COMMERCIAL PRODUCTION OF ALLERGEN AMB A 1 BY MEANS OF TRANSIENT EXPRESSION IN PLANTS

Abstract

A plant cell including a DNA molecule having at least one heterologous nucleotide sequence encoding a preproprotein of a pectate lyase chosen from Amb a 1, the alpha subunit of Amb a 1, the beta subunit of Amb a 1, and homologs of Amb a 1, functionally bound to a strong promoter.

Claims

1-17. (canceled)

18. A plant cell comprising a DNA molecule comprising at least one heterologous nucleotide sequence encoding a preproprotein of a pectate lyase chosen from Amb a 1, the alpha subunit of Amb a 1, the beta subunit of Amb a 1, and homologs of Amb a 1, functionally bound to a strong promoter.

19. The plant cell as claimed in claim 18, wherein said DNA molecule is not integrated into the plant cell genome.

20. The plant cell as claimed in claim 18, wherein the pectate lyase is chosen from Amb a 1.0101, Amb a 1.0201, Amb a 1.0202, Amb a 1.0301, Amb a 1.0302, Amb a 1.0303, Amb a 1.0304, Amb a 1.0305, Amb a 1.0401, Amb a 1.0402, Amb a 1.0501, Amb a 1.0502, the alpha subunit of Amb a 1, the beta subunit of Amb a 1 and proteins of the Asteraceae family which have at least 57% identity, preferably at least 60% identity, preferably at least 64% identity, with one of the isoforms of Amb a 1.

21. The plant cell as claimed in claim 18, wherein the pectate lyase is chosen from Amb a 1.0101, Amb a 1.0201, Amb a 1.0202, Amb a 1.0301, Amb a 1.0302, Amb a 1.0303, Amb a 1.0304, Amb a 1.0305, Amb a 1.0401, Amb a 1.0402, Amb a 1.0501, Amb a 1.0502, the alpha subunit of Amb a 1, the beta subunit of Amb a 1 and proteins of Artemisia vulgaris, Ambrosia psilostachya or Ambrosia trifida which have at least 57% identity, preferably at least 60% identity, preferably at least 64% identity, with one of the isoforms of Amb a 1.

22. The plant cell as claimed in claim 18, wherein the heterologous nucleotide sequence comprises a sequence encoding the signal peptide, a sequence encoding the propeptide and a sequence encoding the mature pectate lyase.

23. The plant cell as claimed in claim 22, wherein: the signal peptide is chosen from the natural pectate lyase signal peptide and the tobacco chitinase signal peptide; and the propeptide is chosen from the natural pectate lyase propeptides and the propeptides of peptidases C1A.

24. The plant cell as claimed in claim 18, wherein the heterologous nucleotide sequence also comprises an intracellular trafficking peptide sequence which targets the pectate lyase in soluble or membrane form to the endoplasmic reticulum or the various compartments which constitute the endomembrane secretory system of the plant cell.

25. The plant cell as claimed in claim 18, which comprises an expression vector comprising: elements of prokaryotic DNA encoding a bacterial origin of replication and a gene for antibiotic resistance; the heterologous nucleotide sequence as previously defined; an expression cassette allowing the expression of a silencing suppressor, preferably p19; and DNA elements which control transcript processing, such as termination/polyadenylation sequences, preferably the Tnos sequence.

26. A plant comprising at least one plant cell as claimed in claim 18.

27. A process for producing a pectate lyase chosen from Amb a 1, the alpha subunit of Amb a 1, the beta subunit of Amb a 1, and homologs of Amb a 1, comprising the expression of said pectate lyase in a plant cell as claimed in claim 18 or in a plant comprising at least one said plant cell.

28. The process for producing a pectate lyase as claimed in claim 27, comprising the following steps: a) transformation of agrobacteria with an expression vector comprising a heterologous nucleotide sequence encoding a preproprotein of a pectate lyase, functionally bound to a strong promoter; and b) transfection of the plant cell or of the plant with the agrobacteria obtained in step a).

29. The process for producing a pectate lyase as claimed in claim 28, wherein the agrobacteria used in step a) are chosen from the strains LBA4404, GV3101, EHA 101/105 and C58, and wherein the transfection of step b) preferably comprises the following steps: b1) culturing the plant cell or plant aeroponically or hydroponically, under LED lighting, preferably for five weeks hydroponically on free floats, b2) agroinfiltration of the plant cell or plant obtained in b1), under vacuum, by the agrobacteria obtained in step a); and b3) returning the plant cell or plant obtained in b2) to culturing, typically for 3 to 6 days.

30. The process for producing a pectate lyase as claimed in claim 28, also comprising a step c) of extraction of the pectate lyase produced, said step c) comprising the following steps: infiltration under vacuum of the plant cell or leaves (i.e. the aerial part) of the plant, in an enzymatic solution comprising a pectinase or maceroenzyme, which does not have proteolytic activity, taking off the infiltrated plant cell or infiltrated leaves from the plant and incubation, in an enzymatic solution of pectinase or maceroenzyme, for a duration of between 2 h 30 and 5 h, at a temperature of between 24 C. and 30 C., then placing the mixture obtained under agitation at between 20 and 30 rpm at room temperature for a duration of between 30 minutes and 2 h, filtration then optional centrifugation of the digestate obtained, and recovery of the supernatant.

Description

[0078] The legends for the figures are as follows:

[0079] FIG. 1: Schematic representation of the different cassettes making it possible to produce a mature and active pectate lyase Amb a 1 (A, B, C, F and G) or the alpha subunit (E and I) or the beta subunit (D and H), using either the signal peptide and the natural propeptide of the protein (A-F) or the tobacco chitinase signal peptide and a propeptide of the peptidase CIA family (G-I) in order to increase yields.

[0080] The pectate lyase is also produced in a form mutated on lysine 180 (K180) in order to limit proteolysis enabling the production of the alpha and beta subunits (F). [0081] A (SEQ ID NO: 1): cDNA encoding the natural preproprotein (of sequence SEQ ID NO: 7). This cDNA may be fused to trafficking signals described in application WO2008/056265, [0082] B (SEQ ID NO: 2): cDNA optimized for use in N. benthamiana encoding the natural preproprotein (of sequence SEQ ID NO: 7). This cDNA may be fused to trafficking signals described in application WO2008/056265, [0083] C (SEQ ID NO: 3): Harmonized cDNA encoding the natural form of the preproprotein (of sequence SEQ ID NO: 7). This cDNA contains codons optimized for use in N. benthamiana, but also comprises rare codons, in order to conserve the rhythm of synthesis of the protein for better preservation of the 3D structure. This cDNA may be fused to trafficking signals described in application WO2008/056265, [0084] D (SEQ ID NO: 4): Native/optimized/harmonized cDNA encoding the beta subunit (of sequence SEQ ID NO: 8). This cDNA may be fused to trafficking signals described in application WO2008/056265. [0085] E (SEQ ID NO: 5): Native/optimized/harmonized cDNA encoding the alpha subunit (of sequence SEQ ID NO: 9). This cDNA may be fused to trafficking signals described in application WO2008/056265. [0086] F: Native/optimized/harmonized cDNA encoding the mutated (K180) form of the protein. This cDNA may be fused to trafficking signals described in application WO2008/056265. [0087] G: Native/optimized/harmonized cDNA encoding the mature form of the protein fused to the tobacco chitinase signal sequence (Neuhaus, J.-M., 1996) and to the propeptide Der p1 (p08176-aa 19 to 98 or SEQ ID NO: 6). This cDNA may be fused to trafficking signals described in application WO2008/056265. [0088] H: Native/optimized/harmonized cDNA encoding the beta subunit of the protein fused to the tobacco chitinase signal sequence (Neuhaus, J.-M., 1996) and to the propeptide Der p1 (p08176-aa 19 to 98 or SEQ ID NO: 6). This cDNA may be fused to trafficking signals described in application WO2008/056265. [0089] I: Native/optimized/harmonized cDNA encoding the alpha subunit of the protein fused to the tobacco chitinase signal sequence (Neuhaus, J.-M., 1996) and to the propeptide Der p1 (p08176-aa 19 to 98 or SEQ ID NO: 6). This cDNA may be fused to trafficking signals described in application WO2008/056265.

[0090] FIG. 2: The AllergoPur platform used for the expression and the production of the different forms of pectate lyase according to the invention.

[0091] FIG. 3: Expression of the protein Amb a 1. The proteins extracted from plants transfected with the native cDNA (F1-F3), with the optimized cDNA (F4-F6) or with the harmonized cDNA (F7-F9) encoding the protein Amb a 1 were separated by SDS-PAGE and analyzed by immunodetection with an antibody directed against a Flag tag. The immunodetection analysis demonstrates the specific production of the protein Amb a 1 and of the alpha subunit. The cleaved beta subunit is not immunodetected on this blot.

[0092] FIG. 4: Purification of the allergen Amb a 1. The proteins extracted from plants were transfected under vacuum with the harmonized cDNA encoding the protein Amb a 1 by IMAC chromatography. The fractions retained on the column were then analyzed by SDS-PAGE electrophoresis. Given the C-terminal position of the Flag, two polypeptides corresponding to the precursor form (preproprotein) (Amb a 1p) and to the alpha subunit are purified from the extract transfected with the total cDNA (lane 3). These two polypeptides (Amb a 1 alpha and Amb a 1p) are then separated (lanes 4 and 5, respectively) by molecular sieve chromatography. The two polypeptides produced have slightly lower electrophoretic mobility than the native proteins. This difference is explained by the presence of the flag at the C-terminal position.

[0093] FIG. 5: Expression of the allergen Amb a 11. The proteins extracted from 3 plants (P1-P3) transfected with the cDNA encoding the allergen Amb a 11 were separated by SDS-PAGE electrophoresis. The protein extracts are analyzed as soon as they are extracted (0 H) or after 12 h incubation (12 H) at room temperature. The incubation at room temperature demonstrates the accumulation of two polypeptides corresponding to Amb a 11, and also the virtually total degradation of the N. benthamiana proteins. The allergen Amb a 11 is therefore produced in active form according to the same process as described presently for Amb a 1.

EXAMPLE:

[0094] Molecular Design and Gene Synthesis

[0095] The cDNAs are synthesized in native form, optimizing the use of the codons for their recognition by the plant system or harmonizing the use of codons (reintroduction of rare codons to set the rhythm of the protein synthesis). Within the context of this invention, the preferred optimization is optimization for expression in Nicotiana benthamiana, as indicated in FIG. 1.

[0096] Preparation of Plasmids

[0097] Restriction sites Xba I/kpn I and Sal I/Sac I are respectively integrated at the 5 and 3 ends of the cDNA during synthesis. These sites are then used to clone the cDNAs in the binary expression vector pAG01 (FIG. 1). The cDNAs are cloned upstream of a 35S promoter (35S) and downstream of a nopaline synthase termination sequence (Tnos); the vector pAG01 also contains an expression vector enabling the expression of the silencing suppressor p19 simultaneously to the recombinant protein in order to increase the production yields. The vectors are then used to transform the strain LBA4404 of Agrobacterium tumefaciens.

[0098] Transient Expression of Pectate Lyases According to the Invention in Leaves of Nicotiana BenthamianaUse of the AllergoPur Platform

[0099] For the production by transient expression, Agrobacterium tumefaciens LBA4404 is used for the transfer of a cDNA encoding pectate lyase without the gene of interest being integrated into the genome of the plant cell; reference is made here to transfection, not to transgenesis. The plants are cultivated in hydroponic conditions in the presence of a nutritive medium (GHE, floragrow, floramicro, florabloom, 10 ml/15 ml/5 ml per 10 l of osmosed water) and under LED lighting.

[0100] The agrobacterium is transferred into the leaf tissue by agroinfiltration according to two processes. For the production of small batches of pectate lyases intended for prototype screening, the agrobacteria are injected manually by virtue of a syringe applied to the epidermis of the lower surface of the leaf. Leaf disks taken from the leaves 4 to 6 days after agroinfiltration are used for the analysis of the different prototypes of pectate lyases. This screening step makes it possible to define the expression vector which will be used to obtain a pectate lyase of optimal quality. The same process is used for large-scale commercial production, but in this case the agroinfiltration is carried out under vacuum, in chambers containing several liters of an agrobacterial culture and in which several tens of plants are infiltrated simultaneously. These plants are then placed back in culture for 4 to 6 days before the purification of the pectate lyases from the leaf extracts (FIG. 2).

[0101] Expression of the Pectate Lyase Amb a 1 and Its Subunits

[0102] The expression of the proteins and also the yields are analyzed by Western blotting and ELISA, respectively. The results are illustrated for the three forms of cDNA encoding the natural protein (FIG. 3).

[0103] The expressed proteins are active. Indeed, the expression of Amb a 1 causes large necroses from the 4th day of expression, compared to control plants. These necroses are due to high pectate lyase activity (Liu et al., 2010).

[0104] Purification and Characterization

[0105] As illustrated in FIG. 4, the pectate lyases are extracted from fresh or frozen biomass then purified on IMAC column (HisTrap Excell).

[0106] This purification enables the production of the precursor form (preproprotein) and of the alpha subunit.

[0107] Finally, as shown in FIG. 5, it is possible to combine the allergens Amb a 1 and Amb a 11 obtained by the process according to the invention, to obtain a composition which may be used in allergy immunotherapy. These allergens may be produced and purified after mechanical extraction as described in application FR1255510, or according to the alternative of extraction by enzymatic infiltration as described in the present application.