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Title:  Process for producing a recombinant protein in a mycobacterium host plasmid pJAM2
United States Patent: 
7,264,815
Issued: 
September 4, 2007

Inventors: 
Jackson; Mary (Paris, FR), Gicquel; Brigitte (Paris, FR)
Assignee: 
Institut Pasteur (Paris, FR)
Appl
. No.: 
11/324,517
Filed: 
January 4, 2006


 

Woodbury College's Master of Science in Law


Abstract

This invention relates to a novel mycobacterial protein named DES, which appears to share significant amino acid sequence homology with soluble stearoyl-ACP desaturases. The results of allelic exchange experiments, indicate that the des gene may be essential to the survival of mycobacteria. These results coupled with the surface localization, the unique structure of DES, and the fact this antigen is expressed in vivo, and DES protein induces a humoral response in human patients, indicate that the DES protein provides a new target for the design of anti-mycobacterial drugs. This invention provides methods of screening molecules that can inhibit the DES enzyme activity of purified DES protein, in order to identify antibiotic molecules that are capable of inhibiting the growth or survival of mycobacteria. These methods may be practiced by using recombinant DES protein obtained from a recombinant mycobacterium host cell that was transformed with a vector containing the des gene, whose expression is controlled by regulatory or promoter sequences that function in mycobacteria. Another aspect of this invention relates to the molecules that have been identified according to the screening methods as having antibiotic activity: against mycobacteria.

SUMMARY OF THE INVENTION

We have characterized a new M. tuberculosis exported protein identified by using the PhoA gene fusion methodology. The des gene, which seems conserved among mycobacterial species, encodes an antigenic protein highly recognized by human sera from both tuberculosis and leprosy patients but not by sera from tuberculous cattle. The results of allelic exchange experiments described in this application, indicate that the des gene is essential to the survival of mycobacteria.

The amino acid sequence of the DES protein contains two sets of motifs that are characteristic of the active sites of enzymes from the class II diiron-oxo protein family. Among this family, the DES protein presents significant homologies to soluble stearoyl-acyl carrier protein (ACP) desaturases. Three dimensional modeling demonstrates that the DES protein and the plant stearoyl-ACP desaturase share a conserved active site.

This invention also provides methods of identifying molecules capable of inhibiting the growth and/or survival of Mycobacteria species. In particular, the methods of this invention include screening molecules that can inhibit the activity of the DES protein. These methods comprise the steps of: a) contacting the molecule with a strain of mycobacteria species containing an active DES protein or a DES like protein or a vector carrying an active DES protein gene or a vector containing a polynucleotide sequence encoding the active site of the DES protein; b) measuring the inhibition of the growth of said mycobacteria strain; and c) identifying the molecule that is reacting with the DES protein or with the active site of said protein carrying conserved residues.

To practice the methods of this invention, the purified DES protein may be a recombinant desaturase protein. The recombinant DES protein can be obtained from a recombinant mycobacterium host cell that was transformed with an expression vector containing a polynucleotide encoding the DES protein whose expression is it controlled by regulatory sequences that function in mycobacteria. In one method of the invention, the recombinant expression vector is a plasmid derived from the pJAM2 plasmid (e.g. pJAM21). The invention also encompasses the pJAM2 and pJAM21 plasmids, as well as recombinant host cells transformed with the pJAM2 and pJAM21 plasmids. A recombinant host cell transformed with pJAM21 has been deposited at Collection Nationale de Cultures de Micro-organisms (CNCM) in Paris, France, on Jun. 23, 1998, under accession number I-2042.

Another aspect of this invention relates to molecules that have been screened according to the methods of this invention and identified as having antibiotic activity against mycobacteria.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

Using the Pho A gene fusion methodology, we identified a new 37 kDa Mycobacterium tuberculosis protein, designated DES. This 37 kDa exported protein contains conserved amino acid residues which are characteristic of class II diiron-oxoproteins. Proteins from that family are all enzymes that require iron for activity. They include ribonucleotide reductases, hydrocarbon hydroxylases and stearoyl-ACP desaturases. The M. tuberculosis DES protein only presents significant homologies to plant stearoyl-ACP desaturases (44% identity at the nucleotide level, and 30% identity at the amino-acid level), which are exported enzymes as they are translocated across the chloroplastic membranes (Keegstra & Olsten, 1989).

Three-dimensional modeling of the DES protein based on homology with the Ricinus communis .DELTA.9 stearoyl-ACP indicates that the DES protein shares significant structural features with the plant stearoyl-ACP desaturases. Most importantly, the active site of the DES protein and the plant .DELTA.9 stearoyl-ACP desaturase are conserved, suggesting that DES is evolutionarily related to the plant desaturases.

The plant stearoyl-ACP desaturase can be used for the screening and the selection of new compounds inhibiting the activity of the enzyme and consequently then tested for the modulation of the properties of DES protein in vivo in a mycobacterial strain, such as M. tuberculosis or in vitro on a purified DES protein. This result suggests that the DES protein could be involved in the mycobacterial fatty acid biosynthesis.

Furthermore, the localization of the protein outside the cytoplasm would be consistent with its role in the lipid metabolism, since lipids represent 60% of the cell wall constituents and that part of the biosynthesis of the voluminous mycolic acids containing 60 to 90 carbon atoms occurs outside the cytoplasm. Among all the different steps of the lipid metabolism, desaturation reactions are of special interest, first because they very often take place at early steps of lipid biosynthesis and secondly because, through the control they have on the unsaturation rate of membranes, they contribute to the adaptation of mycobacteria to their environment (Wheeler & Ratledge, 1994). An enzyme system involving a stearoyl-Coenzyme A desaturase (analog of the plant stearoyl-ACP-desaturases), catalyzing oxydative desaturation of the CoA derivatives of stearic and palmitic acid to the corresponding .DELTA.9 monounsaturated fatty acids has been biochemically characterized in Mycobacterium phlei (Fulco & Bloch, 1962; Fulco & Bloch, 1964; Kashiwabara et al., 1975; Kashiwabara & Sato, 1973). This system was shown to be firmly bound to a membranous structure (Fulco & Bloch, 1964). Thus, M. tuberculosis stearoyl-Coenzyme A desaturase (.DELTA.9 desaturase) is expected to be an exported protein.

Sonicated extracts of E. coli expressing the DES protein were assayed for .DELTA.9 desaturating activity according to the method described by (Legrand and Bensadoun, 1991), using (stearoyl-CoA) .sup.14C as a substrate. However, no .DELTA.9 desaturating activity could be detected. This result is probably linked to the fact that desaturation systems are multi-enzyme complexes involving electron transport chains and numerous cofactors, often difficult to render functional in vitro. Since E. coli and mycobacteria are very different from a lipid metabolism point of view, in E. coli, the M. tuberculosis recombinant .DELTA.9 desaturase might not dispose of all the cofactors and associated enzymes required for activity or might not interact properly with them. Moreover, not all cofactors involved in the .DELTA.9 desaturation process of mycobacteria are known, and they might be missing in the incubation medium.

However, if the DES protein encodes a .DELTA.9 desaturase, an interesting point concerns its primary sequence. Indeed, all animal, fungal, and the only two bacterial .DELTA.9 desaturases sequenced to date (Sakamoto et al., 1994) are integral membrane proteins which have been classified into a third class of diiron-oxo proteins on the basis of their primary sequences involving conserved histidine residues (Shanklin et al., 1994). The plant soluble .DELTA.9 desaturases are the only desaturases to possess the type of primary sequence of class II diiron-oxo proteins (Shanklin & Somerville, 1991). No bacteria have yet been found which have a plant type .DELTA.9 desaturase.

As shown by immunoblotting and ELISA experiments, the DES protein is a highly immunogenic antigen which elicits a B-cell response in 100% of the tuberculosis M. bovis or M. tuberculosis-infected human patients tested, independently of the form of the disease (extrapulmonary or pulmonary). It also elicits an antibody response in lepromatous leprosy patients. Interestingly, although more sera would need to betested, tuberculous cattle do not seem to recognize the DES antigen. Furthermore, the ELISA experiments showed that it is possible to distinguish tuberculosis patients from patients suffering from other pathologies on the basis of the sensitivity of their antibody response to the DES antigen. The DES antigen is therefore a good candidate to be used for serodiagnosis of tuberculosis in human patients. Non-tuberculous patients may recognize the DES protein at a low level because they are all, BCG-vaccinated individuals (BCG expressing the protein), or because of cross-reactivity of their antibody response with other bacterial antigens. It would now be interesting to know whether the DES antigen possesses in addition to its B-cell epitopes, T-cell epitopes, which are the only protective epitopes in the host immunological response against pathogenic mycobacteria. If the DES protein is also a good stimulator of the T-cell response in a majority of tuberculosis patients, it could be used either individually or as part of a cocktail of antigens in the design of a subunit vaccine against tuberculosis.

To gain insights into the precise function of this atypical bacterial enzyme, we attempted to interrupt the des gene in the vaccine strain M. bovis BCG by allelic exchange. In a first experiment, no allelic exchange mutants were obtained, suggesting that the des gene is essential to the viability of mycobacteria. To investigate this hypothesis, the first experiment was repeated using a M. bovis BCG strain transformed with a second wild-type copy of the des gene. Using this transformed M. bovis BCG strain, we obtained allelic exchange mutants, in which a wild-type copy of the des gene was replaced by an inactivated copy of the des gene. Thus, allelic exchange was only possible if a second copy of the wild-type des gene had been inserted into the M. bovis BCG chromosome. This result strongly suggests that des is an essential gene in mycobacteria from the M. tuberculosis complex.

Coupled with the localization of DES at the surface of the tubercle bacilli, and its structural originality (this enzyme's structure differs from all the mammalian and bacterial desaturase structures identified to date), the results of these experiments suggest that the DES protein could be a target for designing new anti-mycobacterial drugs.

Fundamental to the analysis of the biological function and immunological relevance of mycobacterial proteins is their production in a recombinant form that resembles that of their native counterpart. Recent studies analyzing both structure (Garbe et al., 1993; Triccas et al., 1996) and immunogenicity (Garbe et al., 1993; Roche et al., 1996; Triccas et al., 1996) of recombinant proteins obtained from fast growing mycobacterial hosts, such as Mycobacterium smegmatis, have demonstrated superiority over the same protein purified from E. coli expression systems. Although such approaches for the production of recombinant mycobacterial proteins appear advantageous, two major obstacles lie in the way of further improvement to these systems. The first is the inability to regulate high-level expression of foreign genes in M. smegmatis, analogous to systems such as induction of the lac promoter in E. coli (de Boer et al., 1983). Secondly, no simple, efficient and widely adaptable method for the purification of proteins from recombinant mycobacteria has been described.

In this application, we attempt to resolve these two problems. First, we describe the construction of a vector, pJAM2, that utilizes the promoter of the inducible acetamidase enzyme of M. smegmatis to drive high-level expression of foreign genesin M. smegmatis. The 47 kDa acetamidase enzyme of M. smegmatis NCTC 8159 permits the growth of the organism on simple amides as the sole carbon source and is highly inducible in the presence of acetamide (Mahenthiralingam et al., 1993). This property has been previously used to assess luciferase as a reporter of gene expression in mycobacteria (Gordon et al., 1994) and to develop a mycobacterial-conditional antisense mutagenesis system (Parish et al., 1997b). In this study, we constructed a vector that allows for regulated high-level expression of foreign genes in mycobacteria by virtue of the M. smegmatis acetamidase promoter.

Recombinant M. leprae 35 kDa antigen produced in this system represented approximately 8.6% of the total M. smegmatis soluble protein, with the amount of protein produced greater than that when the same gene is placed under the control of the strong mutated .beta.-lactamase promoter of M. fortuitum.

Secondly, we demonstrate the simple and efficient purification of the encoded antigens by use of a poly-histidine tag and one step Ni.sup.++ affinity chromatography. The addition of the histidine tag did not appear to affect the conformation or immunogenicity of the recombinant protein, suggesting the system described may be extremely useful for the purification of structurally and immunologically intact recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

The ability to produce recombinant products in a form that closely resembles their native state is important in the study of microbial antigens and enzymes. Recent studies have highlighted the superiority of recombinant protein purified from mycobacterial hosts compared to E. coli-derived products, as assessed by structural and immunological analysis (Garbe et al., 1993; Roche et al., 1996; Triccas et al., 1996). Previously we have demonstrated that sera from leprosy patients would only recognize the M. leprae 35 kDa. protein if the antigen was produced in a form that resembles the native protein, based on the binding of conformational dependent mAbs and FPLC size exclusion analysis (Triccas et al., 1996). We reconfirm such a finding with protein produced using the acetamidase promoter expression system. Furthermore, the addition of 6 histidine residues to the C-terminus of the recombinant protein does not appear to affect its conformation, as there is little difference in the recognition of leprosy sera by histidine-tagged and nonhistidine-tagged 35 kDa protein. The efficient expression of the 6-histidine tag in mycobacteria and the simple and effective purification of our model protein by Ni-NTA affinity chromatography suggest that this versatile purification system, used successfully in a number of eucaryotic and procaryotic expression systems (Crowe et al., 1994), could be more widely applied to mycobacterial proteins. Furthermore, the histidine purification system overcomes the problems involved with antibody affinity chromatography used in a number of studies to purify recombinant mycobacterial proteins (Roche et al., 1996; Triccas et al., 1996), such as the unavailability of appropriate antibodies or the presence of homologues capable of binding the antibody. Together, these results suggest an application for the pJAM2 expression vector in the production of native-like recombinant mycobacterial proteins that can be exploited to correctly analyze protein function and antigenicity.
 

Claim 1 of 16 Claims

1. A process for producing a recombinant protein in a Mycobacterium host, comprising: a) cloning a DNA sequence comprising all or part of a gene encoding a Mycobacterium protein into plasmid pJAM2, b) introducing the recombinant pJAM2 obtained in a) into a Mycobacterium host, c) selecting transformed colonies of the Mycobacterium host, and d) cultivating the transformed colonies in a medium comprising acetamide to allow expression of the recombinant protein; wherein the recombinant protein comprises a 6.times.HIS tag.
 

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