Use of aprotinin as a carrier to produce a recombinant protein, polypeptide or peptide in algae
20230146589 · 2023-05-11
Inventors
Cpc classification
C07K14/78
CHEMISTRY; METALLURGY
C07K14/8117
CHEMISTRY; METALLURGY
C12N15/8257
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
C07K14/78
CHEMISTRY; METALLURGY
Abstract
Use of aprotinin as a carrier to produce a recombinant protein, polypeptide or peptide in algae The present invention relates to the use of aprotinin as a carrier to produce a recombinant protein, polypeptide or peptide in algae, in particular microalgae, wherein aprotinin and said recombinant protein, polypeptide or peptide are fused together to form a fusion protein. It also relates to a method to produce a recombinant protein, polypeptide or peptide in algae, wherein said method comprises genetic transformation of algae, in particular microalgae, with a recombinant nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises aprotinin and said recombinant protein, polypeptide or peptide. It further relates to a recombinant algae comprising a recombinant nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises aprotinin and a recombinant protein, polypeptide or peptide. The use of said recombinant algae, for producing said fusion protein is also contemplated.
Claims
1.-15. (canceled)
16. The method to produce a recombinant protein, polypeptide or peptide of interest in algae, wherein said method comprises transforming transformation of algae with a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises aprotinin and said recombinant protein, polypeptide or peptide of interest.
17. The method according to claim 16, comprising the following steps: (i) providing a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises aprotinin and said recombinant protein, polypeptide or peptide of interest; (ii) introducing the nucleic acids sequence according to (i) into an expression vector which is capable of expressing the nucleic acid sequence in an algae host cell; and (iii) transforming the genome of algae host cell by the expression vector.
18. The method according to claim 17, further comprising: (iv) identifying the transformed algae host cell; (v) characterizing the microalgae host cell for the production of recombinant fusion protein; (vi) extracting the recombinant fusion protein; and optionally; and (vii) purifying the fusion protein.
19. A recombinant algae comprising a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises aprotinin and a recombinant protein, polypeptide or peptide of interest.
20. The method according to claim 16 to increase accumulation and/or stability and/or solubility and/or folding and/or activity of recombinant proteins peptide, polypeptide or protein of interest in algae, in particular in microalgae, more particularly in the chloroplast of microalgae.
21. The method according to claim 16, wherein said protein, polypeptide or peptide of interest is chosen from, collagen, collagen like and matricins proteins, polypeptides or peptides.
22. The recombinant algae according to claim 19, wherein said protein, polypeptide or peptide of interest is chosen from, collagen, collagen like and matricins proteins, polypeptides or peptides.
23. The method according to claim 21, wherein said matricins proteins, polypeptides or peptides, are chosen from elastin and elastin like proteins.
24. The method according to claim 16, wherein said algae is chosen from the group consisting of Chlorophyta, Chlorophyceae, Pleurastrophyceae, Prasinophyceae, Chromophyta, Bacillariophyceae, Chrysophyceae, Phaeophyceae, Eustigmatophyceae, Haptophyceae, Raphidophyceae, Xanthophyceae, Cryptophyta, Cryptophyceae, Rhodophyta, Porphyridiophycea, Stramenopiles, Glaucophyta, Glaucocystophyceae, Chlorarachniophyceae, Haptophyceae, Dinophyceae, Scenedesmaceae, Euglenophyta, Euglenophyceae and Cyanophyceae.
25. The method according to claim 24, wherein said algae is chosen from the group consisting of Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, Nannochloropsis, Arthrospira platensis, Arthrospira maxima, Anabaena sp. PCC7120, Leptolyngbya sp, Synechocystis sp, and Synechococcus sp.
26. The method according to claim 16, wherein said recombinant protein, polypeptide or peptide of interest is fused to the C-terminus of aprotinin.
27. The method according to claim 16, wherein said fusion protein also comprises an epitope tag.
28. The method according to claim 16, wherein said fusion protein also comprises a signal peptide.
29. The method according to claim 16, wherein said fusion protein also comprises a protease specific cleavage site between the aprotinin and the recombinant protein, polypeptide or peptide of interest.
30. A method for producing a fusion protein, wherein said fusion protein comprises aprotinin and a recombinant protein, polypeptide or peptide of interest, said method comprising the use of a recombinant algae according to claim 19.
31. The recombinant algae according to claim 22, wherein said matricins proteins, polypeptides or peptides, are chosen from elastin and elastin like proteins.
32. The recombinant algae according to claim 19, wherein said algae is chosen from the group consisting of Chlorophyta, Chlorophyceae, Pleurastrophyceae, Prasinophyceae, Chromophyta, Bacillariophyceae, Chrysophyceae, Phaeophyceae, Eustigmatophyceae, Haptophyceae, Raphidophyceae, Xanthophyceae, Cryptophyta, Cryptophyceae, Rhodophyta, Porphyridiophycea, Stramenopiles, Glaucophyta, Glaucocystophyceae, Chlorarachniophyceae, Haptophyceae, Dinophyceae, Scenedesmaceae, Euglenophyta, Euglenophyceae and Cyanophyceae.
33. The recombinant algae according to claim 19, wherein said recombinant protein, polypeptide or peptide of interest is fused to the C-terminus of aprotinin.
34. The recombinant algae according to claim 19, wherein said fusion protein also comprises an epitope tag.
35. The recombinant algae according to claim 19, wherein said fusion protein also comprises a signal peptide.
Description
FIGURES
[0206]
[0207]
[0208]
[0209]
[0210]
[0211]
[0212]
[0213]
[0214]
[0215]
[0216]
[0217]
[0218]
EXAMPLES
Example 1
Material and Methods
[0219] All oligonucleotides and synthetic genes were purchased from Eurofins. All enzymes were purchased from NEB, Promega, Invitrogen and Sigma Aldrich/Merck. All plasmids were built on the pBluescript II backbone.
Algal Strains and Growth Conditions
[0220] The two algal strains used are the Chlamydomonas reinhardtii wild type (137C; mt+) and the cell wall deficient strain CW15 (CC-400; mt+), obtained from the Chlamydomonas Resource Center, University of Minnesota).
[0221] Prior to transformation, all strains were grown in TAP (Tris Acetate Phosphate) medium to mid-logarithmic phase (densities of approximately 1-2×10.sup.6 cell/mL) at a temperature comprised between 23° C. to 25° C. (ideally 25° C.) on a rotary shaker in presence of constant light (70-150 μE/m.sup.2/s).
[0222] Transformants were grown in the same conditions and the same media containing 100 μg/mL of spectinomycin or 100 μg/mL kanamycin, depending of the selectable marker gene present in the transformation vector.
[0223] Growth kinetics was also followed by measuring the optical density at 750 nm using a spectrophotometer.
Algal Transformation
[0224] Chlamydomonas reinhardtii cells were transformed using the helium gun bombardment technique of gold micro-projectiles complexed with transforming DNA, as described in the article Boynton et al., 1988. Briefly, the Chlamydomonas reinhardtii cells were cultivated in TAP medium until midlog phase, harvested by gentle centrifugation, and then resuspended in TAP medium to a final concentration of 1.Math.10.sup.8 cells/mL. 300 μL of this cell suspension was plated onto a TAP agar medium supplemented with 100 μg/mL of spectinomycin or 100 μg/mL of kanamycin, depending of the selectable marker gene present in the transformation vector. The plates were bombarded with gold particles (S550d; Seashell Technology) coated with transformation vector, as described by the manufacturer. The plates were then placed at 25° C. under standard light conditions to allow selection and formation of transformed colonies.
Total DNA Extraction and PCR Screening of Positive Transformants
[0225] Total DNA extraction was performed using the chelating resin Chelex 100 (Biorad) from single colonies (with size of around 1 mm in diameter) of wild type and/or antibiotic resistant transformants Chlamydomonas strains.
[0226] From isolated colonies, a quantity of cells corresponding to about 0.5 mm in diameter was removed with a pick and resuspended in 20 μL of H.sub.2O. 200 μL of ethanol were added and incubated 1 min at room temperature. 200 μL of 5% Chelex were incorporated and vortexed. After an incubation of 8 min at 100° C., the mixture was cooled down and centrifuged 5 min at 13,000 rpm. Finally, the supernatant was collected.
[0227] After transformation, algae colonies growing onto restrictive solid medium plates were expected to have the antibiotic resistant gene and the other transgene(s) incorporated the transgene(s) into their genome.
[0228] In order to identify stable integration of the recombinant genes into the algal genome of the antibiotic resistant transformants were screened by Polymerase Chain Reaction (PCR or PCR amplification) in a thermocycler using 1 μL of total DNA previously extracted as template, two synthetic and specific oligonucleotides (primers) and Taq polymerase (GoTaq, Promega). The cycles of PCR amplification followed the guidelines recommended by the manufacturer. The PCR reactions were subjected to gel electrophoresis in order to check the PCR fragment of interest.
Protein Extraction, Western Blot Analyses
[0229] Chlamydomonas cells (50 mL, 1-2.Math.10.sup.6 cells/mL) were collected by centrifugation. Cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10 mM EDTA). In some embodiments of the example, the lysis buffer didn't contain 10 mM EDTA. After 30 min at room temperature, cell debris were removed by centrifugation at 13000 rpm and the supernatant containing the total soluble proteins was collected.
[0230] Depending on the further analysis step, total soluble proteins were extracted under non denaturing conditions. Cell pellet was resuspended in a buffer containing 50 mM Tris-HCl (pH 6.8 or 8) or 20 mM Tris-HCl (pH 6.8 or 8). The sonication step was carried out with the algal cell suspension held on ice, using a cell disruptor a sonicator FB505 500W (Sonic/Fisher Brand) and a setting of the micro-tip probe to 20% power, with continuous sonication for 5 min. After sonication, cell debris were removed by centrifugation at 13000 rpm, 30 min.
[0231] Total soluble proteins present in the supernatant were quantified using the Pierce BCA protein assay kit, following the instructions of the supplier (Thermofisher).
[0232] Total soluble protein samples (50 or 100 μg or another quantity further mentioned in the example depending of the experiment) were separated in a 12 or 15% Tris-glycine SDS-PAGE prepared according to Laemmli (1970).
[0233] For experiments performed under reducing conditions, samples were prepared in Laemmli sample loading buffer with 50 mM DTT (or more depending of the fusion protein) or 5% Beta-mercaptoethanol, and further denaturated 5 min at 95° C. before loading. The SDS PAGE experiments were carried out using a Protein Gel tank from BioRad.
[0234] After separation, samples were blotted onto a nitrocellulose membrane (GE HealthCare) using standard transfer buffer and a Trans-Blot® Turbo™ Transfer System from Biorad. In order to visualize the transferred proteins, the nitrocellulose membrane were stained by Ponceau S dye. Membranes were further blocked with Tris-buffered saline Tween buffer (TBS-T) (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% Bovin Serum Albumin (BSA). After one hour of saturation at room temperature under gently shaking, membranes were incubated during one night at 4° C. with TTBS buffer containing mouse primary antibody (See Table 1).
[0235] The antibody mentioned in Table 1 below were used as primary antibodies.
TABLE-US-00004 TABLE 1 Primary antibodies Primary antibody Source Dilution Monoclonal ANTI-FLAG ® M2 Sigma 1:1000 antibody produced in mouse Monoclonal ANTI-HA PURIFIED Sigma 1:6000 antiobody produced in mouse IGG Monoclonal ANTI-Aprotinin Abcam 1:2000 or 1:3000 antibody produced in mouse
[0236] After three washes with TBS-T-BSA buffer, membranes were incubated one hour at room temperature with TBS-T-BSA buffer containing secondary antibodies (Anti-Mouse IgG (H+L), HRP Conjugate; Promega). After four washes with TTBS buffer and one wash with TBS buffer, the membranes were incubated in an enhanced chemiluminescence (ECL) substrate (Clarity Max ECL substrate; Biorad). The ECL signals were visualized with the ChemiDoc™ XRS+ system (Biorad).
Protein Purification
[0237] Depending on the protein and on the further steps to which the protein is submitted, the first or second method below is conducted.
1) After centrifugation, algae cell pellets were resuspended in a buffer containing 50 mM Tris-HCl pH8, 500 mM NaCl and 0.1% Tween 20. Approximately, 10 mL of buffer were used per g of wet algal cells, depending of the transformants. The resuspended cells were sonicated in the same conditions as previously described.
2) After centrifugation, algae cell pellets were resuspended in a buffer containing 20 mM Tris-HCl pH 8. The resuspended cells were sonicated in the same conditions as previously described. The total soluble proteins were precipitated with 80% ammonium sulfate as described by Wingfield, 2001. After a 15000 g centrifugation for 30 min at 4° C., the protein pellets were resolubilized in 60 mM Tris-HCl pH8 buffer. These suspensions were dialysed in Slide-A-Lyzer Dialysis Cassettes (3.5 kDa MWCO, Thermo Scientific) as described by the manufacturer against the previous buffer. The next step being an anti-FLAG M2 affinity chromatography, NaCl and Tween 20 were added to the dialyzed samples to adjust the buffer composition to that of the binding buffer hereinafter described.
Affinity Chromatography
[0238] All recombinant proteins were tagged in their N-terminal with a Flag-Tag epitope which will bind specifically on an anti-Flag M2 affinity gel (Sigma/Merck). This resin contains a mouse monoclonal Anti-Flag® M2 antibody that is covalently attached to agarose.
[0239] All steps of this experiment were carried out as described by the manufacturer. Briefly, the samples of total soluble proteins were filtered using a cellulose acetate 0.45 μm filter and mixed with anti-Flag® M2 affinity gel prepared as recommended by the manufacturer and equilibrated in binding buffer (50 mM Tris-HCl pH8, 500 mM NaCl, 0.1% Tween 20). Approximately, 1 mL of resin was used per 4 to 8 g of wet algal cells, depending of the transformants. Binding of the recombinant fusion protein was performed at 4° C. for 4h or overnight with a gently and continuous end-over-end mixing. After incubation, the mixture of soluble protein incubated with resin were loaded by gravity on an empty Bio-rad Econo-pac column or collected by centrifugation, and washed several times with 40 column volumes of TBST and 20 column volumes of TBS. The protein of interest was eluted from the resin using 100 mM Glycine pH 3.5, 500 mM NaCl and neutralized with Tris-HCl pH 8 to a final concentration of 50 mM. Each elution fraction were further analyzed by SDS-PAGE and Western Blot.
[0240] The elution fractions containing the protein of interest were dialyzed in Slide-A-Lyzer Dialysis Cassettes (3.5 kDa MWCO, Thermo Scientific) as described by the manufacturer against the buffer used in the further step, as for instance, for the protease digestion. The dialyzed samples were concentrated using Vivaspin 6 (3 kDa MWCO, GE Healthcare).
Separation of the Protein of Interest from the Carrier
[0241] The separation of the protein of interest from the carrier was made by protease digestion, in particular, in the present invention by enterokinase (light chain) or Tobacco Etch Virus (TEV) Protease from New England BioLabs (NEB).
[0242] Enzymatic digestions were performed as recommended by the manufacturer.
[0243] For example, for enterokinase light chain digestion, reactions combined 25 μg of protein of interest in 20 μL of buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 2 mM CaCl.sub.2)), with 1 μL of enterokinase light chain. Incubation was made at 25° C. for 16h.
[0244] For example, for TEV digestion, typical reaction recommended by the manufacturer combined 15 μg of protein substrate with 5 μL of TEV protease reaction buffer (10×) to make a 50 μL total reaction volume. After addition of 1 μL of TEV Protease, reaction was incubated at 30° C. for 1 hour or 4° C. overnight.
[0245] For example, for Factor Xa digestion, the manufacturer recommended to digest 50 μg of fusion protein with 1 μg of FXa in a volume of 50 μL at 23° C. for 6 h. The reaction buffer consisted in 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 2 mM CaCl.sub.2).
Cleavage of the Polypeptide by Endoproteinases
[0246] The choice of the endoproteinase used to cleave the polypeptide of interest depends of the amino acid sequence of this polypeptide. Endoproteinases can be for instance, endoproteinase Glu-C, endoproteinase Arg-C, endoproteinase Asp-C, endoproteinase Asp-N, or endoproteinase Lys-C.
[0247] Enzymatic digestions were performed as recommended by the manufacturer. For example, for endoproteinase Glu-C digestion (from NEB), the manufacturer recommended to digest 1 μg of substrate protein with 50 ng of endoproteinase Glu-C at 37° C. for 16 h. The reaction buffer consisted in 50 mM Tris-HCl pH 8.0 and 0.5 mM GluC-GluC.
Size Exclusion Chromatography (SEC)
[0248] Size-exclusion chromatography of purified and digested fusion protein was performed using an AKTA Pure system (GE Healthcare) in order to separate the protein of interest from the carrier.
[0249] A Superdex S30 Increase G10/300 GL column (GE Healthcare) and a HiLoad 26/600 Superdex 30 prep grade column were first calibrated using two standards diluted with 2×PBS buffer (or appropriate buffer for the further step): aprotinin (bovine lung; 6.5 kDa), and glycine (75 Da).
[0250] After a washing step in water, the Superdex S30 Increase G10/300 GL column was equilibrated in running buffer (2×PBS, pH 7.4, or 1×PBS, pH 7.4 or appropriate buffer for the further step) and 200 to 500 μL samples were run through the column at a rate of 0.5 mL/min. Elution of protein was detected by measuring optical absorbance at 280, 224 and 214 nm. 0.5 mL fractions were collected and analyzed by SDS-PAGE followed by Western-Blot or stained by Coomassie Blue dye.
[0251] After a washing step in water, the HiLoad 26/600 Superdex 30 prep grade column was equilibrated in running buffer (2×PBS, pH 7.4, or 1×PBS, pH 7.4 or appropriate buffer for the further step) and samples (4 to 30 mL) were run through the column at a rate of 2.6 mL/min. Elution of proteins was detected by measuring optical absorbance at 280, 224 and 214 nm. 4 mL fractions were collected and analyzed by SDS-PAGE followed by Western-Blot.
[0252] In some embodiment, the elution fractions of interest were pooled and evaporated using a SpeedVac (Eppendorf). The peptides or polypeptides or proteins present in these evaporated samples were subjected to Edman degradation to confirm the amino acids sequence at the N-terminus of the protein of interest.
Example 2
[0253] Production of Aprotinin in the Chloroplast of Chlamydomonas reinhardtii by Chloroplast Genome Transformation
Design of Aprotinin
[0254] The nucleic acid and amino acids sequences of the mature Bos Taurus (bovine) aprotinin (SEQ. ID No 1 and 2) are known in the art and were extracted from UniProt (P00974) and the GenBank Accession Number X05274; (Creighton and Charles, 1987).
[0255] In Chlamydomonas reinhardtii, the codon usage has been shown to play a significant role in protein accumulation (Franklin et al., 2002; Mayfield and Schultz, 2004).
[0256] The nucleic acid sequence encoding aprotinin alone or chimeric aprotinin or fused with recombinant peptide/polypeptide/proteins were modified in order to improve its expression in C. reinhardtii host cell.
[0257] Methods for altering nucleic acids for improved expression in host cell are known in the art, particularly in algae cell, particularly in C. reinhardtii.
[0258] A codon usage database is found at http://www.kazusa.or.jp/codon/ (See the codon usage table for chloroplast genome of C. reinhardtii;
[0259] For improving expression in C. reinhardtii chloroplast of the gene of interest of the present invention, codons from the native bovine aprotinin sequence which are not commonly used, were replaced with a codon coding for the same or a similar amino acid residue that is more commonly used in the C. reinhardtii chloroplast codon bias. In addition, other codons were replaced to avoid sequences of multiple or extended codon repeats, or some restriction enzyme site, or having a higher probability of secondary structure that could reduce or interfere with expression efficiency.
[0260] In order to check and to fulfill all criteria mentioned above, the nucleic acid sequence of aprotinin were optimized by the software GENEius of Eurofins and the appropriate codon usage for C. reinhardtii chloroplast genomes.
[0261] After optimization, the gene encoding aprotinin (APRO) were operationally fused at its 5′end to a codon optimized nucleic acid sequence encoding the HA Tag (HA) followed by a signal peptide, the 3×Flag Tag (3F) and the cleavage site recognized by the Factor Xa protease (FX). This fusion protein is named HA-SP-3F-FX-APRO or chimeric aprotinin (SEQ ID No 70 and corresponding nucleic acid sequence SEQ ID No 71).
[0262] The nucleic acid sequence encoding the Signal Peptide (SP; SEQ ID No 72: NNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQA and corresponding nucleic acid sequence SEQ ID No 73) is extracted from the sequence of the E. coli TorA gene encoding the Trimethylamine-N-oxide reductase 1 (UniProt number P33225).
[0263] The codon usage of the HA Tag, the signal peptide, the 3×Flag Tag and the recognition site of Factor Xa were previously optimized for the expression of another transgenes and as described previously using the codon usage for C. reinhardtii chloroplast genome but independently of the aprotinin optimization.
[0264] This fusion gene ha-sp-3f-fx-apro (SEQ ID No 71) was synthesized and cloned by Eurofins Genomics in the vector pEX-A2 resulting in vector pAL60.
Construction of Transformation Vectors
[0265] Several chloroplast transformation vectors for the expression of different chimeric aprotinins and aprotinin were constructed (
[0266] The transformation vector pAU76 allowed the production of the chimeric aprotinin HA-SP-3F-FX-APRO (SEQ ID No 70): aprotinin was fused at its N-terminus to an amino acid sequence containing the HA epitope Tag (HA) followed by the signal peptide (SP), the 3×Flag epitope Tag (3F) and the cleavage site for Factor Xa protease (FX).
[0267] After the production in algae chloroplasts of the chimeric aprotinin HA-SP-3F-FX-APRO, the N-terminus fragment HA-SP will be cleaved during protein translocation into the thylakoids, and the following recombinant protein 3F-FX-APRO will be produced in vivo.
[0268] The protease cleavage site FX allows the in vitro release of aprotinin by adding Factor Xa protease to a sample of the chimeric aprotinin 3F-FX-APRO (SEQ ID No 74).
[0269] The codon usage of the nucleic sequence encoding the HA-SP followed by the 3×Flag epitope Tag (3F) and the cleavage site for the Factor Xa protease (FX) were optimized as described previously using the usage codon for C. reinhardtii chloroplast genome.
[0270] The nucleic acid sequence of the chimeric aprotinin was amplified by PCR from the vector pAL60 using the primers O5′AS_GibaproBE (SEQ ID No 75) and O3′AS_GibaproBE (SEQ ID No 76).
[0271] This PCR fragment of 267 pb containing the chimeric aprotinin was cloned between the psbD promoter/5′UTR and the atpA 3′UTR into the transformation vector pLE63 linearized by BamHI/PmeI digestions to give pAU76. Thus the expression cassette of the goi was PpsbD-5′UTRpsbD-ha-sp-3f-fX-apro-3′UTRatpA (SEQ ID No 77).
[0272] The chloroplast transformation vector pLE63 contained two expression cassettes (
[0273] These two expression cassettes are flanked by a left (LHRR) and right (RHRR) endogenous homologous recombination sequences which are identical to those surrounding the targeted integration site into the C. reinhardtii chloroplast genome. The choice of the insertion site within the chloroplast genome was generally made such as not to disrupt an essential gene or interrupt the expression of a polycistronic unit.
[0274] The chloroplast transformation vector pLE63 allowed the targeted integration of the transgenes into the chloroplast genome of C. reinhardtii between the 5S rDNA and psbA genes (and derived from instance from GenBank Accession Number NC005352).
[0275] The chloroplast transformation vector pAU97 allowed the production of the chimeric aprotinin HA-SP-3F-APRO (SEQ ID No 7 and 8): aprotinin was fused at its N-terminus with the HA Tag followed by the signal peptide, and the 3×Flag Tag. In this case, the release of aprotinin can be performed in vitro by enterokinase which cleaved the protein sequence after the second lysine amino acid in the motif sequence DYKDDDDK (SEQ ID No 60). The gene GNC-SFAPRO encoding this fusion protein was synthetized by Eurofins and cloned directly using Gibson assembly reaction (NEB) into pLE63 linearized by BamHI/PmeI digestions to give pAU97
[0276] The chloroplast transformation vector pAU94 allowed the production of the chimeric aprotinin HA-SP-APRO-LG-FX-NY2-3F (SEQ ID No 78 AND 79). The N-terminus of aprotinin is fused with the HA Tag followed by the signal peptide SP. Its C-terminus is linked to a linker LG followed by the Factor Xa cleavage site, an hexapeptide and the 3×Flag Tag. pAU94 was obtained by a Gibson assembly cloning into pLE63 linearized by BamHI/PmeI of a PCR fragment of 563 bp containing the nucleic sequence encoding the chimeric aprotinin HA-SP-APRO-LG-FX-NY2-3F.
[0277] In the case of algae chloroplasts transformed by pAU97 or pAU94, the N-terminus fragment HA-SP will be cleaved during the translocation of the protein HA-SP-3F-APRO or HA-SP-APRO-LG-FX-NY2-3F, respectively, during protein translocation into the thylakoids, and the following recombinant protein 3F-APRO (SEQ ID No 4) or APRO-LG-FX-NY2-3F (SEQ ID No 80) will be produced in vivo.
[0278] The chloroplast transformation vector pAU27 allowed the production of the chimeric aprotinin into the stroma of algae chloroplast 3F-APRO (SEQ ID No 4). It was obtained using Gibson assembly reaction for cloning into pAU63 linearized by NcoI/PmeI the gene GNC-FAPRO synthetized by Eurofins.
Transformation of Algae
[0279] The transformation vectors pAU76, pAU97, pAU94 and pAU27 were bombarded in Chlamydomonas cell (137c and CW15) as described in the Example 1.
[0280] In order to identify stable integration of the recombinant genes encoding chimeric aprotinin into the chloroplast algal genome, spectinomycin resistant colonies were screened by PCR amplification of the gene of interest using the primers O5′ASTatpA (SEQ ID No 81) or O5′ASTatpA2 (SEQ ID No 82) and O3′SUTRpsbD (SEQ ID No 83) annealing, respectively, in the atpA 3′UTR and in psbD promoter.
Analyses and Results
[0281] Western Blot analysis were performed on total soluble protein samples extracted from several transformants obtained after transformation with pAU76, pAU97, pAU94 or pAU27 (
[0282] The results show that all chimeric aprotinin were produced, being probed by the anti-Flag M2 antibody.
[0283] Moreover, in the case of transformants AU76, AU97 and AU94, the HA Tag and the signal peptide seems to be cleaved because Western Blot analyses performed on total soluble protein extracts show no specific protein recognized by the anti-HA antibody (
Confirmation of the Chimeric Aprotinin Activity Using the Trypsin Binding Assay
[0284] Aprotinin is a 58 amino acids monomeric polypeptide with a three-dimensional conformation maintained by three disulfide bridges.
[0285] Aprotinin, also known as a pancreatic trypsin inhibitor, binds with high affinity and specificity to serine proteases as for instance trypsin. The formed BPTI-trypsin complex is extremely stable.
[0286] 25 μg of total soluble protein or purified protein of interest from transformants were incubated separately for 30 minutes at 37° C. with 1.8 μg of trypsin (Sigma). As a positive control, 100 ng of standard aprotinin mixed with 50 μg of total soluble protein from WT strain were incubated in parallel and in the same conditions. Afterwards, samples were separated by SDS-PAGE under non-reducing conditions.
[0287] Western blot analysis performed using monoclonal antibodies directed against aprotinin showed for standard aprotinin and chimeric aprotinin from the transformant CW-AU76-1, the apparition of a higher molecular weight band as soon as trypsin was added to the samples (
[0288] This experiment demonstrates that the totality of chimeric aprotinin produced in chloroplast transformants with vectors pAU76 has an active conformation able to bind to trypsin. This also strongly suggests that the three disulfide bonds which maintain the structure of aprotinin are formed and correctly paired.
[0289] Chimeric aprotinin extract from the transformant CW-AU76-1 were purified by affinity chromatography using an anti-FLAG M2 affinity resin (
Example 3
[0290] Production of Aprotinin as a Fusion Partner for the Production of a Polypeptide of Elastin in the Chloroplast of Chlamydomonas reinhardtii by Chloroplast Genome Transformation
Construction of Transformation Vectors (pLA01, pLA02, pLA03 and pLA04)
[0291] In chloroplast transformation vector, ELP4, an elastin like polypeptide consisted of a repeat of the VGVAPG hexapeptide (SEQ ID No 34), more particularly of a 4-fold repeat of this hexapeptide (SEQ ID No 39: VGVAPGVGVAPGVGVAPGVGVAPG), was expressed in a fusion protein in which it was fused, at the C-terminus of the chimeric aprotinin HA-SP-3F-FX-APRO. This fusion partner contained aprotinin fused at its N-terminus to an amino acid sequence made of the HA epitope Tag (HA) followed by the signal peptide (SP), the 3×Flag epitope Tag (3F), and the cleavage site for Factor Xa (FX; SEQ ID No 102: IEGR). The Flag epitope Tag sequence (SEQ ID No 60: DYKDDDDK) which is the cleavage site for the enterokinase was inserted between the chimeric aprotinin and ELP4 in order to allow the release of ELP4 from aprotinin by in vitro site specific proteolysis of the fusion protein with enterokinase.
[0292] The nucleic acid sequence encoding ELP4 was first codon-optimized using the same method described in Example 1 and the codon usage in the chloroplast genome of Chlamydomonas. The resulting sequence was used to design two overlapping oligomers O5′Gibs-ELP4 (SEQ ID No 84) and O3′Gibs-ELP4 (SEQ ID No 85) which were used as primers and template to amplify by PCR the fragment FGibs-ELP4 of 194 bp. This amplified DNA were cloned using the Gibson Assembly Master Mix from New England Biolabs (as recommended by the manufacturer) into the chloroplast transformation vector pAU76 described in the Example 2 and linearized by PmeI to form the vector pLA00.
[0293] The nucleic acid sequence encoding ELP4 were amplified by PCR from pLA00 using the primers O5′Gibs501BE (SEQ ID No 86) and O3′Gibs501BE (SEQ ID No 87). The PCR fragment FPCR-AP-FELP4 (SEQ No 125) of 359 pb were cloned using the Gibson Assembly Master Mix into the pAL863 linearized by BamHI and PmeI to form the vector pLA01. The transformation vector pLA01 allowed the production of the fusion protein HA-SP-3F-FX-APRO-F-ELP4 (SEQ ID No 88) containing ELP4 linked at its N-terminus to the chimeric aprotinin HA-SP-3F-FX-APRO followed by the 1× Flag Tag (
[0294] The transformation vector pLA02 was obtained by cloning by Gibson Assembly into pLE63 (linearized by BamHI and PmeI), the PCR fragment FPCR-FELP4-HA (SEQ ID No 89) (359 pb) amplified from pLA00 with primers O5′Gibs02BE (SEQ No 90) and O3′Gibs02BE (SEQ No 91). The transformation vector pLA02 allowed the production of fusion protein HA-SP-3F-1F-ELP4 containing ELP4 linked at its N-terminus to the chimeric sequence HA-SP-3F followed by the 1× Flag Tag (
[0295] In the case of algae chloroplasts transformed by pLA01 or pLA02 and if the signal peptide SP is cleaved after translocation of the fusion protein into the lumen of thylakoids, two other different proteins can be produced in vivo, 3F-FX-APRO-1F-ELP4 or 3F-1F-ELP4 (SEQ ID No 92).
[0296] In both types of transformation vectors, the release of ELP4 from the fusion proteins can be performed in vitro by enterokinase digestion which cleaved the protein sequence after the second lysine amino acid in the motif sequence DYKDDDDK present in the 1× Flag Tag just upstream ELP4.
[0297] The elastin like polypeptide named ELPE4 consisted of a repeat of the VGVAPGE (SEQ ID No 38), a derivative of the peptide VGVAPG, more particularly of a 4-fold repeat of this peptide (SEQ No 40, VGVAPGEVGVAPGEVGVAPGEVGVAPGE). In chloroplast transformation vector, ELPE4 was also expressed in a fusion protein in which it was fused at the C-terminus of the chimeric aprotinin HA-SP-3F-FX-APRO (SEQ ID No 5) (
[0298] In order to separate in vitro by the ELPE4 from the carrier, a flexible linker LGM (SEQ ID No 65: RSGGGGSSGGGGGGSSRS) followed by a cleavage site for TEV protease (TV; SEQ ID No 93: ENLYFQG) or enterokinase (EK; SEQ ID No 94: DDDDK) were added.
[0299] Two types of fusion proteins have been produced from two different chloroplast expression vectors: HA-SP-3F-FX-APRO-LGM-TV-ELPE4 or HA-SP-3F-FX-APRO-LGM-EK-ELPE4
[0300] The nucleic acid sequence encoding LGM-TV-ELPE4 or LGM-EK-ELPE4 were codon-optimized using also the same method described in Example 1 and the codon usage for chloroplast genome of C. reinhardtii. After codon optimization, the different synthetic genes Igm-tv-elpe4 (SEQ ID No 95) and Igm-ek-elpe4 (SEQ ID No 96) were synthetized by Eurofins. These optimized genes were cloned by Gibson assembly downstream the gene encoding the carrier into an expression cassette (SEQ No 97) present in the chloroplast transformation vector pAU76 linearized by PmeI to give respectively, pLA03 and pLA04 (
Transformation of Algae
[0301] The transformation vectors pAL01, pLA02, pLA03 and pLA04 were bombarded in C. reinhardtii cell (137c and CW15) as described in the Example 1.
[0302] In order to identify stable integration of the recombinant genes encoding fusion protein into the chloroplast algal genome, spectinomycin resistant colonies were screened by PCR analysis using the primers O5′ASTatpA2 (SEQ No 82) and O3′SUTRpsbD (SEQ No 83) annealing, respectively, in the atpA 3′UTR and psbD 5′UTR.
Analyses and Results
[0303] Western Blot analysis performed using anti-Flag antibody on total soluble proteins extracted from different independent strains of LA01, LA03 or LA04 transformants revealed that the fusion proteins HA-SP-3F-FX-APRO-F-ELP4 (SEQ ID No 88), HA-SP-3F-FX-APRO-LGM-TV-ELPE4 (SEQ ID No 100) and HA-SP-3F-FX-APRO-LGM-EK-ELPE4 (SEQ ID No 101) were produced in the C. reinhardtii chloroplast.
[0304] As shown in the
[0305] In the LA02 transformants, no recombinant protein were detected (
[0306] Biomass of one transformant CW-LA01 was produced. Cell pellet was resuspended in sonication buffer.
[0307] Fusion protein were purified by anti-Flag M2 affinity chromatography. Elution fraction containing the fusion protein were identify by Western Blot analysis, dialyzed and concentrated. Enterokinase protease digestions were performed followed by a size exclusion chromatography (HiLoad 26/00 Superdex 30) allowing the purification of the polypeptide ELP4.
[0308] The same method was applied for the purification of the ELPE4. The fusion protein were purified by affinity chromatography. Elution fraction containing the fusion protein were identify by Western Blot analysis, dialyzed and concentrated. Enterokinase or TEV protease digestions were performed depending on the transformant followed by a size exclusion chromatography (HiLoad 26/00 Superdex 30) allowing the purification of the polypeptide ELPE4.
[0309] In order to cleave by endoproteinase the polypeptides ELPE4 into peptides VGVAPGE, the SEC elution fractions were evaporated and dialyzed for salts removing and buffer changing, using a dialysis tube with a 1 kDa cutoff.
[0310] After digestion by the Glu-C endoproteinase of the dialyzed samples as described in the Example 1, the released peptides were purified by a size exclusion chromatography.
Example 4
[0311] Production of Aprotinin as a Fusion Partner for the Production of a Mono and Polypeptides of KTTKS and Derivatives in the Chloroplast of Chlamydomonas reinhardtii by Chloroplast Genome Transformation
[0312] Construction of Transformation Vectors (pNY18, pNY19, pNY13, pNY14, pNY15, pNY16)
[0313] Several chloroplast transformation vectors were constructed in order to express the peptides KTTKS (named NY2 of SEQ ID No 18) and GKTTKS (named GNY2 of SEQ ID No 19) and polypeptides of KTTKS derivatives in a fusion protein using aprotinin as a carrier (
[0314] In chloroplast transformation vector, the peptide NY2 and the polypeptides (NY3a)×5 (KTTKSDKTTKSDKTTKSDKTTKSDKTTKSD) (SEQ ID No 26) and (NY3b)×5 (KTTKSEKTTKSEKTTKSEKTTKSEKTTKSE) (SEQ ID No 28) were produced in fusion proteins in which they were fused at the C-terminus of the chimeric aprotinin HA-SP-3F-FX-APRO (SEQ ID No 5). This fusion partner contained aprotinin fused at their N-terminus to an amino acid sequence made of the HA epitope Tag (HA) followed by the signal peptide (SP), the 3×Flag epitope Tag (3F), and the cleavage site for Factor Xa (FX; IEGR (SEQ ID No 102)).
[0315] In the fusion protein, the peptide NY2 or the polypeptides (NY3a)×5 and (NY3b)×5 were separated from the carrier by the flexible linker LGM (SEQ ID No 65: RSGGGGSSGGGGGGSSRS) followed by a cleavage site for TEV protease (TV; SEQ ID No 93; ENLYFQG) or enterokinase (EK; SEQ ID No 94; DDDDK).
[0316] Therefore, different fusion proteins were produced in independent algae transformants, as for instance, the protein called HA-SP-3F-FX-APRO-LGM-EK-NY2 (SEQ ID No 103 and 104), HA-SP-3F-FX-APRO-LGM-TV-NY2 (SEQ ID No 105 and 106), HA-SP-3F-FX-APRO-LGM-EK-(NY3a)×5 (SEQ ID No 107 and 108), HA-SP-3F-FX-APRO-LGM-TV-(NY3a)×5 (SEQ ID No 109 and 110), HA-SP-3F-FX-APRO-LGM-EK-(NY3b)×5 (SEQ ID No 111 and 112), HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 (SEQ ID No 113 and 114) (
[0317] After their production in algae chloroplasts, the signal peptide (SP) targeted these fusion proteins into the thylakoids. During protein translocation, the N-terminus fragment HA-SP were cleaved and the following other recombinant proteins were produced in vivo, 3F-FX-APRO-LGM-TV-(NY3b)×5 (SEQ ID No 115), 3F-FX-APRO-LGM-EK-(NY3b)×5 (SEQ ID No 116), 3F-FX-APRO-LGM-TV-(NY3a)×5 (SEQ ID No 117), 3F-FX-APRO-LGM-EK-(NY3a)×5 (SEQ ID No 118), 3F-FX-APRO-LGM-EK-NY2 (SEQ ID No 119) and 3F-FX-APRO-LGM-TV-NY2 (SEQ ID No 120).
[0318] The release of the peptides and the polypeptides from the chimeric aprotinin has been performed in vitro by site specific proteolysis of the fusion protein with enterokinase or TEV proteases.
[0319] After the cleavage of the fusion protein HA-SP-3F-FX-APRO-LGM-TV-NY2 or 3F-FX-APRO-LGM-TV-NY2 by the TEV protease, the released peptide was GNY2 (GKTTKS). In the case of the TEV digestion of the fusion proteins HA-SP-3F-FX-APRO-LGM-TV-(NY3a)×5 (or 3F-FX-APRO-LGM-TV-(NY3a)×5) and HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 (or 3F-FX-APRO-LGM-TV-(NY3b)×5), the released polypeptides was G((NY3a)×5) and G((NY3b)×5), respectively.
[0320] In Chlamydomonas reinhardtii, the codon usage in the nucleic acid sequence encoding protein of interest has been shown to play a significant role in protein accumulation (Franklin et al., 2002; Mayfield and Schultz, 2004).
[0321] The nucleic acid sequence encoding the chimeric aprotinin were designed and optimized in order to improve their expression in C. reinhardtii host cells
[0322] Methods for altering polynucleotides for improved expression in host cell are known in the art, particularly in algae cell, particularly in C. reinhardtii.
[0323] A codon usage database is found at http://www.kazusa.or.jp/codon/ (See the codon usage for chloroplast genome of C. reinhardtii;
[0324] For improving expression in C. reinhardtii chloroplast of the gene of interest, codons from their native sequence which are not commonly used, were replaced with a codon coding for the same or a similar amino acid residue that is more commonly used in the C. reinhardtii chloroplast codon bias. In addition, other codons were replaced to avoid sequences of multiple or extended codon repeats, or some restriction enzyme sites, or having a higher probability of secondary structure that could reduce or interfere with expression efficiency.
[0325] In order to check and to fulfill all criteria mentioned above, the amino acid sequence of the protein of interest were also optimized by the software GENEius of Eurofins using the appropriate usage codon for C. reinhardtii chloroplast genome.
[0326] After optimization, the gene encoding aprotinin (APRO) were operationally fused at its 5′end to a codon optimized nucleic acid sequence encoding the HA epitope Tag (HA) followed by a signal peptide, the 3×Flag epitope Tag (3F) and the cleavage site recognized by the Factor Xa protease (FX) to form the chimeric aprotinin HA-SP-3F-FX-APRO (SEQ ID No 5 AND 6).
[0327] The nucleic acid sequence encoding the recombinant peptide of KTTKS or GKTTKS, or polypeptide of KTTKS or their derivatives were designed and optimized as mentioned above in order to improve their expression in C. reinhardtii host cells.
[0328] After codon optimization, the different synthetic genes GNC-LENY3a2 (SEQ ID No 121), GNC-LENY3b1 (SEQ ID No 122), GNC-LTNY3a2 (SEQ ID No 123) and GNC-LTNY3b1 (SEQ ID No 124) encoding respectively the polypeptides (NY3a)×5, (NY3b)×5), G((NY3a)×5), G((NY3b)×5) were synthetized by Eurofins. These optimized genes were cloned by Gibson assembly method downstream the gene encoding the carrier into an expression cassette present in the chloroplast transformation vector pAU76 linearized by PmeI to give respectively, pNY16, pNY14, pNY15, and pNY13.
[0329] The chloroplast transformation vectors pNY13 and pNY14 allowed the expression of the polypeptide (NY3b)×5 in the fusion proteins HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 and HA-SP-3F-FX-APRO-LGM-EK-(NY3b)×5, respectively (
[0330] The chloroplast transformation vectors pNY15 and pNY16 allowed the expression of the polypeptide (NY3a)×5 in the fusion proteins HA-SP-3F-FX-APRO-LGM-TV-(NY3a)×5 and HA-SP-3F-FX-APRO-LGM-EK-(NY3a)×5, respectively (
[0331] The optimized genes GNC-ALENY2 (SEQ ID No 99) and GNC-ALTNY2 (SEQ ID No 98) were cloned by Gibson assembly method into an expression cassette present in the chloroplast transformation vector pAU63 linearized by BamHI and PmeI digestions to give respectively, pNY18 and pNY19.
[0332] The chloroplast transformation vectors pNY18 and pNY19 allowed the expression of the peptide NY2 in the fusion proteins HA-SP-3F-FX-APRO-LGM-EK-NY2 and HA-SP-3F-FX-APRO-LGM-TV-NY2, respectively (
[0333] The expression vectors pAU76 and pAU63 for chloroplast genome transformation contained two expression cassettes (
[0334] These two expression cassettes are flanked by a left (LHRR) and right (RHRR) endogenous homologous recombination sequences which are identical to those surrounding the targeted integration site into the C. reinhardtii chloroplast genome. The chloroplast transformation vectors i allow the targeted integration of the transgenes into the chloroplast genome of C. reinhardtii between the 5S rRNA and psbA genes (and derives from instance from Gen Bank Accession Number NC005352).
[0335] The selectable marker gene was the aadA gene coding aminoglycoside 3″-adenylyltransferase and conferring the resistance to spectinomycin and streptomycin. The gene is operationally linked at its 5′ end to the C. reinhardtii 16S rRNA promoter (Prrn) fused to the atpA 5′UTR (SEQ ID No 67) and at its 3′ end to the 3′UTR of the C. reinhardtii rbcL gene (SEQ ID No 69) (
[0336] Stable expression and translation of the fusion protein gene were controlled by the promoter and 5′UTR from the C. reinhardtii psbD (SEQ ID No 66) and the 3′UTR from C. reinhardtii atpA (SEQ ID No 67) (
[0337] The construction of pAU94 was previously described in the Example 2. The chloroplast transformation vector pAU94 allowed the production of the chimeric aprotinin HA-SP-APRO-LG-FX-NY2-3F (SEQ ID No 78 and 79).
Transformation of Algae
[0338] The transformation vectors pNY18, pNY19, pNY13, pNY14, pNY15, pNY16 were bombarded in C. reinhardtii cell (137c and CW15) as described in the Example 1. In order to identify stable integration of the recombinant genes encoding fusion protein into the chloroplast algal genome, spectinomycin resistant colonies were screened by PCR analysis. For positive PCR screens of the fusion protein gene, the primers
[0339] O5′ASTatpA2 5′-CCTACTTAATTAAAAACtgcagtagctagctctgc-3′ (SEQ ID No 82) and O3′SUTRpsbD 5′-cgatgagttgtttttttattttggagatacacgc-3′ (SEQ ID No 83) annealing, respectively, in the atpA 3′UTR and psbD 5′UTR were used.
Analyses and Results
[0340] Western Blot analysis of total soluble proteins extracted from different independent strains transformed with different expression vectors revealed that the fusion proteins HA-SP-3F-FX-APRO-LGM-EK-NY2, HA-SP-3F-FX-APRO-LGM-TV-NY2, HA-SP-3F-FX-APRO-LGM-EK-(NY3a)×5, HA-SP-3F-FX-APRO-LGM-TV-(NY3a)×5, HA-SP-3F-FX-APRO-LGM-EK-(NY3b)×5, HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 and HA-SP-APRO-LG-FX-NY2-3F were produced significantly (
[0341] Moreover, for all transformants, the HA Tag and the signal peptide SP seems to be cleaved because Western Blot analyses performed on total soluble protein extracts show that no specific protein is recognized by the anti-HA antibody.
[0342] The comparison of the fusion protein amounts between the different types of transformants, performed by Western blot analyses, seems to show that the clones CW-AL813-4 and CW-AL818-6 would strongly produce HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 (0.1% of total soluble proteins; TSP) and HA-SP-3F-FX-APRO-LGM-EK-NY2 (0.089% TSP), respectively.
Purification of Peptides and Polypeptides of KTTKS or Derivatives
[0343] About 80 g of cells for each transformants CW-NY13-4 and CW-NY18-6 were produced from several 1 L cascade cultures. Algae cells were resuspended and sonicated as described in the Material and Methods. 850 mL of total soluble protein extract for each transformants CW-NY13-4 and CW-NY18-6 were obtained.
[0344] In order to concentrate these extract volumes of 850 mL, a supplementary steps of ammonium sulfate precipitation and dialysis were added before the affinity chromatography as described in Example 1.
[0345] Fusion protein were purified by anti-Flag M2 affinity chromatography. Elution fractions were analysed by Western Blot analysis. The results, shown in
[0346] The elution fraction from affinity chromatography containing the fusion protein 3F-FX-APRO-LGM-EK-NY2 or 3F-FX-APRO-LGM-TV-(NY3b)×5 were dialyzed in dialysis cassettes (3.5 kDa MWCO) against the buffer used in the next step of protease digestion. and concentrated using centrifugal concentrators (3 kDa MWCO).
[0347] Then the cleavage of the peptide or polypeptide from the carrier was performed with a site specific proteolysis of the fusion protein APRO-LGM-EK-NY2 or APRO-LGM-TV-(NY3b)×5 using enterokinase or TEV protease, respectively.
[0348] After an overnight incubation at 4° C., the digestions of each fusion protein were injected on a HiLoad 26/600 Superdex 30 prep grade column and run at a rate of 2.6 mL/min. These size exclusion chromatography (SEC) experiments allowed the purification of the peptide NY2 and polypeptide (NY3b)×5.
[0349] Further analysis on the SEC purified polypeptide (NY3b)×5 and peptide NY2 were performed by high performance liquid chromatography (HPLC) on C18 and C4 large pore reverse phase columns with 215 nm UV detection and mass spectrometry.
[0350] In order to cleave the polypeptides (NY3b)×5 into peptides by endoproteinase, the SEC elution fractions were evaporated and dialyzed for salts removing and buffer changing, using a dialysis tube with a 1 kDa cutoff.
[0351] After digestion by the Glu-C endoproteinase of the dialyzed samples as described in the Example 1, the released peptides were purified by a size exclusion chromatography.