METHOD FOR ENZYMATICALLY MODIFYING THE TRI-DIMENSIONAL STRUCTURE OF A PROTEIN

Abstract

The invention is directed to a method for producing conformational-stabilized proteins by incorporating a recognition sequence in a protein, the method comprising the steps of (a) generating at least one genetic construct comprising the recognition sequence; (b) expressing in a natural host the at least one genetic construct using oligonucleotide primers, thereby forming a vector; and (c) using a plant-based over-expression system with a constitutive promoter to over-express the vector. The method is remarkable in that the recognition sequence comprises at least one stretch of amino acids with as sequence Phe-x1-x2-Tyr, wherein Phe is phenylalanine, X1 and X2 are amino acid residues and Tyr is tyrosine.

Claims

1-15. (canceled)

16. A method for incorporating a recognition sequence in a protein, said method comprising the steps of: (a) generating at least one genetic construct comprising the recognition sequence; (b) expressing in a natural host the at least one genetic construct using oligonucleotide primers, thereby forming a vector; and (c) using a plant-based over-expression system with a constitutive promoter to over-express the vector, wherein the recognition sequence comprises at least one stretch of amino acids with as sequence Phe-x.sub.1-x.sub.2-Tyr, wherein Phe is phenylalanine, x.sub.1 and x.sub.2 are amino acid residues and Tyr is tyrosine and in that the plant-based over-expression system has an enzymatic activity which converts the phenylalanine residue of the recognition sequence into a didehydro-phenylalanine residue.

17. The method according to claim 16, wherein the plant-based over-expression system is based on at least one of at least one plant or on at least one plant cells suspension.

18. The method according to claim 16, wherein the plant-based over-expression system is based on at least one plant belonging to the group of rosids.

19. The method according to claim 16, wherein the plant-based over-expression system is based on a least one plant belonging to the group of fabids or malvids.

20. The method according to claim 16, wherein the plant-based over-expression system is based on at least one of Medicago sativa, Arabidopsis thaliana or Cannabis sativa.

21. The method according to claim 16, wherein x.sub.1 and x.sub.2 are at least one of polar hydroxyl-containing amino acid or basic amino acid.

22. The method according to claim 16, wherein the at least one genetic construct comprising the recognition sequence is based on a focus sequence of one beta subunit of polygalacturonase of the Medicago sativa represented by SEQ-ID NO: 3, wherein the focus sequence is delimited by the residue numbered 190 and by the residue numbered 219.

23. The method according to claim 22, wherein the residue numbered 203 is phenylalanine and the residue numbered 206 is phenylalanine.

24. Method according to claim 22, wherein the residue numbered 203 is phenylalanine and the residue numbered 206 has been modified from phenylalanine to tyrosine.

25. The method according to claim 22, wherein the residue numbered 203 has been modified from phenylalanine to tyrosine and the residue numbered 206 is phenylalanine.

26. A method for producing a structurally-modified natural protein, said method comprising the steps of: (a) generating at least one genetic construct comprising the recognition sequence; (b) expressing in a natural host the at least one genetic construct using oligonucleotide primers, thereby forming a vector; and (c) using a plant-based over-expression system with a constitutive promoter to over-express the vector, wherein the recognition sequence comprises at least one stretch of amino acids with as sequence Phe-x.sub.1-x.sub.2-Tyr, wherein Phe is phenylalanine, x.sub.1 and x.sub.2 are amino acid residues and Tyr is tyrosine and in that the plant-based over-expression system has an enzymatic activity which converts the phenylalanine residue of the recognition sequence into a didehydro-phenylalanine residue, and at least one subsequent step (d) of isolation from the plant-based over-expression system.

27. A structurally-modified natural protein obtainable by the method for producing a structurally-modified natural protein, comprising the steps of: (a) generating at least one genetic construct comprising the recognition sequence; (b) expressing in a natural host the at least one genetic construct using oligonucleotide primers, thereby forming a vector; and (c) using a plant-based over-expression system with a constitutive promoter to over-express the vector; wherein the recognition sequence comprises at least one stretch of amino acids with as sequence Phe-x.sub.1-x.sub.2-Tyr, wherein Phe is phenylalanine, x.sub.1 and x.sub.2 are amino acid residues and Tyr is tyrosine and in that the plant-based over-expression system has an enzymatic activity which converts the phenylalanine residue of the recognition sequence into a didehydro-phenylalanine residue, and at least one subsequent step (d) of isolation from the plant-based over-expression system.

28. The structurally-modified natural protein according to claim 27 for use as a biocatalyst.

29. The structurally-modified natural protein according to claim 27 for use as protein therapeutics.

Description

DRAWINGS

[0050] FIG. 1 is an exemplary representation of the MS/MS spectrum of the peptide represented by SEQ-ID NO: 1.

[0051] FIG. 2 is an exemplary table comprising the peaks of the MS/MS spectrum of FIG. 1.

DETAILED DESCRIPTION

[0052] The invention proposes the incorporation of a phenylalanine, or a stretch of amino acids containing a phenylalanine, at critical positions in recombinant proteins, a phenylalanine that after enzymatic modification provides a conformationally-restrained bending point in the 3D structure of the protein.

[0053] The conformational determination originates from an enzymatic dehydration of the alpha-beta carbon bond of phenylalanine, making it controllable.

[0054] First of all dynamic modelling of the stabilized recombinant protein (hereafter designated as product) and variations thereof will be done to identify the molecular form with the highest stability while the enzymatic properties of the product are similar or better than that of the wild type protein. The product has to include the determined recognition sequence, including the phenylalanine residue that is to be dehydrated, for the modifying enzyme.

[0055] The recognition sequence consists minimally in a phenylalanine residue followed by a tyrosine residue, separated by two other residues, i.e. Phe-x.sub.1-x.sub.2-Tyr with xand x.sub.2 being amino acid residues, dominantly being polar hydroxyl-containing and/or basic amino acids. The tyrosine at the +3 position is likely to be essential for the modification to occur.

[0056] It is however not excluded that the recognition sequence may be longer.

[0057] SEQ-ID NO: 1 is part of the protein sequence of the beta subunit of polygalacturanose (alfalfa contig 53863). This particular part of the protein sequence has been identified thanks to mass spectrometry analysis, in particular tandem mass spectrometry (MS/MS). General information about the whole protein sequence can be retrieved on http://plantgrn.noble.org/AGED/.

[0058] In SEQ-ID NO: 1, the sequences of interest which are recognized by the modifying enzyme comprised in the natural host are (1) Phe.sub.800 and Tyr.sub.803; and (2) Phe.sub.814 and Tyr.sub.817.

[0059] FIG. 1 shows the MS/MS spectrum of peptide represented by SEQ-ID NO: 1. The dF residues as indicated on the spectrum corresponds to didehydro-phenylalanine (Phe) with a residual mass of 145 Da compared to the residual mass of 147 Da for the unmodified phenylalanine.

[0060] Both recognition sequences (1) and (2) have thus been identified. FIG. 1 specifically indicates the y-ion (i.e., those fragment peaks that appear to extend from the C-terminus) series as well the b-ion (i.e., those fragment peaks that appear to extend from the N-terminus) series.

[0061] FIG. 2 shows a table corresponding to the matching peaks of the MS/MS spectrum given in FIG. 1. The fragment ions given in b2, b3 and in y20, y19 corresponds to Phe (or dF) from the recognition sequence (1) Phe800 and Tyr803. The fragments ions given in b16, b17 and in y5, y6 corresponds to Phe (or dF) from the recognition sequence (2) Phe814 and Tyr817. It indeed illustrates the 145 Da mass compared to the mass of 147 Da normally expected for an unmodified Phe.

[0062] In order to confirm the results obtained by mass spectrometry, the use of the MASCOT software enables the identification of proteins by interpreting mass spectrometry data.

[0063] Searching via MASCOT database thus results in a highly significant match between spectrum and the peptide sequence with Phe. The mascot score is of 148 (a score superior to 47 being considered as significant) and an expected value of 9.3 e-0.12.

[0064] The following experiments, with a coding sequence for the alfalfa beta subunit of polygalacturonase represented by the sequence SEQ-ID NO: 2 and SEQ-ID NO: 3 (known under the reference alfalfa contig Medtr8g064530) or still by the sequences of alfalfa contig 53863 represented by SEQ-ID NO: 4, SEQ-ID NO: 5, and SEQ-ID NO: 6 have been undertaken to confirm the above results, i.e. the determination of the recognition sequence required for a modifying enzyme to change the conformation of a protein.

[0065] General information about the protein sequence alfalfa contig Medtr8g064530 (SEQ-ID NO: 2 and SEQ-ID NO: 3) can be retrieved on http//plantgrn.noble.org/LegumeIPv2/.

[0066] General information about the protein sequence alfalfa contig 53863 (SEQ-ID NO: 4, SEQ-ID NO: 5 and SEQ-ID NO: 6) can be retrieved on http//plantgrn.noble.org/AGED/.

[0067] In order to achieve the introduction of the modified amino acid residues in the chain of recombinant proteins different genetic constructs have to be created.

[0068] Synthetic constructs of the protein sequence alfalfa contig Medtr8g064530 are overexpressed in a plant cell suspension culture and the effect of site directed mutations of phenylalanine and tyrosine residues on the stabilities are studied.

[0069] Site-directed mutants of phenylalanine are generally made by the replacement of the phenylalanine with tyrosine.

[0070] The focus sequence is represented in SEQ-ID NO: 3.

[0071] The advantages of focusing on this sequence provide several benefits, as stated below:

[0072] First, it contains the only Phe (Phe.sub.203) experimentally found to be unmodified when the recognition sequence Phe.sub.206-x-x-Tyr.sub.209 has been exploited by the modifying enzyme.

[0073] The replacement of Phe.sub.203 by Tyr indicates that the recognition sequence Phe.sub.206-x-x-Tyr.sub.209 is still usable for the modifying enzyme.

[0074] Secondly, the replacement of Phe.sub.206 by Tyr abolishes the modification site at residue 206 (i.e., the recognition sequence Phe.sub.206-x-x-Tyr.sub.209 is not anymore usable for the modifying enzyme) but creates the recognition sequence Phe.sub.203-x-x-Tyr.sub.206. These studies establish that the sequence Phe-x-x-Tyr is sufficient as recognition sequence. Potentially, other surrounding amino acids are important in the recognition of the site of modification by the modifying enzyme.

[0075] The following step is the generation of site directed mutants. Different constructs are made: one construct for the full length native protein, one construct for the full length with Phe.sub.203 to Tyr, one construct for the full length with Phe.sub.206 to Tyr, one construct for the full length with Tyr.sub.209 to Phe and one empty vector.

[0076] This protein sequence of the beta subunit of polygalacturanose (alfalfa contig 53863) has been employed to confirm that recognition sequence comprises at least one stretch with as sequence Phe-x.sub.1-x.sub.2-Tyr.

[0077] In the SEQ-ID NO: 4, the sequences of interest which are recognized by the modifying enzyme are (3) Phe.sub.102 and Tyr.sub.106 ; and (4) Phe.sub.130 and Tyr.sub.133.

[0078] In the SEQ-ID NO: 5, the sequences of interest which are recognized by the modifying enzyme are (5) Phe.sub.200 and Tyr.sub.203; (6) Phe.sub.214 and Tyr.sub.217 ; (7) Phe.sub.229 and Tyr.sub.232; and (8) Phe.sub.243 and Tyr.sub.246. It is noted that the residue Phe.sub.236 is not affected at all by the modifying enzyme.

[0079] In the SEQ-ID NO: 6, the sequence of interest which is recognized by the modifying enzyme is (9) Phe.sub.277 and Tyr.sub.280.

[0080] Oligonucleotide primers are generated for the different constructs and are used to generate mutants with the specified amino acid changes. These mutants are generated using according to methods for site directed mutagenesis well known in the art.

[0081] These generated vectors are overexpressed in plant-based expression system (i.e., plant and/or plant cell suspensions) using a constitutive promotor. Since the modifying enzyme that converts phenylalanine into the didehydro form is inherently present in the plant suspension cell culture, no extra modifications of this production system are required. This is achieved in situ.

[0082] The modifying enzyme converts phenylalanine into didehydro-phenylalanine, thereby changing the tri-dimensional structure of the natural protein, thereby stabilizing the protein and making it less sensitive to various change of the environment, like the temperature and/or the pH.

[0083] The synthesis of the stabilized recombinant protein in a system containing the modifying enzyme which is able to interact actively with the recognition sequence is thus performed. The modifying enzyme is co-expressed in the same natural host (in situ) whereby the target phenylalanine is modified prior to the isolation of the product from the appropriate culture system. The used host must also produce the modifying enzyme. For plant species (notably alfalfa, hemp and thale-cress) this activity is naturally present but it may be required to make the modifying activity of the host inducible. For other culture systems no naturally occurring modifying activity is known. In these cases a second construct, containing the modifying enzyme, must be made using the same vector to ensure the concomitant expression of both the product gene and the modifying enzyme.

[0084] By this in situ approach, the stabilized recombinant protein can be obtained directly.

[0085] This process is suitable for the stabilization of natural proteins comprising a large number of amino acids. This process leads in fact to a modified natural protein, the modification being in the tri-dimensional structure of the protein. It is a process for the stabilization and functional customization of proteins through the incorporation of a stable, conformation-determining amino acid in a protein sequence.

[0086] Once the modifying enzyme has performed the structural modification, the product can be isolated from the culture medium using a pull-down approach with antibodies or other techniques currently used in the art to isolate recombinant proteins. Analytical techniques known by the skilled person in the art will be also employed to determine the structure and to check the stability of the structurally modified protein. For instance, mass spectrometry analysis, fluorescence testing and ELISA test, among many other, might be used.

[0087] The stabilized recombinant protein may be used in different systems as biocatalysts (e.g. production of biodiesel by lipases, biomass valorisation, lignin cleavage, etc.) or in protein therapeutics (e.g. stabilized forms of insulins, stabilized forms of antibodies, etc.).