence being located between the PCR primers which have been used to amplify the nucleotide sequence. An oligonucleotide sequence located between said two primers can be determined from
The invention also relates to a kit for enzymatic amplification of a nucleotide sequence by PCR technique and detection of the amplified nucleotide sequence containing
The invention also relates to a process for preparing a polypeptide according to the invention comprising the following steps:
The polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book titled “Methode der organischen chemie” (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared according to the method described by R. D. MERRIFIELD in the article titled “Solid phase peptide synthesis” (J. Am. Chem. Soc., 45, 2149-2154, 1964).
The invention also relates to a process for preparing the nucleic acids according to the invention.
A suitable method for chemically preparing the single-stranded nucleic acids (containing at most 100 nucleotides of the invention) comprises the following steps:
DNA synthesis using the automatic β-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986.
In the case of single-stranded DNA, the material which is obtained at the end of the DNA synthesis can be used as such.
A suitable method for chemically preparing the double-stranded nucleic acids (containing at most 100 bp of the invention) comprises the following steps:
A method for the chemical preparation of nucleic acids of length greater than 100 nucleotides—or bp, in the case of double-stranded nucleic acids—comprises the following steps:
The invention also relates to antibodies themselves formed against the polypeptides according to the invention.
It goes without saying that this production is not limited to polyclonal antibodies.
It also relates to any monoclonal antibody produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the purified polypeptide of the invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by its ability to produce the monoclonal antibodies recognizing the polypeptide which has been initially used for the immunization of the animals.
The invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type.
The peptides which are advantageously used to produce antibodies, particularly monoclonal antibodies, are the following ones gathered in Table 4:
(see
(see
The amino acid sequences are given in the 1-letter code.
Variations of the peptides listed in Table 4 are also possible depending on their intended use. For example, if the peptides are to be used to raise antisera, the peptides may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labeled for use in radioimmune assays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. These peptides possess therefore the primary sequence of the peptides listed in Table 4 but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptides.
The invention also relates to a process for detecting in vitro antibodies related to tuberculosis in a human biological sample liable to contain them, this process comprising
Preferably, the biological medium is constituted by a human serum.
The detection can be carried out according to any classical process.
By way of example a preferred method brings into play an immunoenzymatic process according to ELISA technique or immunofluorescent or radioimmunological (RIA) or the equivalent ones.
Thus the invention also relates to any polypeptide according to the invention labeled by an appropriate label of the enzymatic, fluorescent, radioactive . . . type.
Such a method for detecting in vitro antibodies related to tuberculosis comprises for instance the following steps:
The invention also relates to a process for detecting and identifying in vitro antigens of M. tuberculosis in a human biological sample liable to contain them, this process comprising:
Preferably, the biological medium is constituted by sputum, pleural effusion liquid, broncho-alveolar washing liquid, urine, biopsy or autopsy material.
Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4.
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising the following steps:
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis as above defined, the following necessary or kit can be used, said necessary or kit comprising:
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising:
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis , the following necessary or kit can be used, said necessary or kit comprising:
The invention also relates to an additional method for the in vitro diagnostic of tuberculosis in a patient liable to be infected by M. tuberculosis, comprising the following steps:
Appropriate antibodies are advantageously monoclonal antibodies directed against the peptides which have been mentioned in Table 4.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis, the following necessary or kit can be used, said necessary or kit comprising:
An advantageous kit for the diagnostic in vitro of tuberculosis comprises:
The invention also relates to a kit, as described above, also containing a preparation of one of the polypeptides or peptides of the invention, said antigen of the invention being either a standard (for quantitative determination of the antigen of M. tuberculosis which is sought) or a competitor, with respect to the antigen which is sought, for the kit to be used in a competition dosage process.
The invention also relates to an immunogenic composition comprising a polypeptide or a peptide according to the invention, in association with a pharmaceutically acceptable vehicle.
The invention also relates to a vaccine composition comprising among other immunogenic principles anyone of the polypeptides or peptides of the invention or the expression product of the invention, possibly coupled to a natural protein or to a synthetic polypeptide having a sufficient molecular weight so that the conjugate is able to induce in vivo the production of antibodies neutralizing Mycobacterium tuberculosis, or induce in vivo a cellular immune response by activating M. tuberculosis antigen-responsive T cells.
The peptides of the invention which are advantageously used as immunogenic principle have one of the following sequences:
(see
(see
The amino acid sequences are given in the 1-letter code.
Other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention.
FIGS. 1(A) and 1(B) correspond to the identification of six purified λgt 11 M. tuberculosis recombinant clones.
Arrow (←) indicates fusion protein produced by recombinant λgt11-M-tuberculosis clones. Expression and immunoblotting were as described above. Molecular weight (indicated in kDa) were estimated by comparison with molecular weight marker (High molecular weight-SDS calibration kit, Pharmacia).
DNA was isolated from λgt11 phage stocks by using the Lambda Sorb Phage Immunoadsorbent, as described by the manufacturer (Promega). Restriction sites were located as described above. Some restriction sites (*) were deduced from a computer analysis of the nucleotide sequence. The short vertical bars (|−|) represent linker derived EcoRI sites surrounding the DNA inserts of recombinant clones. The lower part represents a magnification of the DNA region containing the antigen of molecular weight of 32-kDa, that has been sequenced. Arrows indicate strategies and direction of dideoxy-sequencing. (→) fragment subcloned in Bluescribe M13; (⇄) fragment subcloned in mp10 and mp11 M13 vectors; (▪→) sequence determined with the use of a synthetic oligonucleotide.
a and 3b correspond to the nucleotide (SEQ ID NO:34) and amino acid sequences (SEQ ID NO:35) of the general formula of the antigens of the invention.
a and 4b correspond to the nucleotide (SEQ ID NO:36) and amino acid sequences (SEQ ID NO:37) of one of the antigens of the invention.
Two groups of sequences resembling the E. coli consensus promoter sequences are boxed and the homology to the consensus is indicated by italic bold letters. Roman bold letters represent a putative Shine-Dalgarno motif.
The NH2-terminal amino acid sequence of the mature protein which is underlined with a double line happens to be very homologous—29/32 amino acids—with the one of MPB 59 antigen (34). Five additional ATG codons, upstream of the ATG at position 273 are shown (dotted underlined). Vertical arrows (⇓) indicate the presumed NH2 end of clone 17 and clone 24. The option taken here arbitrarily represents the 59 amino acid signal peptide corresponding to ATG183.
a-5c correspond to the nucleotide (SEQ ID NO:38) and amino acid sequences (SEQ ID NO:39) of the antigen of 32-kDa of the invention.
The NH2-terminal amino acid sequence of the mature protein which is underlined with a double line happens to be very homologous—29/32 amino acids—with the one of MPB 59 antigen (34). Vertical arrows (⇓) indicate the presumed NH2 end of clone 17 and clone 24.
a-7b represent the homology between the amino acid sequences of the antigen of 32-kDa (SEQ ID NO:44) of the invention and of antigen α of BCG (SEQ ID NO:45) (revised version).
Identical amino acids; (:) evolutionarily conserved replacement of an amino acid (.), and absence of homology ( ) are indicated. Underlined sequence (=) represents the signal peptide, the option taken here arbitrarily representing the 43-amino acid signal peptide corresponding to ATG91. Dashes in the sequences indicate breaks necessary for obtaining the optimal alignment.
a to 9d represent the nucleic acid sequence alignment of the 32-kDa protein gene of M. tuberculosis of the invention (SEQ ID NO:46) (upper line), corresponding to the sequence in
a represents part of the nucleic acid sequence of the 32-kDA protein including probe region A and probe region B as well as primer region P1.
b represents part of the nucleic acid sequence of the 32-kDA protein including Primer regions P2, P3 and P4 and part of probe region C.
c represents part of the nucleic acid sequence of the 32-kDA protein including part of probe region C, probe regions D and E and primer region P5.
d represents part of the nucleic acid sequence of the 32 kDA protein including probe region F and primer region P6.
Dashes in the sequence indicate breaks necessary for obtaining optimal alignment of the nucleic acid sequence.
The primer regions for enzymatical amplification are boxed (P1 to P6).
The specific probe regions are boxed and respectively defined by probe region A, probe region B, probe region C, probe region D, probe region E and probe region F.
It is to be noted that the numbering of nucleotides is different from the numbering of
a corresponds to the restriction and genetic map of the pIGRI plasmid used in Example IV for the expression of the P32 antigen of the invention in E. coli and contains SEQ ID NO:50.
On this figure, underlined restriction sites are unique.
b-10m correspond to the pIGRI nucleic acid sequence SEQ ID NO:40.
On this figure, the origin of nucleotide stretches used to construct plasmid pIGRI are specified hereafter.
Position
Table 5 hereafter corresponds to the complete restriction site analysis of pIGRI.
List of Non Cutting Selected Enzymes
Aat II, Afl II, Apa I, Asu II, Avr II, Bbv II*, Bcl I, Bql II, Bsp MI*, Bsp MII, Bss HII, Bst Eli, Bst XI, Eco 31I*, Eco RI, Esp I, Hpa I, Mlu I, Mme I, Nde I, Not I, Nsi I, Pma CI, Pvu I, Pvu II, Rsr II, Sac I, Sac II Sau I, Sca I, Sci I, Sfi I, Sma I, Sna BI, Spe I Spl I, Ssp I, Stu I, Taq IIA, Taq IIA*, Tth 111I, Vsp I Xca I, Xho I, Xma I, Total number of selected enzymes which do not cut: 45
a corresponds to the restriction and genetic map of the pmTNF MPH plasmid used in Example V for the expression of the P32 antigen of the invention in E. coli and contains SEQ ID NO:51 and SEQ ID NO:52.
b-11m correspond to the pmTNF-MPH nucleic acid sequence SEQ ID NO:41.
On this figure, the origin of nucleotide stretches used to construct plasmid pmTNF-MPH is specified hereafter.
Position
Table 6 hereafter corresponds to the complete restriction site analysis of pmTNF-MPH.
a corresponds to the restriction and genetic map of the plasmid pIG2 used to make the intermediary construct pIG2 Mt32 as described in Example IV for the subcloning of the P32 antigen in plasmid pIGRI and contains SEO ID NO:53.
b-12l correspond to the pIG2 nucleic acid sequence (SEQ ID NO:42).
On this figure, the origin of nucleotide stretches used to construct plasmid pIG2 is specified hereafter.
Position
Table 7 corresponds to the complete restriction site analysis of pIG2.
On this figure:
a and
On
On
Screening of the λqt11 M. tuberculosis Recombinant DNA Library with Anti-32-kDa Antiserum
A λ.gt11 recombinant library constructed from genomic DNA of M. tuberculosis (Erdman strain), was obtained from R. Young (35). Screening was performed as described (14, 35) with some modifications hereafter mentioned. λgt11 infected E. coli Y1090 (105 pfu per 150 mm plate) were seeded on NZYM plates (Gibco) (16) and incubated at 42° C. for 24 hrs. To induce expression of the β-galactosidase-fusion proteins the plates were overlaid with isopropyl β-D-thiogalactoside (IPTG)-saturated filters (Hybond C extra, Amersham), and incubated for 2 hrs at 37° C. Screening was done with a polyclonal rabbit anti-32-kDa antiserum. Said polyclonal antiserum rabbit anti-32-kDa antiserum was obtained by raising antiserum against the P32 M. bovis BCG (strain 1173P2—Institut Pasteur Paris) as follows: 400 μg (purified P32 protein of M. bovis BCG) per ml physiological saline were mixed with one volume of incomplete Freund's adjuvant. The material was homogenized and injected intradermally in 50 μl doses, delivered at 10 sites in the back of the rabbits, at 0, 4, 7 and 8 weeks (adjuvant was replaced by the diluent for the last injection). One week later, the rabbits were bled and the sera tested for antibody level before being distributed in aliquots and stored at −80° C.
The polyclonal rabbit anti-32-kDa antiserum was pre-absorbed on E. coli lysate (14) and used at a final dilution of 1:300. A secondary alkaline-phosphatase anti-rabbit IgG conjugate (Promega), diluted at 1:5000 was used to detect the β-galactosidase fusion proteins. For color development nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were used. Reactive areas on the filter turned deep purple within 30 min. Usually three consecutive purification steps were performed to obtain pure clones. IPTG, BCIP and NBT were from Promega corp. (Madison Wis.).
Plaque Screening by Hybridization for Obtaining the Secondary Clones BY1, By2 and By5 Hereafter Defined
The procedure used was as described by Maniatis et al. (14).
Preparation of Crude Lysates from λgt11 Recombinant Lysogens
Colonies of E. coli Y1089 were lysogenized with appropriate λgt11 recombinants as described by Hyunh et al. (14). Single colonies of lysogenized E. coli Y1089 were inoculated into LB medium and grown to an optical density of 0.5 at 600 nm at 30° C. After a heat shock at 45° C. for 20 min., the production of β-galactosidase-fusion protein was induced by the addition of IPTG to a final concentration of 10 mM. Incubation was continued for 60 min. at 37° C. and cells were quickly harvested by centrifugation. Cells were concentrated 50 times in buffer (10 mM Tris pH 8.0, 2 mM EDTA) and rapidly frozen into liquid nitrogen. The samples were lysed by thawing and treated with 100 μg/ml DNase I in EcoRI restriction buffer, for 5-10 minutes at 37° C.
Immunoblotting (Western Blotting) Analysis:
After SDS-PAGE electrophoresis, recombinant lysogen proteins were blotted onto nitrocellulose membranes (Hybond C, Amersham) as described by Towbin et al. (29). The expression of mycobacterial antigens, fused to β-galactosidase in E. coli Y1089 was visualized by the binding of a polyclonal rabbit anti-32-kDa antiserum (1:1000) obtained as described in the above paragraph “Screening of the λgt11 M. tuberculosis recombinant DNA library with anti-32-kDa antiserum” and using a monoclonal anti-β-galactosidase antibody (Promega). A secondary alkaline-phosphatase anti-rabbit IgG conjugate (Promega) diluted at 1:5000, was used to detect the fusion proteins.
The use of these various antibodies enables to detect the β-galactosidase fusion protein. This reaction is due to the M. tuberculosis protein because of the fact that non fused β-galactosidase is also present on the same gel and is not recognized by the serum from tuberculous patients.
In order to identify selective recognition of recombinant fusion proteins by human tuberculous sera, nitrocellulose sheets were incubated overnight with these sera (1:50) (after blocking aspecific protein binding sites). The human tuberculous sera were selected for their reactivity (high or low) against the purified 32-kDa antigen of M. bovis BCG tested in a Dot blot assay as previously described (31). Reactive areas on the nitrocellulose sheets were revealed by incubation with peroxidase conjugated goat anti-human IgG antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 hrs and after repeated washings color reaction was developed by adding peroxidase substrate (α-chloronaphtol) (Bio-Rad) in the presence of peroxidase and hydrogen peroxide.
Recombinant DNA Analysis
Initial identification of M. tuberculosis DNA inserts in purified λgt11 clones was performed by EcoRI restriction. After digestion, the excised inserts were run on agarose gels and submitted to Southern hybridization. Probes were labeled with α32P-dCTP by random priming (10). Other restriction sites were located by single and double digestions of recombinant λgt11 phage DNA or their subcloned EcoRI fragments by HindIII, PstI, KpnI, AccI and Sphl.
Sequencing
Sequence analysis was done by the primer extension dideoxy termination method of Sanger et al. (25) after subcloning of specific fragments in Bluescribe-M13 (6) or in mp10 and mp11 M13 vectors (Methods in Enzymology, vol. 101, 1983, p. 20-89, Joachim Messing, New M13 vectors for cloning, Academic Press). Sequence analysis was greatly hampered by the high GC content of the M. tuberculosis DNA (65%). Sequencing reactions were therefore performed with several DNA polymerases: T7 DNA polymerase (“Sequenase” USB), Klenow fragment of DNA polymerase I (Amersham) and in some cases with AMV reverse transcriptase (Super RT, Anglian Biotechnology Ltd.) and sometimes with dITP instead of dGTP. Several oligodeoxynucleotides were synthesized and used to focus ambiguous regions of the sequence. The sequencing strategy is summarized in
Routine computer aided analysis of the nucleic acid and deduced amino acid sequences were performed with the LGBC program from Bellon (2). Homology searches used the FASTA programs from Pearson and Lipman (23) and the Protein Identification Resource (PIR) from the National Biomedical Research Fundation—Washington (NBRF) (NBRF/PIR data bank), release 16 (March 1988).
Results
Screening of the λgt11lM, M. tuberculosis Recombinant DNA Library with Polyclonal Anti-32-kDa Antiserum
Ten filters representing 1.5.times. 106 plaques were probed with a polyclonal rabbit anti-32-kDa antiserum (8). Following purification, six independent positive clones were obtained.
Analysis of Recombinant Clones
EcoRI restriction analysis of these 6 purified λgt11recombinant clones DNA, (
Finally, clone 23 (not shown) and clone 24 both contained an insert of 4 kb with one additional EcoRI restriction site giving two fragments of 2.3 kb and 1.7 kb. Southern analysis (data not shown) showed that the DNA inserts of clones 15, 16, 19 and the small fragment (1.7 kb) of clone 24 only hybridized with themselves whereas clone 17 (2.7 kb) hybridized with itself but also equally well with the 2.3 kb DNA fragment of clone 24. Clones 15, 16 and 19 are thus distinct and unrelated to the 17, 23, 24 group. This interpretation was further confirmed by analysis of crude lysates of E. coli Y1089 lysogenized with the appropriate λgt11 recombinants and induced with IPTG. Western blot analysis (
As clone 17 was incomplete, the same λgt11 recombinant M. tuberculosis DNA library was screened by hybridization with a 120 bp EcoRI-KpnI restriction fragment corresponding to the very 5′ end of the DNA insert of clone 17 (previously subcloned in a Blue Scribe vector commercialized by Vector cloning Systems (Stratagene Cloning System) (
DNA Sequencing
The 1358 base pairs nucleotide sequence derived from the various λkg11 overlapping clones is represented in
Six ATG codons were found to precede the TTT at position 360 in the same reading frame. Usage of any of these ATGs in the same reading frame would lead to the synthesis of signal peptides of 29, 42, 47, 49, 55 and 59 residues.
Hydropathy Pattern
The hydropathy pattern coding sequence of the protein of 32-kDa of the invention and that of the antigen α of BCG (17) were determined by the method of Kyte and Doolittle (15). The nonapeptide profiles are shown in
Homology
Matsuo et al. (17) recently published the sequence of a 1095 nucleotide cloned DNA corresponding to the gene coding for the antigen a of BCG. The 978 bp coding region of M. bovis antigen α as revised in Infection and Immunity, vol. 58, p. 550-556, 1990, and 1017 bp coding regions of the protein of 32-kDa of the invention show a 77.5% homology, in an aligned region of 942 bp. At the amino acid level both precursor protein sequences share 75.6% identical residues. In addition, 17.6% of the amino acids correspond to evolutionary conserved replacements as defined in the algorithm used for the comparison (PAM250 matrix, ref 23).
Human Sera Recognize the Recombinant 32-kDa Protein
The invention has enabled to prepare a DNA region coding particularly for a protein of 32-kDa (cf.
Six ATG codons were found to precede this TTT at position 220 in the same reading frame. Usage of any of these ATGs in the same reading frame would lead to the synthesis of signal peptides of 43, 48, 50, 56 or 60 residues. Among these various possibilities, initiation is more likely to take place either at ATG91 or ATG52 because both are preceded by a plausible E. coli-like promoter and a Shine-Dalgarno motif.
If initiation takes place at ATG91, the corresponding signal peptide would code for a rather long peptide signal of 43 residues. This length however is not uncommon among secreted proteins from Gram positive bacteria (5). It would be preceded by a typical E. coli Shine-Dalgamo motif (4/6 residues homologous to AGGAGG) at a suitable distance.
If initiation takes place at ATG52, the corresponding signal peptide would code for a peptide signal of 56 residues but would have a less stringent Shine-Dalgarno ribosome binding site sequence.
The region encompassing the: translation termination triplet was particularly sensitive to secondary structure effects which lead to so-called compressions on the sequencing gels. In front of the TAG termination codon at position 1105, 22 out of 23 residues are G-C base pairs, of which 9 are G's.
Upstream ATG130, a sequence resembling an E. coli promoter (11) comprising an hexanucleotide (TTGAGA) (homology 5/6 to TTGACA) and a AAGAAT box (homology 4/6 to TATAAT) separated by 16 nucleotides was observed. Upstream the potential initiating codon ATG91, one could detect several sequences homologous to the E. coli“-35 hexanucleotide box”, followed by a sequence resembling a TATAAT box. Among these, the most suggestive is illustrated on
Searching the NBRF data bank (issue 16.0) any significant homology between the protein of 32-kDa of the invention and any other completely known protein sequence could not be detected. In particular no significant homology was observed between the 32-kDa protein and α and β subunits of the human fibronectin receptor (1). The NH2-terminal sequence of the 32-kDa protein of the invention is highly homologous—29/32 amino acids—to that previously published for BCG MPB 59 antigen (34) and to that of BCG α-antigen—31/32 amino acids—(Matsuo, 17) and is identical in its first 6 amino acids with the 32-kDa protein of M. bovis BCG (9). However, the presumed initiating methionine precedes an additional 29 or 42 amino acid hydrophobic sequence which differs from the one of α-antigen (cf.
Interestingly, no significant homology between the nucleic acid (1-1358) of the invention (cf.
In the previous example, in
Step 1: Preparation of the DNA Fragments:
1) The plasmid BS-By5-800 obtained by subcloning HindIII fragments of By5 (cf.
2) The plasmid BS-4.1 obtained by subcloning the 2.7 kb EcoRI insert from λgt11-17) into the Blue Scribe M13+ plasmid (Stratagene) (see
3) Blue Scribe M13+ was digested with HindIII and SphI, and treated with calf intestine alkaline phosphatase (special quality for molecular biology, Boehringer Mannheim) as indicated by the manufacturer.
Step 2: Ligation:
The ligation reaction contained:
125 ng of the 800 bp HindIII fragment (1)
125 ng of the 1500 bp SphI-HindIII insert (2)
50 ng of the HindIII-SphI digested BSM13+ vector (3)
(3)
2 μl of 10 ligation buffer (Maniatis et al., 1982)
1 μl of (=2.5 U) of T4 DNA ligase (Amersham)
4 μl PEG 6000, 25% (w/v)
8 μl H2O
The incubation was for 4 hours at 16° C.
Step 3: Transformation:
100 μl of DH5α E. coli (Gibco BRL) were transformed with 10 μl of the ligation reaction (step 2) and plated on IPTG, X-Gal ampicillin plates, as indicated by the manufacturer. About 70 white colonies were obtained.
step 4:
As the 800 bp fragment could have been inserted in both orientations, plasmidic DNA from several clones were analyzed by digestion with PstI in order to select one clone (different from clone 11), characterized by the presence of 2 small fragments of 229 and 294 bp. This construction contains the HindIII-HindIII-SphI complex in the correct orientation. The plasmid containing this new construction was called: “BS.BK.P32 complet”.
The DNA sequence coding for a polypeptide, or part of it, can be linked to a ribosome binding site which is part of the expression vector, or can be fused to the information of another protein or peptide already present on the expression vector.
In the former case the information is expressed as such and hence devoid of any foreign sequences (except maybe for the aminoterminal methionine which is not always removed by E. coli).
In the latter case the expressed protein is a hybrid or a fusion protein.
The gene, coding for the polypeptide, and the expression vector are treated with the appropriate restriction enzyme(s) or manipulated otherwise as to create termini allowing ligation. The resulting recombinant vector is used to transform a host. The transformants are analyzed for the presence and proper orientation of the inserted gene. In addition, the cloning vector may be used to transform other strains of a chosen host. Various methods and materials for preparing recombinant vectors, transforming them to host cells and expressing polypeptides and proteins are described by Panayatatos, N., in “Plasmids, a practical approach (ed. K. G. Hardy, IRL Press) pp. 163-176, by Old and Primrose, principals of gene manipulation (2d Ed, 1981) and are well known by those skilled in the art.
Various cloning vectors may be utilized for expression. Although a plasmid is preferable, the vector may be a bacteriophage or cosmid. The vector chosen should be compatible with the host cell chosen.
Moreover, the plasmid should have a phenotypic property that will enable the transformed host cells to be readily identified and separated from those which are not transformed. Such selection genes can be a gene providing resistance to an antibiotic like for instance, tetracycline carbenicillin, kanamycin, chloramphenicol, streptomycin, etc.
In order to express the coding sequence of a gene in E. coli the expression vector should also contain the necessary signals for transcription and translation.
Hence it should contain a promoter, synthetic or derived from a natural source, which is functional in E. coli. Preferably, although usually not absolutely necessary, the promoter should be controllable by the manipulator. Examples of widely used controllable promoters for expression in E. coli are the lac, the trp, the tac and the lambda PL and PR promoter.
Preferably, the expression vector should also contain a terminator of transcription functional in E. coli. Examples of used terminators of transcription are the trp and the rrnB terminators.
Furthermore, the expression vector should contain a ribosome binding site, synthetic or from a natural source, allowing translation and hence expression of a downstream coding. sequence. Moreover, when expression devoid of foreign sequences is desired, a unique restriction site, positioned in such a way that it allows ligation of the sequence directly to the initiation codon of the ribosome binding site, should be present.
A suitable plasmid for performing this type of expression is pKK233-2 (Pharmacia). This plasmid contains the trc promoter, the lac Z ribosome binding site and the rmB transcription terminator.
Also suitable is plasmid pIGRI (Innogenetics, Ghent, Belgium). This plasmid contains the tetracycline resistance gene and the origin of replication of pAT153 (available from Bioexcellence, Biores B. V., Woerden, The Netherlands), the lambda PL promoter up to the MboII site in the 5′ untranslated region of the lambda N gene (originating from pPL(λ); Pharmacia).
Downstream from the PL promoter, a synthetic sequence was introduced which encodes a “two cistron” translation casette whereby the stop codon of the first cistron (being the first 25 amino acids of TNF, except for Leu at position 1 which is converted to Val) is situated between the Shine-Dalgamo sequence and the initiation codon of the second ribosome binding site. The restriction and genetic map of pIGRI is represented in
b and Table 5 represent respectively the nucleic acid sequence and complete restriction site analysis of pIGRI.
However, when expression as a hybrid protein is desired, then the expression vector should also contain the coding sequence of a peptide or polypeptide which is (preferably highly) expressed by this vector in the appropriate host.
In this case the expression vector should contain a unique cleavage site for one or more restriction endonucleases downstream of the coding sequence.
Plasmids pEX1, 2 and 3 (Boehringer, Mannheim) and pUEX1, 2 and 2 (Amersham) are useful for this purpose.
They contain an ampicillin resistance gene and the origin of replication of pBR322 (Bolivar at al. (1977) Gene 2, 95-113), the lac Z gene fused at its 5′ end to the lambda PR promoter together with the coding sequence for the 9 first amino acids of its natural gene cro, and a multiple cloning site at the 3′ end of the lac Z coding sequence allowing production of a beta galactosidase fused polypeptide.
The pUEX vectors also contain the CI857 allele of the bacteriophage lambda CI repressor gene.
Also useful is plasmid pmTNF MPH (Innogenetics). It contains the tetracycline resistance gene and the origin of replication of pAT13 (obtainable from Bioexcellence, Biores B. ., Woerden. The Netherlands), the lambda PL promoter up to the MboII site in the N gene 5′ untranslated region (originating from pPL (λ); Pharmacia), followed by a synthetic ribosome binding site (see sequence data), and the information encoding the first 25 AA of mTNF (except for the initial Leu which is converted to Val). This sequence is, in turn, followed by a synthetic polylinker sequence which encodes six consecutive histidines followed by several proteolytic sites (a formic acid, CNBr, kallikrein, and E. coli protease VII sensitive site, respectively), each accessible via a different restriction enzyme which is unique for the plasmid (SmaI, NcoI, BspMII and StuI, respectively; see restriction and genetic map,
Table 6 gives a complete restriction site analysis of pmTNF MPH.
The presence of 6 successive histidines allows purification of the fusion protein by Immobilized Metal Ion Affinity Chromatography (IMAC).
After purification, the foreign part of the hybrid protein can be removed by a suitable protein cleavage method and the cleaved product can then be separated from the uncleaved molecules using the same IMAC based purification procedure.
In all the above-mentioned plasmids where the lambda PL or PR promoter is used, the promoter is temperature-controlled by means of the expression of the lambda cI ts 857 allele which is either present on a defective prophage incorporated in the chromosome of the host (K12ΔH, ATCC no 33767) or on a second compatible plasmid (pACYC derivative). Only in the pUEX vectors is this cI allele present on the vector itself.
It is to be understood that the plasmids presented above are exemplary and other plasmids or types of expression vectors maybe employed without departing from the spirit or scope of the present invention.
If a bacteriophage or phagemid is used, instead of plasmid, it should have substantially the same characteristics used to select a plasmid as described above.
Fifteen μg of plasmid “BS-BK-P32 complet” (see Example II) was digested with EclXI and BstEII (Boehringer, Mannheim) according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of DNA. EclXI cuts at position 226 (
The DNA coding for the “P32 antigen fragment” (as defined above) is subcloned in pIGRI (see
Five μg of plasmid pIG2 is digested with NcoI. Its 5′ sticky ends are filled in prior to dephosphorylation.
Therefore, the DNA was incubated in 40 μl NB buffer (0.05 M Tris-Cl pH 7.4; 10 mM MgCl2; 0.05% β-mercaptoethanol) containing 0.5 mM of all four dXTP (X=A, T, C, G) and 2 μl of Klenow fragment of E. coli DNA polymerase I (5 U/μl, Boehringer, Mannheim) for at least 3 h at 15° C.
After blunting, the DNA was once extracted with one volume of phenol equilibrated against 200 mM Tris-Cl pH 8, twice with at least two volumes of diethylether and finally collected using the “gene clean kit™” (Biol101) as recommended by the supplier. The DNA was then dephosphorylated at the 5′ ends in 30 μl of CIP buffer (50 mM TrisCl pH 8, 1 mM ZnCl2) and 20 to 25 units of calf intestine phosphatase (high concentration, Boehringer, Mannheim). The mixture was incubated at 37° C. for 30 min, then EGTA (ethyleneglycol bis(β-aminoethylether)-N,N,N′,N′tetraacetic acid) pH 8 is added to a final concentration of 10 mM. The mixture was then extracted with phenol followed by diethylether as described above, and the DNA was precipitated by addition of 1/10 volume of 3 M KAc (Ac═CH3COO) pH 4.8 and 2 volumes of ethanol followed by storage at −20° C. for at least one hour.
After centrifugation at 13000 rpm in a Biofuge A (Hereaus) for 5 min the pelleted DNA was dissolved in H2O to a final concentration of 0.2 μg/μl.
The EclXI-BstEII fragment, coding for the “P32 antigen fragment” (see above) was electrophoresed on a 1% agarose gel (BRL) to separate it from the rest of the plasmid and was isolated from the gel by centrifugation over a Millipore HVLP filter (Φ2 cm) (2 min, 13000 rpm, Biofuge at room temperature) and extracted with Tris equilibrated phenol followed by diethylether as described above.
The DNA was subsequently collected using the “Gene clean kit™” (Bio101) as recommended by the supplier.
After that, the 5′ sticky ends were blunted by treatment with the Klenow fragment of E. coli DNA polymerase I as described above and the DNA was then again collected using the “Gene clean kit™” in order to dissolve it in 7 μl of H2O.
One μl of vector DNA is added together with one μl of 10× ligase buffer (0.5 M TrisCl pH 7.4, 100 mM MgCl2, 5 mM ATP, 50 mM DTT (dithiothreitol)) and 1 μl of T4 DNA ligase (1unit/μl, Boehringer, Mannheim). Ligation was performed for 6 h at 13° C. and 5 μl of the mixture is then used to transform strain DH1 (lambda) [strain DH1—ATCC No 33849—lysogenized with wild type bacteriophage λ] using standard transformation techniques as described for instance by Maniatis et al. in “Molecular cloning, a laboratory manual”, Cold Spring Harbor Laboratory (1982).
Individual transformants are grown and lysed for plasmid DNA preparation using standard procedures (Experiments with gene fusions, Cold Spring Harbor Laboratory (1984) (T. J. Silhavy, H. L. Berman and L. W. Enquist, eds) and the DNA preparations are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis.
A check for correct blunting is done by verifying the restoration of the NcoI site at the 5′ and 3′ end of the antigen coding sequence. One of the clones containing the P32 antigen fragment in the correct orientation is kept for further work and designated pIG2-Mt32. In this intermediary construct, the DNA encoding the antigen is not in frame with the ATG codon. However, it can now be moved as a NcoI fragment to another expression vector.
15 μg of pIG2-Mt32 is digested with NcoI. The NcoI fragment encoding the P32 antigen is gel purified and blunted as described above. After purification, using “gene clear kit TM” it is dissolved in 7 μl of H2O.
5 μg of plasmid pIGRI is digested with NcoI, blunted and dephosphorylated as described above. After phenol extraction, followed by diethylether and ethanolprecipitation, the pellet is dissolved in H2O to a final concentration of 0.2 μg/μl.
Ligation of vector and “antigen fragment” DNA is carried out as described above. The ligation mixture is then transformed into strain DH1 (lambda) and individual transformants are analysed for the correct orientation of the gene within the plasmid by restriction enzyme analysis. A check for correct blunting is done by verifying the creation of a new NsiI site at the 5′ and 3′ ends of the antigen coding sequence. One of the clones containing the P32 antigen fragment in the correct orientation is kept for further work and designated pIGRI.Mt32.
Fifteen μg of the plasmid pIG2 Mt32 (see example IV) was digested with the restriction enzyme NcoI (Boehringer, Mannheim), according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of DNA.
After digestion, the reaction mixture is extracted with phenol equilibrated against 200 mM TrisCl pH 8, (one volume), twice with diethylether (2 volumes) and precipitated by addition of 1/10 volume of 3 M KAc (Ac═CH3COO) pH 4.8 and 2 volumes of ethanol followed by storage at −20° C. for at least one hour.
After centrifugation for 5 minutes at 13000 rpm in a Biofuge A (Hereaus) the DNA is electrophoresed on a 1% agarose gel (BRL).
The DNA coding for the “P32 antigen fragment” as described above, is isolated by centrifugation over a Millipore HVLP filter (Φ2 cm) (2 minutes, 13000 rpm, Biofuge at room temperature) and extracted one with triscl equilibrated phenol and twice with diethylether. The DNA is subsequently collected using “Gene clean kit™” (Bio 101) and dissolved in 7 μl of H2O.
The 5′ overhanging ends of the DNA fragment generated by digestion with NcoI were filled in by incubating the DNA in 40 μl NB buffer (0.05 M Tris-HCl, pH 7.4; 10 mM Mgcl2; 0.05% β-mercaptoethanol) containing 0.5 mM of all four dXTPS (X=A, T, C, G) and 2 μl of Klenow fragment of E. coli DNA polymerase I (5 units/μl Boehringer Mannheim) for at least 3 h at 15° C. After blunting, the DNA was extracted with phenol, followed by diethylether, and collected using a “gene clean kit™” as described above.
Five μg of plasmid pmTNF MPH is digested with StuI, subsequently extracted with phenol, followed by diethylether, and precipitated as described above. The restriction digest is verified by electrophoresis of a 0.5 μg sample on an analytical 1.2% agarose gel.
The plasmid DNA is then desphosphorylated at the 5′ ends to prevent self-ligation in 30 μl of CIP buffer (50 mM TrisCl pH 8, 1 mM ZnC12) and 20 to 25 units of calf intestine phosphatase (high concentration, Boehringer Mannheim). The mixture is incubated at 37° C. for 30 minutes, then EGTA (ethyleneglycol bis(β-aminoethylether)-N,N,N′,N′tetraacetic acid) pH8 is added to a final concentration of 10 mM. The mixture is extracted with phenol followed by diethylether and the DNA is precipitated as described above. The precipitate is pelleted by centrifugation in a Biofuge A (Hereaus) at 13000 rpm for 10 min at 4° C. and the pellet is dissolved in H2O to a final DNA concentration of 0.2 μg/μl.
One μl of this vector DNA is mixed with the 7 μl solution containing the DNA fragment coding for the “P32antigen fragment” (as defined above) and 1 μl 10× ligase buffer (0.5 M TrisCl pH7.4, 100 mM MgCl2, 5 mM ATP, 50 mM DTT (dithiothreitol)) plus 1 μl T4 DNA ligase (1 unit/μl, Boehringer Mannheim) is added. The mixture is incubated at 13° C. for 6 hours and 5 μl of the mixture is then used for transformation into strain DH1 (lambda) using standard transformation techniques are described by for instance Maniatis et al. in “Molecular cloning, a laboratory manual”, Cold Spring Harbor Laboratory (1982).
Individual transformants are grown and then lysed for plasmid DNA preparation using standard procedures (Experiments with gene fusions, Cold Spring Harbor Laboratory 1984 (T. J. Silhavy, M. L. Berman and L. W. Enquist eds.)) and are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis.
One of the clones containing the DNA sequence encoding the antigen fragment in the correct orientation was retained for further work and designated pmTNF-MPH-Mt32. It encodes all information of the P32 antigen starting from position +4 in the amino acid sequence (see
A—Material and Methods
DNA of pmTNF-MPH-Mt32 is transformed into E. coli strain K12ΔH (ATCC 33767) using standard transformation procedures except that the growth temperature of the cultures is reduced to 28° C. and the heat shock temperature to 34° C.
A culture of K12.DELTA.H harboring pmTNF-MPH-Mt32, grown overnight in Luria broth at 28° C. with vigorous shaking in the presence of 10 μg/ml tetracycline, is inoculated into fresh Luria broth containing tetracycline (10 μg/ml) and grown to an optical density at 600 nanometers of 0.2 in the same conditions as for the overnight culture.
When the optical density at 600 nanometers has reached 0.2 half of the culture is shifted to 42° C. to induce expression while the other half remains at 28° C. as a control. At several time intervals aliquots are taken which are extracted with one volume of phenol equilibrated against M9 salts (0.1% ammonium chloride, 0.3% potassium dihydrogenium phosphate, 1.5% disodium hydrogenium phosphate, 12 molecules of water) and 1% SDS. At the same time, the optical density (600 nm) of the culture is checked. The proteins are precipitated from the phenol phase by addition of two volumes of acetone and storage overnight at −20° C. The precipitate is pelleted (Biofuge A, 5 min., 13000 rpm, room temperature) dried at the air, dissolved in a volume of Laemmli (Nature (1970) 227:680) sample buffer (+β-mercapto ethanol) according to the optical density and boiled for 3 min.
Samples are then run on a SDS polyacrylamide gel (15%) according to Laemmli (1970). Temperature induction of mTNF-His6-P32 is monitored by both Coomassie Brilliant Blue (CBB) staining and immunoblotting. CBB staining is performed by immersing the gel in a 1/10 diluted CBB staining solution (0.5 g CBB-R250 (Serva) in 90 ml methanol: H2O (1:1 v/v) and 10 ml glacial acetic acid) and left for about one hour on a gently rotating platform. After destaining for a few hours in destaining solution (30% methanol, 7% glacial acetic acid) protein bands are visualised and can be scanned with a densitometer (Ultroscan XL Enhanced Laser Densitometer, LKB).
For immunoblotting the proteins are blotted onto Hybond C membranes (Amersham) as described by Townbin et al (1979). After blotting, proteins on the membrane are temporarily visualised with Ponceau S (Serva) and the position of the molecular weight markers is indicated. The stain is then removed by washing in H2O. A specific protein binding sites are blocked by incubating the blots in 10% non-fat dried milk for about 1 hour on a gently rotating platform. After washing twice with NT buffer (25 mM Tris-HCl, pH 8.0; 150 mM NaCl) blots are incubated with polyclonal rabbit anti-32-kDa antiserum (1:1000), obtained as described in example I (“screening of the λgt11 M. tuberculosis recombinant DNA library with anti-32-kDa antiserum”) in the presence of E. coli lysate or with monoclonal anti-hTNF-antibody which crossreacts with mTNF (Innogenetics, no 17F5D10) for at least 2 hours on a rotating platform. After washing twice with NT buffer+0.02% Triton.X.100, blots are incubated for at least 1 hour with the secondary antiserum alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins ( 1/500; Prosan) in the first case, and alkaline phosphatase conjugated rabbit anti-mouse immunoglobulins ( 1/500; Sigma) in the second case.
Blots are washed again twice with NT buffer+0.02% Triton X100 and visualisation is then performed with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) from Promega using conditions recommended by the supplier.
B. Results
Upon induction of K12.DELTA.H cells containing pmTNF-MPH-Mt32, a clearly visible band of about 35-kDa appears on CBB stained gels, already one hour after start of induction (
The hybrid protein mTNF-His6-P32 (amino acid sequence, see
a. Preparation of the Crude Cell Extract:
12 l of E. coli cells K12AH containing plasmid pmTNF-MPH-Mt32 were grown in Luria Broth containing tetracycline (10 μg/ml) at 28° C. to an optical density (600 nm) of 0.2 and then induced by shifting the temperature to 42° C. After 3 hours of induction, cells were harvested by centrifugation (Beckman, J A 10 rotor, 7,500 rpm, 10 min). The cell paste was resuspended in lysis buffer (10 mM KCl, 10 mM Tris-HCl pH 6.8, 5 mM EDTA) to a final concentration of 50% (w/v) cells.
ε-NH2-capronic acid and dithiotreitol (DTT) were added to a final concentration of resp. 20 mM and 1 mM, to prevent proteolytic degradation. This concentrated cell suspension was stored overnight at −70° C.
Cells were lysed by passing them three times through a French press (SLM-Aminco) at a working pressure of 800-1000 psi. During and after lysis, cells were kept systematically on ice.
The cell lysate was cleared by centrifugation (Beckman, J A 20, 18,000 rpm, 20 min, 4° C.). The supernatant (SN) was carefully taken off and the pellet, containing membranes and inclusion bodies, was kept for further work since preliminary experiments had shown that the protein was mainly localised in the membrane fraction.
7 M guanidinium hydrochloride (GuHCl, marketed by ICN) in 100 mM phosphate buffer pH 7.2 was added to the pellet volume to a final concentration of 6 M GuHCl. The pellet was resuspended and extracted in a bounce tissue homogenizer (10 cycles).
After clearing (Beckman, J A 20, 18,000 rpm, 20 min, 4° C.), about 100 ml of supernatant was collected (=extract 1) and the removing pellet was extracted again as described above (=extract 2, 40 ml).
The different fractions (SN, EX1, EX2) were analysed on SDS-PAGE (Laemmli, Nature 1970; 227:680) together with control samples of the induced culture. Scanning of the gel revealed that the recombinant protein makes up roughly 25% of the total protein content of the induced cell culture. After fractionation most of the protein was found back in the extracts. No difference was noticed between reducing and non-reducing conditions (plus and minus β-mercaptoethanol).
b. Preparation of the Ni++ IDA (Imino Diacetic Acid) Column:
5 ml of the chelating gel, Chelating Sepharose 6B (Pharmacia) is washed extensively with water to remove the ethanol in which it is stored and then packed in a “Econo-column” (1×10 cm, Biorad). The top of the column is connected with the incoming fluid (sample, buffer, etc) while the end goes to the UV280 detector via a peristaltic jump. Fractions are collected using a fraction collector and, when appropriate, pH of the fractions is measured manually.
The column is loaded with Ni++ (6 ml NiCl2.6H2O; 5 μg/μl) and equilibrated with starting buffer (6 M guanidinium hydrochloride, 100 mM phosphate buffer, pH 7.2).
After having applied the sample, the column is washed extensively with starting buffer to remove unbound material.
To regenerate the column, which has to be done after every 2-3 runs, 20 ml (about 5 column volumes) of the following solutions are pumped successively through the column:
0.05 M EDTA-0.5 M NaCl
0.1 M NaOH
H2O
6 ml NiCl2.6 H2O (5 mg/ml).
After equilibrating with starting buffer the column is ready to use again.
c. Chromatography:
All buffers contained 6 M guanidinium hydrochloride throughout the chromatography. The column was developed at a flow rate of 0.5 ml/min at ambient temperature. Fractions of 2 ml were collected and, when appropriate, further analysed by SDS-PAGE and immunoblotting. Gels were stained with Coomassie Brilliant Blue R250 and silver stain, as described by ANSORGE (1985). Immunoblotting was carried out as described in example I. The primary antiserum used was either polyclonal anti-32kDa-antiserum ( 1/1000) obtained as described in example I (“screening of the λgt11 M. tuberculosis recombinant DNA library with anti-32kDa-antiserum”) or anti-E. coli-immunoglobulins ( 1/500; PROSAN), or monoclonal anti-hTNF-antibody which cross-reacts with mTNF (Innogenetics, No 17F5D 10). The secondary antiserum was alkaline phosphatase conjugated swine anti-rabbit immunoglobulins ( 1/500, PROSAN), or alkaline phosphatase conjugated rabbit-anti-mouse immunoglobulins ( 1/500, Sigma).
C1. Elution with Decreasing-pH:
Solutions used:
A: 6 M GuHCl 100 mM phosphate pH 7.2
B: 6 M GuHCl 25 mM phosphate pH 7.2
C: 6 M GuHCl 50 mM phosphate pH 4.2
After applying 3 ml of extract 1 (OD280=32.0) and extensively washing with solution A, the column is equilibrated with solution B and then developed with a linear pH gradient from 7.2 to 4.2 (25 ml of solution B and 25 ml of solution C were mixed in a gradient former). The elution profile is shown in
From SDS-PAGE analysis (Coomassie and silverstain) it was clear that most of the originally bound recombinant protein was eluted in the fractions between pH 5.3 and 4.7.
Screening of these fractions on immunoblot with anti-32-kDa and the 17F5D10 monoclonal antibody showed that, together with the intact recombinant protein, also some degradation products and higher aggregation forms of the protein were present, although in much lower amount. Blotting with anti-E. coli antibody revealed that these fractions (pH 5.3-4.7) still contained immunodetectable contaminating E. coli proteins (75, 65, 43, 35 and 31 kDa bands) and lipopolysaccharides.
C2. Elution with Increasing Imidazol Concentration:
Solutions used:
A: 6 M GuHCl 100 mM phosphate pH 7.2
B: 6 M GuHCl 50 mM imidazol pH 7.2
C: 6 M GuHCl 100 mM imidazol pH 7.2
D: 6 M GuHCl 15 mM imidazol pH 7.2
E: 6 M GuHCl 25 mM imidazol pH 7.2
F: 6 M GuHCl 35 mM imidazol pH 7.2
Sample application and washing was carried out as in C1, except that after washing, no equilibration was necessary with 6 M GuHCl 25 mM phosphate. The column was first developed with a linear gradient of imidazol going from 0 to 50 mM (25 ml of solution A and 25 ml of solution B were mixed in a gradient former) followed by a step elution to 100 mM imidazol (solution C). During the linear gradient, proteins were gradually eluted in a broad smear, while the step to 100 mM gave rise to a clear peak (
SDS-PAGE analysis of the fractions revealed that in the first part of the linear gradient (fr 1-24) most contaminating E. coli proteins were washed out, while the latter part of the gradient (fr 25-50) and the 100 mM peak contained more than 90% of the recombinant protein.
As in C1, these fractions showed, besides a major band of intact recombinant protein, some minor bands of degradation and aggregation products. However, in this case, the region below 24-kDa seemed nearly devoid of protein bands, which suggests that less degradation products co-elute with the intact protein. Also, the same contaminating E. coli proteins were detected by immunoblotting, as in C1, although the 31-kDa band seems less intense and even absent in some fractions.
In a second stage, we developed the column with a step gradient of increasing imidazol concentrations. After having applied the sample and washed the column, 2 column volumes (about 8 ml) of the following solutions were brought successively onto the column solution D, E, F and finally 4 column volumes of solution C. The stepgradient resulted in a more concentrated elution profile (
In conclusion, the mTNF-His6-P32 protein has been purified to at least 90% by IMAC. Further purification can be achieved through a combination of the following purification steps:
IMAC on chelating superose (Pharmacia)
ion exchange chromatography (anion or cation)
reversed phase chromatography
gel filtration chromatography
immunoaffinity chromatography
elution from polyacrylamide gel.
These chromatographic methods are commonly used for protein purification.
The plasmids of
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP90/01593 | Sep 1990 | WO | international |
| 89402571.7 | Sep 1989 | EP | regional |
This application is a divisional application of and claims priority to U.S. Ser. No. 10/329,087, filed on Dec. 23, 2002, now U.S. Pat. No. 7,083,797, which is a divisional of U.S. Ser. No. 09/342,673, filed on Jun. 29, 1999, now U.S. Pat. No. 6,531,138, which is a continuation of U.S. Ser. No. 08/447,430, filed on May 22, 1995, now U.S. Pat. No. 5,916,558, which is continuation of U.S. Ser. No. 07/690,949, filed on Jul. 8, 1991, which claims priority to, and is the U.S. National Phase of, PCT Application PCT/EP90/01593, filed on Sep. 19, 1990, which claims priority to British Patent Application No. 89402571.7, filed on Sep. 19, 1989, the contents of which are all incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10329087 | Dec 2002 | US |
| Child | 11472812 | Jun 2006 | US |
| Parent | 09342673 | Jun 1999 | US |
| Child | 10329087 | Dec 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08447430 | May 1995 | US |
| Child | 09342673 | Jun 1999 | US |
| Parent | 07690949 | Jul 1991 | US |
| Child | 08447430 | May 1995 | US |
