RECOMBINANT MICROALGAE ABLE TO PRODUCE KTTKS PEPTIDES, POLYPEPTIDES, OR PROTEINS AND THEIR DERIVATIVES AND ASSOCIATED METHOD AND USES THEREOF
20230137630 · 2023-05-04
Assignee
Inventors
Cpc classification
C12N15/79
CHEMISTRY; METALLURGY
C07K14/78
CHEMISTRY; METALLURGY
C07K2319/01
CHEMISTRY; METALLURGY
A61K8/64
HUMAN NECESSITIES
C12N15/8257
CHEMISTRY; METALLURGY
A61K8/645
HUMAN NECESSITIES
International classification
A61K8/64
HUMAN NECESSITIES
Abstract
The present invention concerns a recombinant microalgae comprising a nucleic acid sequence encoding a recombinant peptide of KTTKS (SEQ ID No 1); a recombinant peptide, polypeptide or protein consisting in repeated units of SEQ ID No 1; or a derivative thereof, said nucleic acid sequence being located in the chloroplast genome of microalgae. It also relates to a method for producing a recombinant peptide of SEQ ID No 1; a recombinant peptide, polypeptide or protein consisting in repeated units of SEQ ID No 1; or a derivative thereof, wherein said method comprises the chloroplast genome transformation of a microalgae with a nucleic acid sequence encoding said recombinant protein, polypeptide or peptide. It further relates to the use of said recombinant peptide, polypeptide or protein for the cosmetic industry.
Claims
1. A recombinant microalgae comprising a nucleic acid sequence encoding: (a) a recombinant peptide of SEQ ID NO:1; (b) a recombinant peptide, polypeptide or protein consisting in repeated units of SEQ ID NO:1; or (c) a derivative of (a) or (b); the nucleic acid sequence being located in the chloroplast genome of microalgae.
2. A method for producing: (a) a recombinant peptide of SEQ ID NO:1; (b) a recombinant peptide, polypeptide or protein consisting in repeated units of SEQ ID NO:1; or (c) a derivative of (a) or (b); in the chloroplast of microalgae, wherein the method comprises transformation of the chloroplast genome of microalgae with a nucleic acid sequence encoding the recombinant protein, polypeptide or peptide.
3. The method according to claim 2, comprising: (i) providing a nucleic acid sequence encoding the recombinant protein, polypeptide or peptide; (ii) introducing the nucleic acid sequence according to (i) into an expression vector which is capable of expressing the nucleic acid sequence in microalgae host cell; (iii) transforming the chloroplast genome of microalgae host cell by the expression vector.
4. The method according to claim 3, further comprising: (iv) identifying the transformed microalgae host cell; (v) characterizing the microalgae host cell for the production of recombinant protein, polypeptide or peptide expressed from the nucleic acid sequence; and (vi) extracting the recombinant protein, polypeptide or peptide; and optionally (vii) purifying the recombinant protein, polypeptide or peptide.
5. The according to claim 3, wherein the expression vector also comprises at least one expression cassette, the at least one expression cassette comprising the nucleic acid sequence encoding the recombinant protein, polypeptide or peptide.
6. The recombinant microalgae according to claim 1, wherein the nucleic acid sequence encoding the protein, polypeptide or peptide is codon optimized for expression in the chloroplast genome of the microalgae host cell.
7. The recombinant microalgae according to claim 1, wherein the derivative of (a) or (b) consists in an amino acid sequence at least 80% identical to the amino acid sequence of the recombinant peptide of SEQ ID NO:1 or of the recombinant peptide, polypeptide or protein consisting in repeated units of SEQ ID NO:1.
8. The recombinant microalgae according to claim 1, wherein the nucleic acid sequence encoding a recombinant protein, polypeptide or peptide is fused operationally at its 5′ or 3′ end to a nucleic acid sequence encoding a carrier.
9. The recombinant microalgae according to claim 1 wherein the nucleic acid sequence encoding a recombinant protein, polypeptide or peptide is operably linked to at least one regulatory sequence chosen from the psbD promoter and 5′UTR or the 16S rRNA promoter (Prrn) promoter fused with the atpA 5′UTR, the psaA promoter and 5′UTR, the atpA promoter and 5′ UTR, the atpA and rbcL 3′UTRs.
10. The method according to claim 5, wherein the at least one expression cassette further comprises a nucleic acid sequence encoding an epitope Tag peptide fused operationally at its 5′ or 3′end to the nucleic acid sequence encoding the recombinant protein, polypeptide or peptide.
11. The method according to claim 5 wherein the at least one expression cassette further comprises a nucleic acid sequence encoding a signal peptide.
12. The method according to claim 5, wherein the at least one expression cassette further comprises a nucleic acid sequence encoding an amino acid sequence allowing the production of the recombinant protein, polypeptide or peptide in specific cell compartment.
13. The recombinant microalgae according to claim 1, wherein the microalgae is selected 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.
14. The recombinant microalgae or method according to claim 13, wherein the microalgae is selected from the group consisting of Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, and Nannochloropsis.
15. A method for manufacturing a cosmetic composition, comprising adding as a component the peptide and/or polypeptide and/or protein aqueous mixture produced according to the method of claim 2.
16. A cosmetic non-therapeutical treatment comprising as a component the peptide and/or polypeptide and/or protein aqueous mixture produced according to the method of claim 2 and free of algae debris.
17. (canceled)
Description
FIGURES
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EXAMPLES
Example 1
[0202] Material and Methods
[0203] 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
[0204] 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).
[0205] 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 comprise 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).
[0206] 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.
[0207] Growth kinetics was also followed by measuring the optical density at 750 nm using a spectrophotometer.
Algal Transformation
[0208] Chlamydomonas reinhardtii cells are 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 1.10.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
[0209] 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.
[0210] 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.
[0211] After transformation, algae colonies growing onto restrictive solid medium plates were expected to have the antibiotic resistant gene and the other transgene(s) incorporated into their genome.
[0212] In order to identify stable integration of the recombinant genes into the algal genome, 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
[0213] Chlamydomonas cells (50 mL, 1-2.10.sup.8 cells/mL) were collected by centrifugation. Cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 6.8, 2% SDS and 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.
[0214] Depending on the further analysis step, total soluble proteins were extracted under non denaturing conditions. Cell pellet was resuspended in a buffer containing 20 mM Tris-HCl pH 6.8. The sonication step was carried out with the algal cell suspension held on ice, using a cell disruptor sonicator FB505 500W (Sonic/FisherBrand) 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.
[0215] Total soluble proteins present in the supernatant were quantified using the Pierce BCA protein assay kit, following the instructions of the supplier (Thermofisher).
[0216] 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).
[0217] 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.
[0218] 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 3).
[0219] The antibody mentioned in Table 3 below were used as primary antibodies.
TABLE-US-00003 TABLE 3 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 antibody produced in mouse or 1:3000
[0220] 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
[0221] Depending on the protein and on the further steps to which the protein is submitted, the first or second method below is conducted.
[0222] 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.
[0223] 2) After centrifugation, algae cell pellets were resuspended in a buffer containing 20 mM Tris-HCl pH 6.8. 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
[0224] 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.
[0225] 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 4 h 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 TBST and 20 column volumes 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 was further analyzed by SDS-PAGE and Western Blot.
[0226] 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
[0227] 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).
[0228] Enzymatic digestions were performed as recommended by the manufacturer.
[0229] 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 16 h.
[0230] 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.
[0231] 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
[0232] 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.
[0233] 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)
[0234] 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.
[0235] 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).
[0236] 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 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.
[0237] 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 the 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.
[0238] 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 acid sequence at the N-terminus of the protein of interest.
Example 2
[0239] Production of Mono and Polypeptides of KTTKS or Derivatives in a Fusion Protein Using Aprotinin as Carrier, in the Chloroplast of Chlamydomonas Reinhardtii by Chloroplast Genome Transformation
[0240] Construction of Transformation Vectors (pNY18, pNY19, pNY13, pNY14, pNY15, pNY16)
[0241] Several chloroplast transformation vectors were constructed in order to express the peptides KTTKS (named NY2) and GKTTKS (named GNY2) and polypeptides of KTTKS derivatives in a fusion protein using aprotinin as a carrier (
[0242] In chloroplast transformation vector, the peptide NY2 and the polypeptides (NY3a)×5 (KTTKSDKTTKSDKTTKSDKTTKSDKTTKSD) (SEQ ID No 9) and (NY3b)×5 (KTTKSEKTTKSEKTTKSEKTTKSEKTTKSE) (SEQ ID No 11) were produced in fusion proteins in which they were fused, as examplified in the present invention, at the C-terminus of the chimeric aprotinin HA-SP-3F-FX-APRO. 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 61)).
[0243] 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 (RSGGGGSSGGGGGGSSRS) followed by a cleavage site for TEV protease (TV; SEQ ID No 32; ENLYFQG) or enterokinase (EK; SEQ ID No 33; DDDDK).
[0244] 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 33 and 34), HA-SP-3F-FX-APRO-LGM-TV-NY2 (SEQ ID No 35 and 36), HA-SP-3F-FX-APRO-LGM-EK-(NY3a)×5 (SEQ ID No 37 and 38), HA-SP-3F-FX-APRO-LGM-TV-(NY3a)×5 (SEQ ID No 39 and 40), HA-SP-3F-FX-APRO-LGM-EK-(NY3b)×5 (SEQ ID No 41 and 42), HA-SP-3F-FX-APRO-LGM-TV-(NY3b)×5 (SEQ ID No 43 and 44) (
[0245] After their production in algae chloroplasts, the signal peptide (SP) will target these fusion proteins into the thylakoids. During protein translocation, the N-terminus fragment HA-SP will be cleaved and the following other recombinant proteins will be produced in vivo, 3F-FX-APRO-LGM-TV-(NY3b)×5 (SEQ ID No 62), 3F-FX-APRO-LGM-EK-(NY3b)×5 (SEQ ID No 63), 3F-FX-APRO-LGM-TV-(NY3a)×5 (SEQ ID No 64), 3F-FX-APRO-LGM-EK-(NY3a)×5 (SEQ ID No 65), 3F-FX-APRO-LGM-EK-NY2 (SEQ ID No 66) and 3F-FX-APRO-LGM-TV-NY2 (SEQ ID No 67).
[0246] The release of the peptides and the polypeptides from the chimeric aprotinin will be performed in vitro by site specific proteolysis of the fusion protein with enterokinase or TEV proteases.
[0247] 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 will be 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 will be G((NY3a)×5) and G((NY3b)×5), respectively.
[0248] 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).
[0249] The nucleic acid sequence encoding the chimeric aprotinin were designed and optimized in order to improve their expression in C. reinhardtii host cells
[0250] Methods for altering polynucleotides for improved expression in host cell are known in the art, particularly in algae cell, particularly in C. reinhardtii.
[0251] A codon usage database is found at http://www.kazusa.or.jp/codon/. (See the codon usage for chloroplast genome of C. reinhardtii;
[0252] For improving expression in C. reinhardtii chloroplast of the gene of interest in the present invention, 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 codon bias from the C. reinhardtii chloroplast genome. 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.
[0253] 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.
[0254] 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 26 AND 27).
[0255] 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.
[0256] After codon optimization, the different synthetic genes GNC-LENY3a2 (SEQ ID No 68), GNC-LENY3b1 (SEQ ID No 69), GNC-LTNY3a2 (SEQ ID No 70) and GNC-LTNY3b1 (SEQ ID No 71) encoding respectively the polypeptides (NY3a)×5, (NY3b)×5), G((NY3a)×5), G((NY3b)×5) were synthetized by Eurofins. These optimized genes were cloned by the 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.
[0257] 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 (
[0258] 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 (
[0259] The optimized genes GNC-ALENY2 (SEQ ID No 72) and GNC-ALTNY2 (SEQ ID No 73) were cloned by Gibson assembly method into an expression cassette present in the chloroplast transformation vector pLE63 linearized by BamHI and PmeI digestions to give respectively, pNY18 and pNY19.
[0260] 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 (
[0261] The expression vectors pAU76 and pLE63 for chloroplast genome transformation contained two expression cassettes (
[0262] 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. In a preferred embodiment, the chloroplast transformation vectors in the present invention 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 GenBank Accession Number NC005352).
[0263] 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 76) and at its 3′ end to the 3′UTR of the C. reinhardtii rbcL gene (SEQ ID No 77) (
[0264] 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 78) and the 3′UTR from C. reinhardtii atpA (SEQ ID No 79) (
[0265] Transformation of Algae
[0266] 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 O5′ASTatpA2 5′-CCTACTTAATTAAAAACTGCAGTAGCTAGCTCTGC-3′ (SEQ ID No 74) and O3′SUTRpsbD 5′-CGATGAGTTGTTTTTTTATTTTGGAGATACACGC-3′ (SEQ ID No 75) annealing, respectively, in the atpA 3′UTR and psbD 5′UTR were used.
[0267] Analyses and Results
[0268] 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 were well produced (
[0269] Moreover, in all transformants, the HA epitope Tag and the signal peptide seems to be cleaved because Western blots performed on the same total soluble protein extracts showed that the primary anti-HA antibody didn't recognize any fusion protein. Thus, the fusion proteins produced in the different transformants would be 3F-FX-APRO-LGM-EK-NY2, 3F-FX-APRO-LGM-TV-NY2, 3F-FX-APRO-LGM-EK-(NY3a)×5, 3F-FX-APRO-LGM-TV-(NY3a)×5, and 3F-FX-APRO-LGM-EK-(NY3b)×5.
[0270] The comparison of the fusion protein amounts between the different types of transformants, performed by Western blot analyses, showed that among all producing transformants, the clones CW-NY13-4 and CW-NY18-6 produced high levels of 3F-FX-APRO-LGM-TV-(NY3b)×5 (0.1% of total soluble proteins; TSP) and 3F-FX-APRO-LGM-EK-NY2 (0.089% TSP), respectively. Thus, these two transformants were used for larger scale production.
Example 3
[0271] Purification of Peptides and Polypeptides of KTTKS or Derivatives
[0272] 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.
[0273] 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.
[0274] Fusion protein were purified by anti-Flag M2 affinity chromatography. Elution fractions were analysed by Western Blot analysis. The results, shown in
[0275] 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).
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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.