WO2008010096A2 - Procédé de préparation de polypeptides mycobactériens recombinés de levure et leur utilisation pour diagnostiquer des maladies associées à des mycobactéries - Google Patents

Procédé de préparation de polypeptides mycobactériens recombinés de levure et leur utilisation pour diagnostiquer des maladies associées à des mycobactéries Download PDF

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WO2008010096A2
WO2008010096A2 PCT/IB2007/002936 IB2007002936W WO2008010096A2 WO 2008010096 A2 WO2008010096 A2 WO 2008010096A2 IB 2007002936 W IB2007002936 W IB 2007002936W WO 2008010096 A2 WO2008010096 A2 WO 2008010096A2
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Prior art keywords
amino acid
seq
polypeptide
acid sequence
acid residues
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PCT/IB2007/002936
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WO2008010096A3 (fr
Inventor
Mohamed Dahmani Fathallah
Mohamed Ridha Barbouche
John L. Ho
Chaouki Benabdesselem
Koussay Dellagi
Beya Largueche
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Institut Pasteur
Institut Pasteur De Tunis
Cornell Research Foundation Inc.
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Publication of WO2008010096A2 publication Critical patent/WO2008010096A2/fr
Publication of WO2008010096A3 publication Critical patent/WO2008010096A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag

Definitions

  • the present invention relates to a method for preparing a mycobacterial polypeptide qualitatively similar to the native mycobacterial protein.
  • the present invention also relates to the mycobacterial polypeptide obtained by the method of the invention and its use in method and kit for the diagnosis of mycobacterial related diseases, such as tuberculosis.
  • the serum recognition of even the most promising TB antigens by sera can vary widely depending upon the country of origin of the studied cohort, as well as the AFB smear status and disease manifestations of the individuals within the population (18, 27, 37).
  • M. tuberculosis M. tuberculosis
  • CFP32 is one out of the 30 culture filtrate (CF) proteins that have emerged as potential candidates for the serological diagnosis of tuberculosis [37].
  • the CFP32 coding gene is found exclusively in the M. tuberculosis complex as assessed by PCR and southern blot analysis of the genome of several mycobacterial strains [15].
  • CFP32 was identified by mass spectroscopy, N-terminal sequencing and immunodetection [42, 43].
  • CFP32 is expressed by M.
  • tuberculosis complex members including BCG [14].
  • Native CFP32 was detected in the sputum of patients with active pulmonary tuberculosis [14]. Furthermore, the virulence-related neutral red character typical of virulent mycobacteria, was recently shown by Andreu et al [3] to be associated with the cfp32 gene upon gene transfer into M. smegmatis. Moreover CFP32 level in the lung of active TB patients was positively correlated with amounts of the immunosuppressive cytokine IL10 [14]. These observations suggest that CFP32 may play a role in tuberculosis pathogenesis, and likely interact with the immune system, as shown previously by the detection of serologic reaction to E. coli expressed rCFP32 in approximately 30% of tuberculosis patients [14].
  • M. smegmatis and BCG [54, 55, 56] were also used to express mycobacterial genes by heterologous complementation.
  • M. smegmatis and BCG [54, 55, 56] were also used to express mycobacterial genes by heterologous complementation.
  • these are not conventional systems for the production of high levels of recombinant proteins.
  • heterologous complementation of MTB genes into BCG or M. smegmatis may be associated with gene modifications and loss of protein activity.
  • these hosts will need to be manipulated in level 3 standard biosafety containment, if transfected with virulence-associated genes.
  • level 3 standard biosafety containment if transfected with virulence-associated genes.
  • more adapted expression systems need to be explored.
  • the invention relates to a method for preparation of a purified recombinant mycobacterial polypeptide containing immunogenic epitopes substantially similar to the ones of its corresponding native Mycobacterium species protein, said method comprising : a) culturing a recombinant yeast cell expressing said polypeptide in a culture medium under conditions sufficient to effect expression of the polypeptide; and b) separating the polypeptide from the yeast cell or culture medium.
  • the invention further relates to a purified recombinant mycobacterial polypeptide or a functional fragment thereof obtained by the above mentioned method.
  • the invention also relates to a method for diagnosing a mycobacterial related infectious disease in a subject, comprising : a) contacting a sample of a subject with a recombinant mycobacterial polypeptide of the present invention, for a time and under conditions sufficient to form a polypeptide-mycobacterial specific antibody complex; and b) detecting the presence or absence of the complex formed in a).
  • the invention also relates to a yeast host cell comprising a polynucleotide encoding a recombinant mycobacterial polypeptide of the present invention.
  • the invention further relates to a kit for diagnosing a mycobacterial species related infectious disease in a subject, the kit comprising: a) a recombinant mycobacterial polypeptide of the present invention or a yeast host cell of the present invention; and b) reagents to detect a polypeptide-mycobacterial specific antibody immune complex.
  • the invention further relates to an immunogenic composition capable to induce antibodies recognizing specifically the polypeptide of the invention.
  • (C) rCFP32 produced in yeast is relatively more antibody reactive than rCFP32 trans-expressed in E. coli.
  • Western blot was performed using varying indicated input amounts of rCFP32 produced in yeast or E. coli as detected by (/) anti-E. coli rCFP32 (rabbit) antisera and (H) anti-(His)6-tag mAb.
  • the figure is derived from two different blots probed with either anti-E. coli rCFP32 (rabbit) antisera or anti-(His)6-tag mAb.
  • band sizes of molecular weight markers (kDa) are indicated at left in the panel.
  • FIG. 2 Sera from human test subjects show greater reactivity to yeast- expressed rCFP32 than E. co//-produced rCFP32.
  • TB active TB case patients
  • BCG-vaccin. BCG-vaccin.
  • the results of statistical analyses of the mean serological responses are shown (calculated using either the paired t test or unpaired t test, as indicated in the text). Each data point represents one patient.
  • Solid horizontal lines represent the mean of each category while dashed horizontal lines represent the cut-off values above which serological responses to rCFP32 were deemed positive. Cut-off values were determined using the mean for the healthy BCG vaccinated persons plus 2 standard deviations per the E. coli rCFP32 (cut-off A) or the yeast rCFP32 (cut-off B). OD492 nm values above the cut-off are taken to indicate the presence of serum antibodies to native CFP32 resulting from an immune response to an ongoing tubercle bacillus infection. As described in the text, statistical analyses of the data (Fisher's exact test) indicated that the rCFP32 produced in P. pastoris was superior to that trans-expressed in E.
  • Solid horizontal lines represent the mean of each category while dashed horizontal lines represent the cut-off values above which serological responses to rCFP32 were deemed positive and were determined using the mean for the healthy PPD-negative/BCG- na ⁇ ve infants (cut-off A), or the healthy BCG-vaccinated persons (cut-off B), plus 2 standard deviations.
  • OD492 nm values above cut-off B are taken to indicate the minimal presence of serum antibodies to native CFP32 resulting from an immune response to an ongoing tubercle bacillus infection.
  • the associated results of statistical analysis of the data using Fisher's exact test are shown.
  • the results of statistical analysis of the mean serological responses (calculated using the unpaired t test) are proved in the text and supported that there was a statistically significant difference in the means of each control group compared to the TB cohort.
  • Figure 4 Junction of the Saccharomyces cerevisiae ⁇ mating factor pre- propeptide (underlined) and the CFP32 protein sequence (bold italic) (SEQ ID NO: 13).
  • the Kex2 and Ste13 sites for signal sequence cleavage in P. pastohs are indicated with arrows. Arrow in bold indicate the actual cleavage of rCFP32.
  • the four amino acids added to the NH2 terminus of CFP32 are boxed.
  • FIG. 6 Expression of the CFP32 gene in yeast (Pichia pastoris). Heterologous expression of the CFP32 gene by pPICZ.577-transformed yeast as shown by Western blot. Ten ng of purified yeast rCFP32 run in 15% SDS-PAGE transferred to nitro cellulose and revealed using :
  • Figure 8 Amino acid sequence (SEQ ID NO:1) of rCFP32.
  • Figure 9 Nucleotide sequence (SEQ ID NO:2) encoding the mycobacterial polypeptide of Fig. 8.
  • Figure 10 Amino acid sequence (SEQ ID NO:3) of the mycobacterial peptide CFP10.
  • Figure 11 Nucleotide sequence (SEQ ID NO:4) encoding the mycobacterial peptide of Figure 10.
  • Figure 12 Amino acid sequence (SEQ ID NO:5) of the mycobacterial peptide ESAT-6.
  • Figure 13 Nucleotide sequence (SEQ ID NO:6) encoding the mycobacterial peptide of Figure 12.
  • Figure 14 Amino acid sequence (SEQ ID NO:7) of the mycobacterial peptide DES.
  • Figure 15 Nucleotide sequence (SEQ ID NO:8) encoding the mycobacterial peptide of Figure 14.
  • Figure 16 Amino acid sequence (SEQ ID NO:9) of the mycobacterial peptide APA.
  • Figure 17 Nucleotide sequence (SEQ ID NO: 10) encoding the mycobacterial peptide of Figure 16.
  • Figure 18 Amino acid sequence (SEQ ID NO:11) of the mycobacterial peptide EXP.
  • Figure 19 Nucleotide sequence (SEQ ID NO: 12) encoding the mycobacterial peptide of Figure 18.
  • Figure 20 Control gel of the purification of CFP10 and ESAT-6 recombinant proteins produced in Pichia pastohs.
  • Figure 21 A Control gels before and after deglycosylation with tunicamycin of recombinant CFP32 produced in Pichia in vivo.
  • Figure 21 B Control gels before and after deglycosylation with endoglycosylase F of recombinant CFP32 produced in Pichia in vitro.
  • Figure 22 Reactivity of the serums of tuberculous patients and healthy controls vaccinated with BCG with the rCFP32 product in Pichia before and after deglycosylation.
  • “Acceptable carrier” means a vehicle for containing, for instance, a recombinant mycobacterial polypeptide obtained by the method of the invention that can be injected into a mammalian host without adverse effects.
  • Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions.
  • Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i. e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
  • Epitope refers to the site on an antigen or hapten to which a specific antibody molecule binds or is recognized by T cells.
  • the term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.”
  • “Fragment” refers to an isolated portion of a polypeptide, wherein the portion of the polypeptide is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods well known to one of skill in the art.
  • the fragment of a polypeptide may be obtained by cleaving the polynucleotide encoding the recombinant polypeptide into multiple pieces, using restriction endonucleases, or a portion of the encoding polynucleotide may be synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, and then after, the resulting piece may be expressed in the yeast host expression system of this invention by recombinant nucleic acid technology well known to one of skill in the art.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the method of making monoclonal antibodies is common knowledge to a person skilled in the art.
  • “Purified polypeptide” or “purified protein” means a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in a “substantially purified” form.
  • “Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion- exchange chromatography, affinity chromatography and sedimentation according to density.
  • Recombinant broadly describes various technologies whereby genes can be cloned, DNA can be sequenced, and protein products can be produced. As used herein, the term also describes proteins or polypeptides that have been produced following the transfer of genes into the cells of host systems, such as a yeast host system.
  • Sample refers to a variety of sample types obtained from an individual and can be used in a diagnostic or detection assay. The definition encompasses blood and other liquid samples of biological origin such as saliva, sputum, and pulmonary lavage fluid.
  • Sequence identity means that an amino acid or nucleotide at a particular position in a first polypeptide or polynucleotide is identical to a corresponding amino acid or nucleotide in a second polypeptide or polynucleotide that is in an optimal global alignment with the first polypeptide or polynucleotide.
  • amino acid or nucleotide sequence "identity” are determined from an optimal global alignment between the two sequences being compared. An optimal global alignment is achieved using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. MoI. Biol. 48:443-453).
  • Substantially similar means that a first amino acid or nucleotide sequence has an amino acid or nucleotide sequence which is at least 90% identical to amino acid residues or nucleotides of a second amino acid sequence or of a nucleic acid sequence of a polypeptide of the present invention.
  • “Therapeutically effective amount” of a recombinant mycobacterial polypeptide in the present invention is that amount necessary to allow the same to perform its immunological role without causing, overly negative effects in the host to which the composition is administered. The exact amount of recombinant mycobacterial polypeptide to be used and the composition to be administered will vary according to factors such as the type of condition being treated, the mode of administration, as well as the other ingredients in the composition.
  • Transformation and transfection refer to the process of inserting a nucleic acid into a host.
  • Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules.
  • salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules.
  • immunogenic it refers to the property of a molecule or compound, such as a protein/polypeptide to induce in vivo or in vitro a cellular or humoral immune response.
  • the term "recognizing specifically” refers to anti- bodies that bind with a relatively high affinity to one or more epitopes of a recombinant mycobacterial polypeptide obtained by the method of the invention, but which do not substantially recognize and bind molecules other than the one(s) of the invention.
  • the term "relatively high affinity” means a binding affinity between the antibody and the protein of interest, namely a recombinant mycobacterial polypeptide obtained by the method of the invention, of at least 10 6 M “1 , and preferably of at least about 10 7 M "1 and even more preferably 10 8 M “1 to 10 10 M "1 . Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which are common knowledge to one skilled in the art.
  • a yeast host cell such as Pichia pastoris
  • a yeast host cell is superior to the commonly used bacteria Escherichia coli for the production of high levels of recombinant mycobacterial antigens (such as the Mycobacterium tuberculosis culture filtrate antigen CFP32, the polypeptides CFP10, ESAT-6, acyl- carrier protein desaturase DESA1 (DES), fibronectin attachment protein (APA) and cell surface protein EXP53 (EXP)) that are further qualitatively closer to the native ones, for instance in terms of immunogenic properties such as epitope configuration.
  • mycobacterial antigens such as the Mycobacterium tuberculosis culture filtrate antigen CFP32, the polypeptides CFP10, ESAT-6, acyl- carrier protein desaturase DESA1 (DES), fibronectin attachment protein (APA) and cell surface protein EXP53 (EXP)
  • An aspect of the present invention thus relates to a method for preparing such a recombinant mycobacterial polypeptide by using a yeast host cell having the ability of producing higher levels of a recombinant mycobacterial polypeptide.
  • the invention also relates to the recombinant mycobacterial polypeptide obtained by the afore mentioned method and its use in immunogenic compositions, and in methods and kits for diagnosing a Mycobacterial related disease, such as tuber- culosis.
  • the present invention provides a method for preparing a recombinant mycobacterial polypeptide containing immunogenic epitopes substantially similar to the ones of its corresponding native Mycobacterial species protein.
  • the method comprises : a) culturing a recombinant yeast cell expressing a polypeptide in a culture medium under conditions sufficient to effect expression of the polypeptide; and b) separating the polypeptide from the yeast cell or culture medium.
  • said yeast cell consists of a Pichia pastoris cell.
  • the recombinant mycobacterial polypeptide is a polypeptide derived from Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • mycobacterial polypeptide is derived from Mycobacterium tuberculosis, it is selected in the group consisting of: CFP32 peptide, CFP 10 peptide, ESAT-6 peptide, DES peptide, APA peptide and EXP peptide.
  • the CFP32 peptide comprises an amino acid sequence substantially similar to amino acid residues 5 to 264 of SEQ ID NO:1 or an amino acid sequence at least 90% identical to amino acid residues 5 to 264 of SEQ ID NO:1 ;
  • the CFP10 peptide comprises an amino acid sequence substantially similar to amino acid residues 5 to 103 of SEQ ID NO:3 or an amino acid sequence at least 90% identical to amino acid residues 5 to 103 of SEQ ID NO:3;
  • the ESAT-6 peptide comprises an amino acid sequence substantially similar to amino acid residues 5 to 98 of SEQ ID NO:5 or an amino acid sequence at least 90% identical to amino acid residues 5 to 98 of SEQ ID NO:5;
  • the DES peptide comprises an amino acid sequence substantially similar to amino acid residues 1 to 338 of SEQ ID NO:7 or an amino acid sequence at least 90% identical to amino acid residues 1 to 338 of SEQ ID NO:7;
  • the APA peptide comprises an amino acid sequence substantially similar to amino acid residues 1 to 325 of SEQ ID NO:9 or an amino acid sequence at least 90% identical to amino acid residues 1 to 325 of SEQ ID NO:9;
  • the EXP peptide comprises an amino acid sequence substantially similar to amino acid residues 1 to 284 of SEQ ID NO: 11 or an amino acid sequence at least 90% identical to amino acid residues 1 to 284 of SEQ ID NO:11.
  • the culture medium is the medium appropriate for growth of a strain or yeast cell.
  • the culture medium may include, but is not limited to Buffered Glycerol Complex Media (BMGY: 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% Yeast Nitrogen Base (YNB), 4.10 "5 % biotine, 1% glycerol) and Buffered Methanol Complex Medium (BMMY: 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% YNB, 4.10 '5 biotine, 1% methanol).
  • BMGY Buffered Glycerol Complex Media
  • YNB Yeast Nitrogen Base
  • BMMY 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% YNB, 4.10 '5 biotine, 1% methanol
  • the yeast cell as per the present invention preferably consists of a yeast cell of a Pichia pastoris cell.
  • the Pichia pastoris cell according to the present invention was deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on July 18, 2006, under accession number I-3655.
  • the recombinant yeast cell as per the present invention is the yeast deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on June 27, 2007, under accession number I-3777 or the yeast cell deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on June 27, 2007, under accession number I-3778.
  • the recombinant yeast cell is a yeast cell that has been transformed with any polypeptide according to the present invention.
  • protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest.
  • the starting material is usually a biological tissue or a microbial culture.
  • the various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.
  • the methods used in protein purification can roughly be divided into analytical and preparative methods.
  • Analytical methods aim to detect and identify a protein in a mixture
  • preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use.
  • the preparative methods can be used in analytical applications, but not the other way around.
  • Purification may be achieved by column purification, ultracentrifugation, chromatography, high performance liquid chromatography (HPLC), and electrophoresis, but a person skilled in the art will understand that other methods may be used to achieve the desired effect.
  • a chelating Sepharose Fast Flow (Amersham, Biosciences, Uppsala, Sweden) column with nickel, that selectively retains proteins containing a histidine tag is used.
  • separating the polypeptide from the yeast cell or culture medium may be achieved by gel electrophoresis, but a person skilled in the art will understand that other methods may be used to achieve the desired result.
  • the present invention concerns a recombinant mycobacterial polypeptide or a functional fragment thereof obtained by the method of the invention.
  • the polypeptide or fragment thereof of the invention has immunogenic epitopes substantially similar to the ones of its corresponding native mycobacterial protein.
  • the recombinant mycobacterial polypeptide is selected from the group consisting of a polypeptide derived from Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis. More specifically, the recombinant mycobacterial polypeptide is the antigen CFP32 peptide which derives from the Mycobacterium tuberculosis culture filtrate.
  • the CFP32 peptide comprises an amino acid sequence substantially similar to amino acid residues 5 to 264 of SEQ ID NO:1.
  • the polypeptide of the invention may be fused to an epitope tag.
  • Such an epitope tag comprises a c-myc amino acid sequence.
  • An epitope tag contemplated by the present invention consists of a c-myc-(HIS) 6 tag.
  • the c-myc-(HIS) 6 tag has an amino acid sequence consisting of amino acid residues 267 to 287 of SEQ ID NO:1.
  • polypeptide of the invention may comprise an amino acid sequence substantially similar or at least 90% identical to any one of the following: - amino acid residues 5 to 264 of SEQ ID NO: 1 ;
  • the recombinant mycobacterial polypeptide consists of the Mycobacterium tuberculosis CFP10 peptide; it preferably comprises an amino acid sequence substantially similar or at least 90% identical to amino acid residues 5 to 103 of SEQ ID NO:3.
  • the CFP10 peptide may be fused to an epitope tag.
  • the epitope tag comprises an amino acid sequence consisting of amino acid residues 107 to 126 of SEQ ID NO:3.
  • the polypeptide of the invention may comprise an amino acid sequence substantially similar or at least 90% identical to any one of the following amino acid residues 7 to 107 of SEQ ID NO:3, or amino acid residues 5 to 126 of SEQ ID NO:3.
  • the recombinant mycobacterial polypeptide consists of the Mycobacterium tuberculosis ESAT-6 peptide; it preferably comprises an amino acid sequence substantially similar or at least 90% identical to amino acid residues 5 to 98 of SEQ ID NO:5.
  • an epitope tag contemplated by the present invention consists of an amino acid sequence consisting of amino acid residues 102 to 121 of SEQ ID NO:5.
  • the polypeptide of the invention may comprise an amino acid sequence substantially similar or at least 90% identical to any one of the following: amino acid residues 5 to 102 of SEQ ID NO:5, or amino acid residues 5 to 121 of SEQ ID NO:5.
  • the recombinant mycobacterial polypeptide consists of the Mycobacterium tuberculosis peptide DES; it preferably consists of an amino acid sequence substantially similar or at least 90% identical to amino acid residues 1 to 338 of SEQ ID NO:7 .
  • the recombinant mycobacterial polypeptide consists of the Mycobacterium tuberculosis peptide APA; it preferably consists of an amino acid sequence substantially similar or at least 90% identical to amino acid residues 1 to 325 of SEQ ID NO:9.
  • the recombinant mycobacterial polypeptide consists of the Mycobacterium tuberculosis peptide EXP; it preferably consists of an amino acid sequence substantially similar or at least 90% identical to amino acid residues 1 to 284 of SEQ ID NO:1 1.
  • the recombinant mycobacterial polypeptide consists of a combination of at least two of the Mycobacterium tuberculosis peptides as chosen among the group consisting of: CFP32, CFP10, ESAT-6, DES, APA, and EXP as detailed herein above.
  • a further aspect of the invention concerns transformed or transfected yeast cells which comprises a polynucleotide encoding a polypeptide and functional fragments thereof as described above.
  • An example of such a cell is a Pichia pasto ⁇ s host cell and deposited at the CNCM on July 18, 2006, under accession number I-3655.
  • Another example of such a cell is a Pichia pastoris host cell deposited at the CNCM on June 27, 2007, under accession number I-3777.
  • a further example of such a cell is the one deposited at the CNCM on June 27, 2007, under accession number I-3778.
  • the contemplated polynucleotide of the present invention may comprise a sequence substantially similar to nucleotides 13 to 794 of SEQ ID NO:2, or a sequence substantially similar to nucleotides 13 to 828 of SEQ ID NO:2, or a sequence substantially similar to nucleotides 13 to 864 of SEQ ID NO:2.
  • the polynucleotide consists of nucleotides 1 to 864 of SEQ ID NO:2.
  • the polynucleotide of the present invention comprises a sequence substantially similar to: - nucleotides 13 to 311 of SEQ ID NO:4;
  • the polynucleotide consists of nucleotides 1 to 381 of SEQ ID NO:4.
  • polynucleotide of the present invention may also comprise a sequence substantially similar to:
  • nucleotides 13 to 296 of SEQ ID NO:6 - nucleotides 13 to 330 of SEQ ID NO:6;
  • the polynucleotide consists of nucleotides 1 to 366 of SEQ ID NO:6.
  • the polynucleotide of the present invention may further comprise a sequence substantially similar to nucleotides 1 to 1017 of SEQ ID NO:8, nucleotides 1 to 978 of SEQ ID NO: 10 or nucleotides 1 to 855 of SEQ ID NO: 12.
  • said yeast host cell it preferably comprises a polynucleotide comprising a sequence similar or at least 90% identical to:
  • yeast host cells have been deposited at the CNCM
  • Another aspect of the invention is to provide a method for diagnosing a mycobacterial related infectious disease in a subject, comprising : a) contacting a sample of a subject with a recombinant mycobacterial polypeptide of the present invention, for a time and under conditions sufficient to form a polypeptide-mycobacterial specific antibody complex; and b) detecting the presence or absence of the complex formed in a).
  • the mycobacterial related infectious disease is tuberculosis.
  • any assay system capable of detecting a complex of the polypeptide of the invention bound to mycobacterial specific antibody is suitable for this aspect of the present invention, including, but not limited to, an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent assay, a radioimmunoassay, a fluorescent immunoassay and an enzyme-linked immunosorbent as
  • kits for diagnosing a mycobacterial related infectious disease in a subject comprising: a) a recombinant mycobacterial polypeptide of the present invention or a yeast host cell of the invention; and b) reagents to detect a polypeptide-mycobacterial specific antibody immune complex.
  • the kit of the invention may optionally comprise a biological reference sample lacking polypeptides that immunologically bind with the mycobacterial specific antibody and a comparison sample comprising polypeptides which can specifically bind to the mycobacterial specific antibody.
  • one of the reagents is the monoclonal antibody to anti-CFP32 as deposited at the CNCM on June 27, 2007, under accession number 1-3779.
  • the inventors have produced a monoclonal antibody (mAb) recognizing the CFP32 protein and which could be, inter alia, used for a direct diagnostic test by antigenic capture in view of identifying the protein in biological liquids; ex. the sputum of tuberculous patients in which it was demonstrated that CFP32 is present and that the level of the latter is correlated with the level of IL-10 which is an immunosuppressive cytokine.
  • mAb monoclonal antibody
  • Yet another aspect of the invention concerns an immunogenic composition capable of inducing antibodies recognizing specifically a polypeptide obtained by the method of the invention.
  • the immunogenic composition of the invention may comprise an acceptable carrier.
  • the amount of a recombinant mycobacterial polypeptide present in the compositions of the present invention is preferably a therapeutically effective amount.
  • Example 1 Enhanced patient serum immunoreactivity to recombinant
  • Mycobacterium tuberculosis CFP32 produced in the yeast Pichia pastoris was produced in the yeast Pichia pastoris:
  • CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples of patients with active tuberculosis (TB) and to stimulate serum antibody production. Therefore, CFP32 was considered a good candidate target antigen for the rapid serodiagnosis of TB.
  • the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant (r)CFP32 produced in Escherichia coli was used as the test antibody-capture antigen, a potential limitation stemming from differences in bacterial protein post- translational modifications.
  • yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies.
  • yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Bacillus Calmette-Guerin (BCG)-vaccinated individuals, by enzyme-linked immunosorbant assay (ELISA), than E. coli rCFP32.
  • the trans-production of rCFP32 in P. pasto ⁇ s significantly improved the serologic detection of CFP32 specific antibody in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.
  • rCFP32 was produced in the yeast P. pastoris as recently described (4). Briefly, cfp32 (rvO577) cDNA was expressed in Pichia pastoris strain KM71 H as a fusion c-Myc-epitope, six histidine [(His)6]- tag recombinant protein using the plasmid pPICZ ⁇ under the control of the strong AOX1 promoter (Invitrogen Corporation, Carlsbad, Calif.). This construct also contained a Saccharomyces cerevisiae alpha mating factor pre-propeptide secretion signal that is cleaved during protein processing.
  • rCFP32 was purified using an Ni+25 -Sepharose Fast Flow column (Amersham, Piscataway, N.J.) and dialyzed against phosphate buffered saline (PBS). As a control for the potential effects due to gene trans-expression and antibiotic pressure, a second near identical plasmid was created, but with the cfp32 cassette out of frame, resulting in a failure to produce recombinant protein (data not shown).
  • Western blot analysis of native M. tuberculosis CFP32, yeast-produced rCFP32, and rCFP32 generated in E. coli. The preparation of M. tuberculosis culture filtrate, expression and purification of E.
  • coli rCFP32 as well as the generation of anti-E. coli rCFP32 in rabbit were previously described.
  • Murine antiserum to yeast-produced rCFP32 was also generated using a classical immunization protocol. Protein assays and Western blot analysis were performed as previously reported (14). Briefly, following electrophoresis of native CFP32 (present in M. tuberculosis culture filtrate), as well as yeast and E. coli rCFP32 proteins, and transfer to nitrocellulose, the membrane was reacted with rabbit anti-E.
  • coli rCFP32 sera (1 :3000 dilution in blocking buffer), mouse anti-yeast rCFP32 sera (1:2000 dilution), or anti-(His)6-tag monoclonal antibody (mAb) (Qiagen, Valencia, Calif.) (2 ⁇ g in 5 ml blocking buffer), and then reacted with the appropriate mAb-linked horseradish peroxidase (Amersham, Piscataway, N.J.). Images were developed using ECL Western blot detection reagents (Amersham), and then exposed to Kodak BioMax film. Protein quantification of M. tuberculosis lysate, as well as yeast and E.
  • BCG vaccination is nearly universal in Tunisia and given first at birth and then a second time upon entry into primary school. Only persons with suspected or with known immunodeficiency are not given BCG.
  • Stored sera from 4 infants who were M. tuberculosis uninfected (PPD-negative) and BCG vaccine na ⁇ ve were used as negative controls. All of the serum specimens were from Tunisian individuals with no HIV infection.
  • the ELISA results from TB patients were analyzed using a cut-off value equal to the mean OD for the serum samples from the healthy BCG- vaccinated controls plus 2 standard deviations (SD).
  • SD standard deviations
  • the differences between groups of TB patients and healthy BCG-vaccinated controls, or PPD negative/BCG-na ⁇ ve healthy controls were compared using the paired t test, unpaired t test, or Fisher's exact test, as indicated. Differences were considered statistically significant if the P value was ⁇ 0.05.
  • pasto ⁇ s cfp32-missense cassette transformant had been grown.
  • Antisera raised in rabbit against E. coli- produced rCFP32 recognized both the native CFP32 in the CF of M. tuberculosis strain H37Rv and the yeast-produced rCFP32, but not the yeast culture medium (BMMY) negative control (Fig. 1A).
  • the band for native CFP32 ran at approximately 32 kDa, hence its name, while that of yeast rCFP32 appeared at 35 kDa.
  • the higher band size of the yeast rCFP32 is likely attributed to the c-Myc and (His)6 tag (4), but may also have potentially received contribution from P. pasto ⁇ s-specific post-translational modifications as well (4).
  • the presence of the c-Myc epitope in the purified yeast rCFP32 product was previously confirmed by Western blot using a specific anti-Myc mAb (4).
  • the immunogenicity of the CFP32 proteins from various sources was then comparatively evaluated in Western blot experiments using different specific reactive antibody preparations. These included the polyclonal rabbit antiserum raised against E. coli expressed rCFP32, a polyclonal mouse antiserum raised against yeast-expressed rCFP32, and a commercially available anti-(His)6-tag mAb of murine origin (Fig. 1 B). Each antibody preparation reacted more strongly towards the yeast rCFP32 than the E. co//-expressed rCFP32 for the same calculated amount of input protein, regardless of the immunogen originally used to derive the antibodies or the respective species in which the antibodies were raised (Fig. 1 B).
  • the sera from every TB patient and healthy BCG vaccinee that was tested reacted more strongly to the yeast rCFP32 than the E. coli rCFP32.
  • there was a greater difference in mean OD492 nm ( ⁇ OD492 nm) response to yeast rCFP32, as compared to E. coli rCFP32, in TB patient cases than there was in BCG-vaccinated persons ( ⁇ OD492 nm 0.75 versus 0.31 , respectively).
  • the mean OD492 nm of TB patients to yeast rCFP32 was also greater than that of the sera from BCG-vaccinated healthy persons (respectively, 1.19 versus 0.39) (P ⁇ 0.0001 , unpaired t test) (Fig. 2).
  • P ⁇ 0.0001 unpaired t test
  • this cohort included each of the samples previously evaluated (Fig. 2) in order to prove consistency of results.
  • the average serological response to yeast rCFP32 in OD492 nm units was significantly higher in TB patients (P ⁇ 0.0001 , unpaired t test).
  • the mean OD492 nm value for TB patients and PPD-negative/BCG-na ⁇ ve controls was 1.28 and 0.23, respectively (with a mean ⁇ OD492 nm of 1.05).
  • yeast rCFP32 was able to diagnose 85.0% of TB patients and misclassified only one BCG-vaccinated healthy serum sample as being reactive (or positive) for M. tuberculosis infection, resulting in a specificity of 97.5%. This donor was also positive in the preliminary evaluation (Fig. 2). All TB patients previously determined to be serologically positive for anti- CFP32 antibodies in Figure 2 were positive in the secondary evaluation illustrated in Figure 3. The positive predictive (97.1%) and negative predictive (90.4%) values of the yeast rCFP32 ELISA were very good as well. Therefore, even though a single antigen was utilized in the assay, the sensitivity and specificity of the yeast rCFP32 TB test approached or surpassed the values of these measures from multi-antigen serodiagnostic tests for TB 1 (17, 18, 37).
  • TB is a leading cause of morbidity and mortality worldwide. Indeed, the World Health Organization recently declared a new TB emergency for Africa, where the incidence rate of infections has tripled in many countries since 1990 (39). The lack of a low-cost, easy to perform, rapid, sensitive, and specific TB diagnostic test impedes patient treatment and transmission control measures, especially in resource-poor countries. Standard bacteriologic culture is slow, the sensitivity of AFB morphologic identification is suboptimal, and molecular methods for diagnosis of TB based on nucleic acid amplification remain out of reach of most high TB-burdened countries due to cost and technical restrictions. On the other hand, a diagnostic method for TB that directly detects patient antibodies to M.
  • tuberculosis components has the technological potential to overcome these limitations and may as well provide additional benefits as well.
  • a low cost, real time, point-of-care serologic test could theoretically be developed into a simple dipstick format for both serum and urinary antibody detection, thereby minimizing infectious material handling and laboratory infrastructure.
  • Such a TB test may also be applied to situations where diagnosis can be problematic, as with extrapulmonary TB patients, children, the elderly, and immucompromised individuals or other smear-negative culture-negative cases.
  • the serodiagnostic methods for TB are not adequately sensitive, poorly specific, or both (16). It is generally accepted that the most likely format of a truly robust serological assay for TB will incorporate a combination of several different M.
  • tuberculosis antigens including proteins such as ESAT-6 and CFP10 that are absent in BCG, thereby allowing a necessary differentiation of M. tuberculosis- ' mfected persons from BCG vaccinees.
  • the inventors investigated whether the means of antigen production may be impeding the development of an optimized serodiagnostic test for TB.
  • M. tuberculosis In the case of M. tuberculosis, its slow growth, coupled with safety concerns, precludes the commercial viability of an immunoassay incorporating mass-produced native protein(s) as the antibody capture antigen(s). In so being, in order to produce a robust serological assay to detect specific, and possibly conformationally sensitive, antibodies, a source of high yield, highly pure, and correctly folded, recombinant protein(s) that would give additional discrimination advantages in the serodiagnosis of TB, needs be identified and evaluated (7). With each of these considerations in mind, the inventors synthesized a recombinant form of a M. tuberculosis CF protein in the yeast P. pastoris and then evaluated its immunogenicity and serodiagnostic potential as compared to the same protein expressed in E. coli.
  • CF proteins are secreted or released by growing M. tuberculosis into the culture medium.
  • One such M. tuberculosis CF protein is CFP32, a 32- kDa putative bimodular glyoxalase of unknown function.
  • CFP32 has been shown to be expressed in the lungs of TB patients and is known to stimulate a humoral antibody response (14, 27).
  • CFP32 is restricted to members of the MTC and has not been identified in environmental mycobacteria (14, 15). Recently, CFP32 was recognized as the enzymatic mediator of M.
  • tuberculosis-spec ⁇ f ⁇ c neutral red dye cytochemical staining a classical test once used to differentiate virulent M. tuberculosis from non- tuberculous mycobacteria (3). Therefore, in being a conserved MTC-restricted antigenic CF protein, the inventors believed CFP32 to be a prime antigen for use in a specific serodiagnostic test for TB.
  • coli rCFP32 as the screening antigen (14) and also performed better than the parallel E. coli rCFP32-based immunoassay to which it was compared in the present study. Moreover, not only was the serologic response of TB patients and BCG-vaccinated persons significantly greater for yeast rCFP32 than E. coli rCFP32, but the differential mean reactivity between these groups was greater in the case of rCFP32 produced in yeast, elevating the usefulness and appeal of this antigen for use as a serodiagnostic marker. Because CFP32 is expressed by all MTC subspecies, including BCG (14), it was not unexpected that residual antibodies were present in individuals BCG- vaccinated over two decades previous.
  • the sensitivity of the yeast rCFP32-based assay dropped by 15%.
  • the BCG-vaccinated cohort arguably represents a more relevant control than the PPD-negative/BCG-na ⁇ ve group in a TB serodiagnosic test applied in certain contexts such as Tunisia; the important point being, that even in this situation, the yeast rCFP32-based assay remained highly sensitive and specific.
  • the inventors did not evaluate a cohort comprised of healthy PPD-positive, presumably M. tuberculosis-exposed, persons. Such a cohort of latently infected persons will be included in a wider evaluation currently in the planning stages. At present, the inventors do not know if the yeast rCFP32 will be able to segregate latent TB from active disease. The inventors hope that the assay will allow us to predict which persons are likely to reactivate latent TB or are manifesting early active disease. In any case, based upon the present data, the rCFP32 produced in P. pastoris seems to have good potential for the serological diagnosis of TB and underscores the need to produce recombinant protein in systems that approximate the native antigen. It is further worth mentioning that in a separate study, and unlike the E. coli version of rCFP32, the yeast-generated rCFP32 exhibited functional properties similar to those of native CFP32 (61).
  • a multivalent serological assay incorporating yeast rCFP32 may overcome these restrictions and serve as a confirmation or replacement of PPD screening.
  • the combined data support that BCG expresses immunogenic CFP32 in vivo but, as indicated by the data from the other excluded individual who was BCG-vaccinated for a third time and had a good serological response to yeast rCFP32, this response wanes over time.
  • BCG is known to protect against certain forms of childhood TB (2).
  • coli rCFP32 both by laboratory raised antibodies and by sera from TB patients or persons vaccinated with BCG. Testing with yeast rCFP32 resulted in a 85.0% sensitivity in diagnosing TB patients and a specificity of 97.5% when using healthy BCG- vaccinated persons to establish a cut-off.
  • Our data provide a sound basis for larger scale comparison between rCFP32 produced by P. pastoris, E. coli, and mycobacteria as serologic test antigens to discriminate active TB from latent M. tuberculosis infection or prior BCG vaccination. Together with CFP32, a panel of M. tuberculosis antigens expressed in yeast may provide a rapid and low cost approach to diagnosis TB with a sensitivity that is at least comparable to mycobacterial culture.
  • Example 2 High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris. Unavailability of large quantities for Mycobacterium tuberculosis (MTB) proteins still remain a major obstacle to the development of subunit vaccine and diagnostic reagents for tuberculosis. E. Coli has proven not to be an optimal host for the expression of MTB genes. In this work, the inventors used the yeast Pichia pastoris to express high level of CFP32, a culture filtrate protein restricted to MTB complex and a potential target antigen, for the sero diagnostic of tuberculosis. The inventors generated a P.
  • Plasmids The pPICZ ⁇ A (Invitrogen Corporation, Carlsbad, CA, USA) vector was used to transfer the CFP32 expression cassette to Pichia pastoris.
  • CFP32 cDNA was amplified by PCR using plasmid pQE31/RvO577 as template.
  • KM71 H strain (Invitrogen Corporation, Carlsbad, CA, and USA.) was used to express a secreted rCFP32.
  • the amplified DNA was ligated to the transfer vector pPICZ ⁇ A, to be fused to the secretion signal of the alpha matting factor from Saccharomycess cerevisea under the control of the alcohol oxidase 1 promoter (AOX1).
  • AOX1 alcohol oxidase 1 promoter
  • the resulting expression cassette was verified by DNA sequencing. Standard techniques (Sambrook et al. 1989) [59] were used for DNA modification, ligation and plasmid transformation. Restriction endonucleases and other enzymes were used as recommended by the supplier (Invitrogen Corporation, Carlsbad, CA, USA.)
  • Plasmid DNA was isolated from the selected transformants E.coli clone. It was digested with Bst X I restriction enzyme (Amersham). The digested DNA was used for the transformation of Pichia pastoris KM71 H strain by electroporation. Linear DNA can generate stable transformants of Pichia pastoris via homologous recombination between the transforming DNA and the DNA cassette flanked by regions of homology within the genome.
  • Genomic DNA of a number of transformants was analyzed by PCR.
  • the isolation of genomic DNA and PCR amplification were then carried out according to the Pichia manual, Invitrogen Corporation, Carlsbad, CA, USA. Primers complementary to the 5' and 3' region of the Aox 1 gene were used for PCR amplification.
  • the insertion of the expression cassette into yeast genome was verified by DNA sequencing using a conventional Big Dye Terminator cycle sequencing ready reaction kit (Perkin Elmer, Applied Biosystems, Foster city, CA, USA) and an ABI373 automated DNA sequencer.
  • a recombinant Pichia pastohs clone was inoculated into 5 ml of YPD media with 5 ⁇ g of Zeocin and incubated at 30 c C in a shaker at 250 rpm over night.
  • the culture was transferred into 250 ml of Buffered Glycerol Complex Media (BMGY: 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% Yeast Nitrogen Base, 4.10 "5 % biotine, 1 % glycerol) in a 2 L baffled flask and incubated at 30 0 C in a shaking incubator at 250 rpm over night.
  • BMGY Buffered Glycerol Complex Media
  • BMGY 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% Yeast Nitrogen Base, 4.10 "5 % biotine, 1 % glycerol) in a 2 L baffled flask and incuba
  • BMMY 1% yeast extract, 2% peptone, 10OmM potassium phosphate pH 6, 1.34% YNB, 4.10 "5 boitine,1% methanol
  • Cultures were centrifuged at 120Og for 10 min and the supernatant was collected.
  • proteins were transferred into a nitrocellulose membrane (Amersham, Biosciences, Uppsala, Sweden) using a Multiphor Il Electrophoresis System (Bio-Rad Laboratories, California, USA).
  • the transblotted membrane was blocked with PBS-5% fat milk- 0.1% Tween 20 (PBS-T-Fat milk) overnight at 4°C.
  • Two types of western blot were performed. In the first one a monoclonal anti-myc HRP antibody (Invitrogen, R951-25) was used. For the second one, membrane was probed with a rabbit polyclonal antibody directed against CFP32.
  • the membrane was incubated for one hour with the anti-myc-HRP antibody and then incubated for one minute with ECL solution (Amersham Biosciences, Uppsala, Sweden).
  • ECL solution Amersham Biosciences, Uppsala, Sweden
  • DAKO anti- rabbit HRP conjugate
  • the amount of the purified fusion protein CFP32-myc-(His) 6 was quantified after dialysis for 48 hours against PBS buffer, using a protein kit from Sigma (Ref.BCA-1) according to the manufacturer's instruction.
  • the recombinant CFP32 was immobilized in a 96 wells ELISA plate overnight at 4°C (2ug per well) and incubated with serum from patients with tuberculosis diluted 1 :200 in PBS.
  • the bound immunoglobulins were detected by adding 100 ⁇ l of horseradish peroxidase (HRP)-conjugate sheep anti-human IgG. Binding was revealed by the addition of 150 ⁇ l of 1mg/ml OPD substrate.
  • the plates were incubated for colour development then blocked with 3 N acid sulphuric and the absorbance's were determined at 492 nm with a microtiter plate reader.
  • the inventors have inserted at the 3' end of the cfp32 cDNA an additional codon specific of alanine [A].
  • A alanine
  • two additional residues, Glutamic acid (E) and Phenylalanine (F) were also inserted at the junction of the ⁇ mating factor and the CFP32 amino acid sequence (FIG.4).
  • This expression cassette was inserted into Pichia pastoris strain KM71 H genome by homologous recombination. The inventors have selected 3 clones that resisted 2000 ⁇ g/ml of zeocin and retained the highest expressing one for the production of rCFP32 in shake-flask.
  • the protein was eluted as a single peak at 500 mM Imidazole concentration, and was seen as a single band with diffused staining pattern when analyzed by SDS-PAGE (FIG.5).
  • This band likely corresponds to the recombinant CFP32 that has lost the myc-(His) 6 tag due to spontaneous cleavage or proteolysis, a feature that the inventors have already observed with fusion proteins produced in P. pastohs KM71 H. [60] Furthermore, a preliminary ELISA study using the rCFP32 produced in P. pastoris and seven sera from tuberculosis patients showed a strong reactivity with all the sera as compared to four healthy BCG vaccinated individuals [Figure 7].
  • N-terminal sequencing of HPLC purified rCFP32 was performed to determine if cleavage of the Saccharomyces cerevisiae ⁇ mating factor, signal peptide was properly processed. A single N terminus end was found and corresponded to the Glu-Ala-Glu-Phe-Pro-Lys-Arg-Ser-Tyr-Arg-Gln sequence (SEQ ID NO:27). The four amino acids Glu-Ala-Glu-Phe (SEQ ID NO:26) at the N-terminus correspond to the two last residues of the ⁇ factor [GIu-AIa] followed by the two residues [Glu-Phe] introduced at the cloning site.
  • Proline is the first residue of CFP32 originally expressed by Mycobacterium (FIG.4).
  • CFP32 including the extra NH 2 Glu-Ala-Glu-Phe (SEQ ID NO:27) residues and the extra Ala at the COOH terminus was analyzed using several protein analysis softwares for the presence of postranslational modification motif. As shown in
  • the inventors have generated a P. Pastoris clone that produces a high level of rCFP32 as a myc-(His) 6 tagged protein. Because of cloning constraints, the inventors designed an expression cassette that introduced two extra residues, glutamic acid and phenylalanine at the NH2 terminus of the molecule as well as an alanine at the COOH end.
  • Alanine was chosen because it's a neutral amino acid that is unlikely to affect the proper folding of the protein. Purification was carried out in one single step and the average yield obtained was 0.5gr per litre of culture supernatant. This is a high production yield considering that the culture was performed in shake flask and that no specific optimization procedure was carried out in any of the production steps.
  • the higher apparent molecular weight observed for the rCFP32 produced in P. pastoris is probably due to the production of this molecule as a fusion protein containing a myc-(His) 6 tag at the COOH end.
  • Yeast specific post translational modifications probably contribute to the size difference as suggested by the diffused banding pattern observed in SDS-PAGE.
  • the identification of putative acylation and glycosylation sites in the CFP32 amino acid sequence argue in favor of post translational modification of the recombinant form produced in Pichia pastoris.
  • N-terminal sequencing suggested that cleavage of the ⁇ mating factor signal peptide, was essentially carried out by the Ste13 protease.
  • the Saccharomyces cerevisiae gmating factor pre-propeptide can be cleaved at different sites by two proteases, namely Kex2 and Ste13 and variation of cleavage was observed for a number of recombinant protein expressed in P. pastoris [19, 60].
  • Proper cleavage of the heterologous signal peptide is important to yield a recombinant protein that is as close as possible to the native protein.
  • Recombinant CFP32 produced in P. pastoris is recognized by sera from patients with tuberculosis suggesting that yeast rCFP32 is similar to the native CFP32 and thereby have immunogenic epitopes for antibodies developed in the patient to native CFP32. These immunogenic epitopes could be due either to a protein folding nearly identical to that of the native CFP32 and/or to post translational modifications that are common in Mycobacterial antigens.
  • the identification in the CFP32 amino acid sequence of a number of putative motifs for post translational modifications, that can be performed by P. pastohs, [57, 58] also argue in favor of this possibility.
  • the high solvent accessibility index observed in rCFP32 is an indicator of high antigenicity and could explain the good reactivity of rCFP32 with tuberculosis patients' sera (100% in our preliminary study as compared to the 30% previously reported with rCFP32 produced in E. CoIi [14]).
  • the rCFP32 produced in P. pastohs could be very useful in conducting further analysis of the structure-function of the protein mainly, its potential role in the pathogenesis of tuberculosis as suggested earlier [3, 14]. Furthermore, reactivity of this rCFP32 with tuberculosis patients' sera is being investigated with a larger panel of patients (4).
  • Table 1 Putative postranslational modification sites present in the amino aid sequence of Mycobacterium tuberculosis culture filtrate protein CFP32. [PKC Protein Kinase C 1 CK2 Casein kinase II, Single letter amino acid nomenclature is used]. One glycosylation and two phosphorylation sites were identified. Six acylation [myristoylation] motifs are scattered throughout the sequence.
  • ⁇ means any amino acid
  • Example 3 High level expression of recombinant Mycobacterium tuberculosis proteins CFP10 and ESAT-6 in Pichia pastoris.
  • Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neural red character when expressing the RvO577 gene.
  • FEMS Microbiol. Lett. 231 :283-289. Benabdesselem, C 1 Barbouche, M. R., Jarboui, M.A., Dellagi, K., Ho, J. L., Fathallah, D. M. 2007. High Level Expression of Recombinant Mycobacterium tuberculosis Culture Filtrate Protein CFP32 in Pichia pastoris. MoI. Biotechnol. 35(1): 41-50.
  • the Mycobacterium tuberculosis complex- restricted gene cfp32 encodes an expressed protein that is detectable in tuberculosis patients and is positively correlated with pulmonary interleukin- 10. Infect. Immun. 71 :6871-83.
  • Electrophoresis 21 , 3740-3756. 44. Korepanova, A., Gao, F.P., Hua, Y., Qin, H., Nakamoto, R.K., Cross, T.A. (2005) Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli. Prot Sci. 14, 1 , 148-58.

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Abstract

L'invention concerne un procédé de préparation d'un polypeptide mycobactérien qualitativement analogue à la protéine mycobactérienne native. L'invention concerne également le polypeptide bactérien obtenu par le procédé de l'invention et son utilisation dans un procédé et dans un kit destinés à diagnostiquer des maladies associées à des mycobactéries, notamment la tuberculose.
PCT/IB2007/002936 2006-07-04 2007-07-04 Procédé de préparation de polypeptides mycobactériens recombinés de levure et leur utilisation pour diagnostiquer des maladies associées à des mycobactéries WO2008010096A2 (fr)

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WO2015168112A1 (fr) * 2014-04-29 2015-11-05 Conagen, Inc. Bioxynthèse d'ergothionéine microbienne
CN110862969A (zh) * 2019-11-28 2020-03-06 扬州大学 分泌抗cfp-10抗体的杂交瘤细胞株、其抗体及其应用

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CEREGHINO JOAN LIN ET AL: "Heterologous protein expression in the methylotrophic yeast Pichia pastoris" FEMS MICROBIOLOGY REVIEWS, ELSEVIER, AMSTERDAM, NL, vol. 24, no. 1, January 2000 (2000-01), pages 45-66, XP002195512 ISSN: 0168-6445 *

Cited By (5)

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WO2015168112A1 (fr) * 2014-04-29 2015-11-05 Conagen, Inc. Bioxynthèse d'ergothionéine microbienne
CN106661585A (zh) * 2014-04-29 2017-05-10 科纳根公司 微生物麦角硫因生物合成
US10544437B2 (en) 2014-04-29 2020-01-28 Conagen Inc. Microbial ergothioneine biosynthesis
CN110862969A (zh) * 2019-11-28 2020-03-06 扬州大学 分泌抗cfp-10抗体的杂交瘤细胞株、其抗体及其应用
CN110862969B (zh) * 2019-11-28 2021-08-24 扬州大学 分泌抗cfp-10抗体的杂交瘤细胞株、其抗体及其应用

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