Recombinant microalgae able to produce peptides, polypeptides or proteins of collagen, elastin and their derivatives in the chloroplast of microalgae and associated method thereof
20230093611 · 2023-03-23
Inventors
Cpc classification
International classification
Abstract
The present invention concerns a recombinant microalgae comprising a nucleic acid sequence encoding a recombinant protein, polypeptide or peptide comprising repeat units of amino acids, said recombinant protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives, and said nucleic acid sequence being located in the chloroplast of microalgae. It further relates to a method for producing a recombinant protein, polypeptide or peptide comprising repeat units of amino acids in the chloroplast of microalgae, said recombinant protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives wherein said method comprises transforming the chloroplast genome of a microalgae with a nucleic acid sequence encoding said recombinant protein, polypeptide or peptide.
Claims
1. A recombinant microalgae comprising a nucleic acid sequence encoding a recombinant protein, polypeptide or peptide comprising repeat units of amino acids, said protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives, and said nucleic acid sequence being located in the chloroplast genome of microalgae.
2. A method for producing a recombinant protein, polypeptide or peptide comprising repeat units of amino acids in the chloroplast of microalgae, said protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives, wherein said method comprises transformation of the chloroplast genome of microalgae with a nucleic acid sequence encoding said recombinant protein, polypeptide or peptide.
3. The method according to claim 2, comprising: (i) providing a nucleic acid sequence encoding said 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; and (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 said nucleic acid sequence; (vi) extracting the recombinant protein, polypeptide or peptide; and optionally (vii) purifying the recombinant protein, polypeptide or peptide.
5. The method according to claim 3, wherein said expression vector also comprises at least one expression cassette, said at least one expression cassette comprising the nucleic acid sequence encoding said recombinant protein, polypeptide or peptide.
6. The method according to claim 2, 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 method according to claim 1, wherein said protein, polypeptide or peptide derivative consists in an amino acid sequence at least 80% identical to the amino acid sequence of the recombinant peptide, polypeptide or protein.
8. The method according to claim 3, wherein said 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 method according to claim 3, wherein said 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 and the atpA and rbcL 3′UTRs.
10. The method according to claim 5, wherein said 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 said nucleic acid sequence encoding the recombinant protein, polypeptide or peptide.
11. The method according to claim 5, wherein said at least one expression cassette further comprises a nucleic acid sequence encoding a signal peptide.
12. The method according to claim 5, wherein said at least one expression cassette further comprises a nucleic acid sequences encoding an amino acid sequence allowing the production of the said recombinant protein, polypeptide or peptide in specific cell compartment.
13. The method according to claim 2, wherein said microalgae is chosen from the group consisting of Chlorophyta, Chlorophyceae, Pleurastrophyceae, Prasinophyceae, Chromophyta, Bacillariophyceae (diatoms), Chrysophyceae, Phaeophyceae, Eustigmatophyceae, Haptophyceae, Raphidophyceae, Xanthophyceae, Cryptophyta, Cryptophyceae, Rhodophyta, Porphyridiophycea, Stramenopiles, Glaucophyta, Glaucocystophyceae, Chlorarachniophyceae, Haptophyceae, Dinophyceae, Scenedesmaceae, Euglenophyta, Euglenophyceae.
14. The method according to claim 13, wherein said microalgae is chosen from the group consisting of Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, and Nannochloropsis.
15. A method for producing a recombinant protein, polypeptide or peptide comprising repeat units of amino acids, said protein, polypeptide or peptide being chosen from collagen, elastin and their derivatives comprising the use of a recombinant microalgae according to claim 1.
16. 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.
17. The recombinant microalgae according to claim 1, wherein said protein, polypeptide or peptide derivative consists in an amino acid sequence at least 80% identical to the amino acid sequence of the recombinant peptide, polypeptide or protein.
18. The recombinant microalgae according to claim 1, wherein said 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.
19. The recombinant microalgae according to claim 1, wherein said 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 and the atpA and rbcL 3′UTRs.
20. The recombinant microalgae according to claim 1, wherein said microalgae is chosen from the group consisting of Chlorophyta, Chlorophyceae, Pleurastrophyceae, Prasinophyceae, Chromophyta, Bacillariophyceae (diatoms), Chrysophyceae, Phaeophyceae, Eustigmatophyceae, Haptophyceae, Raphidophyceae, Xanthophyceae, Cryptophyta, Cryptophyceae, Rhodophyta, Porphyridiophycea, Stramenopiles, Glaucophyta, Glaucocystophyceae, Chlorarachniophyceae, Haptophyceae, Dinophyceae, Scenedesmaceae, Euglenophyta, Euglenophyceae.
Description
FIGURES
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EXAMPLES
Example 1
[0166] Material and Methods
[0167] All oligonucleotides and synthetic genes were purchased from Eurof ins. 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
[0168] 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).
[0169] 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).
[0170] 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.
[0171] Growth kinetics was also followed by measuring the optical density at 750 nm using a spectrophotometer.
Algal Transformation
[0172] 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 1.10.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
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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
[0177] 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, 10 mM EDTA). As further detailed below, 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.
[0178] Depending on the further analysis step, total soluble proteins were extracted under non denaturing conditions in different buffers. 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/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.
[0179] Total soluble proteins present in the supernatant were quantified using the Pierce BCA protein assay kit, following the instructions of the supplier (Thermofisher).
[0180] 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).
[0181] 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.
[0182] 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).
TABLE-US-00002 TABLE 1 Primary antibodies Primary antibody Source Dilution Monoclonal ANTI-FLAG ® M2 antibody Sigma 1:1000 produced in mouse Monoclonal ANTI-HA PURIFIED antiobody Sigma 1:6000 produced in mouse IGG Monoclonal ANTI-Aprotinin antibody Abeam 1:2000 produced in mouse or 1:3000
[0183] 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
[0184] After centrifugation, algae cell pellets were resuspended in different buffers depending on the protein and on the further steps to which the protein is submitted. If the next step was an anti-FLAG M2 affinity chromatography, the buffer contained 50 mM Tris-HCl pH8, 500 mM NaCl and 0.1% Tween 20. If the next step was an anti-HA affinity chromatography, the buffer contained 20 mM Tris-HCl pH8. 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.
Affinity Chromatography
[0185] 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.
[0186] 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 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.
[0187] Some recombinant proteins were tagged with a HA epitope Tag which will bind specifically on an anti-HA agarose resin (Pierce/Thermo Scientific). All steps of this experiment were carried out as described by the manufacturer. Briefly, the filtered samples of total soluble proteins were mixed with an anti-HA agarose resin prepared as recommended by the manufacturer and equilibrated in TBS and incubated overnight at 4° C. with a gently and continuous end-over-end mixing or a rocking platform. After incubation, the resin was pelleted 5 to 10 second at 12,000 g (repeat 3 times). The supernatant was kept for further analysis. The pelleted resin was washed several times with 10 bed volumes of TBST. The protein of interest was eluted from the resin after incubation 15 min at 30° C. of the resin with 10 bed volumes of 1 mg/ml Pierce HA peptide. The resin was pelleted by centrifugation (5 to 10 second at 12,000 g). The supernatant containing the fusion protein was collected. This elution step was repeat 3 additional times.
[0188] Each elution fractions of affinity chromatography were further analyzed by SDS-PAGE and Western Blot.
[0189] Depending of the further step, 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
[0190] 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).
[0191] Enzymatic digestions were performed as recommended by the manufacturer.
[0192] 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.
[0193] 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.
[0194] 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 Endoproteinase
[0195] 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.
[0196] 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)
[0197] 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.
[0198] 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).
[0199] 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.
[0200] 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 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.
[0201] 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
[0202] Production of Collagen Like Protein, Collagen Like Domain and Collagen Like Polypeptide in the Chloroplast of Chlamydomonas reinhardtii by Chloroplast Genome Transformation
Construction of Transformation Vector for the Expression of the Collagen-Like Protein
[0203] In order to produce an innovating collagen-like protein (CLP) and/or collagen-like domain, we screened databases to find collagen-like genes encoding collagen-like domain from different origins. One of the sequence founded was the CCMP2712 protein from the microalgae Guillardia theta (G. theta). The amino acid sequence of the CCMP2712 protein from G. theta (called GtCLP SEQ ID N.sup.o 51) contained collagen like domain and was extracted from GenBank Accession Number XM 005827950.
[0204] Nothing was described about the capability of the protein encoded by the identified gene to form a collagen-like triple-helical structure.
[0205] 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).
[0206] The nucleic acid sequence encoding CCMP2712 protein was designed and optimized in order to improve its expression in C. reinhardtii host cell. Methods for altering nucleic acid sequence for improved expression in host cell are known in the art, particularly in algae cell, particularly in C. reinhardtii.
[0207] A codon usage database was found at http://www.kazusa.or.jp/codon/ (See the codon usage for chloroplast genome of C. reinhardtii;
[0208] 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 site, or having a higher probability of secondary structure that could reduce or interfere with expression efficiency.
[0209] 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 codon usage for C. reinhardtii chloroplast.
[0210] After its codon optimisation, the gene Gtclp encoding the native CCMP2712 protein from G. theta (called “recombinant GtCLP” or 3F-TV-GtCLP-TV-HA) was designed to be operationally fused at its 5′end to the codon optimized nucleic acid sequences encoding an amino acid sequence containing the 3×Flag epitope Tag (SEQ. DYKDDDDKDYKDDDDKDYKDDDDK; SEQ ID N.sup.o 25) followed by the recognition site of the TEV protease (SEQ ENLYFQG; SEQ ID N.sup.o 52). At its 3′end, the optimized gene Gtclp were operationally fused to the optimized nucleic acid sequence coding for the recognition site of the TEV protease followed by the HA epitope Tag (SEQ ID N.sup.o 26). The recombinant GtCLP produced in vivo in C. reinhardtii chloroplast was also called 3F-TV-GtCLP-TV-HA. The two recognition sites of the TEV protease allowed the elimination of the 3×Flag and HA Tags by in vitro protease digestions.
[0211] This resulting fusion gene 3f-tv-Gtclp-tv-ha (SEQ ID N.sup.o 10) encoding the recombinant GtCLP called 3F-TV-GtCLP-TV-HA (SEQ ID N.sup.o 55) was synthesized and cloned by Eurofins Genomics into the vector pEX-A258 resulting in vector pAL70.
[0212] After PCR amplification from the vector pAL70 using the primers O5′SCL70 (SEQ ID N.sup.o 56) and O3′SCL70 (SEQ ID N.sup.o 57), the PCR fragment FPCR-SCL70 of 1317 bp (SEQ ID N.sup.o 58) was cloned using the Gibson Assembly of New England Biolabs (as recommended by the manufacturer) into the expression cassette of the gene of interest (goi) present in the chloroplast transformation vector pLE56 linearized by NcoI and SalI to form the vector pCO86 (
[0213] The chloroplast expression vector pLE56 contained two expression cassettes for the expression of the genes encoding the selectable marker (gos) and the recombinant protein of interest (goi). The selection cassette contained the selectable marker aadA gene coding aminoglycoside 3″-adenylyltransferase and conferring the resistance to spectinomycin and streptomycin. This gene was operationally linked at its 5′ end to the C. reinhardtii 16S rRNA promoter (Prrn) fused to the atpA 5′UTR and at its 3′end to the 3′UTR of the C. reinhardtii rbcL gene. In the second cassette, stable expression of the recombinant goi was controlled by the promoter and 5′UTR from the C. reinhardtii psbD and the 3′UTR from the C. reinhardtii atpA.
[0214] These two expression cassettes were flanked by a left (LHRR) and right (RHRR) endogenous homologous recombination sequences which were 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. In a preferred embodiment, the chloroplast transformation vectors in the present invention allowed the targeted integration of the transgenes into the chloroplast genome of C. reinhardtii between the 5S rDNA and psbA genes (and derives from instance from the sequence GenBank Accession Number NC005352).
Construction of Chloroplast Transformation Vector for the Expression of the Chimeric Collagen-Like Domain of G. theta CCMP2712 CLP.
[0215] The collagen-like domain from G. theta CCMP2712 protein (SEQ ID N.sup.o 59; named GtCLD) was designed in order to fuse at its N-terminus with an amino acid sequence containing a Cys Knot and CR4 Repeat sequences (SEQ. GPCCGPPGPPGPPGPP, SEQ ID N.sup.o 60). The Cys Knot and CR4 Repeat sequences were known in the art as adding conformational rigidity of sequence. At its C-terminus, GtCLD was fused with a CR4 Repeat followed by Cys Knot sequences and the Foldon fibritin of the T4 phage (SEQ. GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID N.sup.o 61). The resulting recombinant protein was named chimeric collagen-like domain from G. theta or GtCCLD (SEQ ID N.sup.o 62). The GtCCLD protein was also designed to be fused at its C-terminus a 3×HA epitope Tag (called 3HA; SEQ ID N.sup.o 63: YPYDVPDYAYPYDVPDYAYPYDVPDYA).
[0216] The synthetic codon optimized gene Gtccld-3ha (SEQ ID N.sup.o 84) were synthetized and cloned in the vector pEX-A258 by Eurofins Genomics to form the pAL81. The gene Gtccld-3ha was subcloned in the goi expression cassette of the chloroplast transformation vector pLE63 previously linearized by BamHI and PmeI digestions to give pCO96. More precisely, Gtccld-3ha was subcloned downstream the nucleic acid sequence ha-sp-3F encoding the HA epitope Tag (HA) linked to the signal peptide (SP) followed by the 3×Flag epitope Tag (HA-SP-3F) (
[0217] Then, in C. reinhardtii chloroplast, the recombinant GtCCLD-3HA (SEQ ID N.sup.o 64) was produced fused at its N-terminus with the amino acid sequence HA-SP-3F and was called HA-SP-3F-GtCCLD-3HA (SEQ ID N.sup.o 83). This recombinant protein was encoded by the nucleic acid sequence called ha-sp-3f-Gtccld-3ha (SEQ ID N.sup.o 19). The chloroplast expression vector pLE63 contained the same expression cassette for the selectable marker as pLE56 (
[0218] In order to remove the nucleic sequence encoding the 3×HA Tag, the PCR fragment Gtccld-2 (SEQ ID N.sup.o 65) containing the nucleic sequence encoding the recombinant chimeric collagen-like domain from the G. theta GtCCLD was amplified by PCR from pAL81 and cloned by the Gibson assembly method between the psbD promoter/5′UTR and the atpA 3′UTR into pLE63 linearized by BamHI and PmeI to form pCO26 (
[0219] Then, in C. reinhardtii chloroplast, the recombinant chimeric collagen-like domain from the G. theta GtCCLD was produced fused at its N-terminus with the amino acid sequence HA-SP-3F and was called HA-SP-3F-GtCCLD (SEQ ID N.sup.o 85) encoded by the nucleic acid sequence ha-sp-3f-Gtccld (SEQ ID N.sup.o 20).
[0220] In order to produce only the collagen-like domain from G. theta without Cys Knot and CR4 Repeat sequences, the PCR fragment Gtcld (SEQ ID N.sup.o 66) containing the nucleic sequence the gene was amplified by PCR from pAL81 and cloned by the Gibson assembly method between the psbD promoter/5′UTR and the atpA 3′UTR into pLE63 linearized by BamHI and PmeI to form pCO28 (
[0221] Then, in the C. reinhardtii chloroplast, the recombinant collagen-like domain from the G. theta GtCLD was produced fused at its N-terminus with the amino acid sequence HA-SP-3F and was called HA-SP-3F-GtCLD (SEQ ID N.sup.o 86) encoded by the nucleic acid sequence ha-sp-3f-Gtcld (SEQ ID N.sup.o 21).
[0222] After their production in algae chloroplast, the signal peptide (SP) will be cleaved in vivo from the recombinant proteins HA-SP-3F-GtCCLD-3HA, HA-SP-3F-GtCCLD and HA-SP-3F-GtCLD during their translocations into the thylakoids. Thus, 3 others proteins could be produced 3F-GtCCLD-3HA, 3F-GtCCLD and 3F-GtCLD.
[0223] The 3×Flag Tag will be cleaved by in vitro enterokinase digestion of these recombinant proteins.
Transformation of Algae
[0224] The transformation vectors pCO86, pCO96, pCO26 and pCO28 were bombarded in C. reinhardtii cells (137c and CW15) as described in the example 1.
[0225] 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 in CO96, CO26 and CO28 transformants, the primers O5′ASTatpA2 (SEQ ID N.sup.o 67) and O3′SUTRpsbD (SEQ ID N.sup.o 68) annealing, respectively, in the atpA 3′UTR and psbD 5′UTR were used. For CO86 transformants, two other primers were used O5′SCL70 (SEQ ID N.sup.o 69) and O5′PpsbDCla2 (SEQ ID N.sup.o 70), annealing, respectively, in the gene of interest and psbD promoter.
Analyses and Results
[0226] Western Blot analysis were performed in reducing conditions on total soluble protein samples extracted from several transformants obtained after transformation with pCO86, pCO96, pCO26 and pCO28 (from 137c and CW15 strains).
[0227] The results showed that the four recombinant proteins were produced, being probed by the anti-Flag antibody and/or anti-HA antibody.
[0228] The
[0229] In order to release the recombinant proteins of interest from the N-terminus epitope Tag fused or not with the signal peptide SP, total soluble protein samples extracted from one clone of CO96, CO26 and CO28 transformants were digested in vitro by enterokinase digestion as described in example 1. The
[0230] In the case of CO86 transformants producing the recombinant protein 3F-TV-GtCLP-TV-HA, total soluble protein samples were digested in vitro either by enterokinase to give the protein TV-GtCLP-TV-HA or by TEV protease to give the protein GtCLP.
[0231] 137c-CO96-4 cells were produced from around 900 mL cultures. Algae cells (around 3 g) were resuspended and sonicated as described in the example 1. 14.6 mL of total soluble protein extract was obtained.
[0232] Recombinant protein from 6.5 mL protein extract from 137c-CO96-4 were purified by affinity chromatography using anti-HA resin (150 μl). Elution fractions were analysed by Western Blot analysis. As an example, the results, shown in
[0233] Western Blot analysis performed in non-reducing conditions (without DDT and boiling or not the protein sample before loading onto the polyacrylamide gel) showed that the chimeric collagen like domain produced in CO96 transformants formed in vivo multimeric structures of high apparent molecular weight in contrast to an apparent molecular weight of the protein in reducing conditions.
Example 3
[0234] Production of Elastin Like Peptides or Polypeptides or Derivatives in a Fusion Protein Using Aprotinin as a Carrier in the Chloroplast of Chlamydomonas reinhardtii by Chloroplast Genome Transformation
Construction of Transformation Vectors (pLA01, pLA02, pAL03 and pAL04)
[0235] In chloroplast transformation vector, ELP4, an elastin like polypeptide consisted of a repeat of the VGVAPG hexapeptide (SEQ ID N.sup.o 5), more particularly of a 4-fold repeat of this hexapeptide (SEQ ID N.sup.o 81: 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 (SEQ ID N.sup.o 41 and 42). 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 N.sup.o 71: IEGR). The Flag epitope Tag sequence (SEQ ID N.sup.o 24: 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.
[0236] After the production in algae chloroplasts of the fusion protein HA-SP-3F-FX-APRO-F-ELP4 (SEQ ID N.sup.o 87), the N-terminus fragment HA-SP will be cleaved during protein translocation into the thylakoids, and the following recombinant protein 3F-FX-APRO-F-ELP4 will be produced in vivo.
[0237] As explained in Example 2, 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.
[0238] The nucleic acid sequence encoding the aprotinin were designed and optimized as described in Example 2 in order to improve their expression in C. reinhardtii host cells. After optimization, the gene encoding aprotinin (APRO) were operationally fused at its 5′end to a codon optimized nucleic acid sequences 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 gene ha-sp-3f-fx-apro (SEQ ID N.sup.o 42).
[0239] The nucleic acid sequence encoding ELP4 was first codon-optimized using also the same method described in Example 1 and the codon usage of the C. reinhardtii chloroplast genome. The resulting sequence was used to design two overlapping oligomers O5′Gibs-ELP4 (SEQ ID N.sup.o 72) and O3′Gibs-ELP4 (SEQ ID N.sup.o 73) 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 and linearized by PmeI to form the vector pLA00.
[0240] The expression vector pAU76 for chloroplast genome transformation contained two expression cassettes for the expression of the genes encoding the selectable marker (identical to previous transformation vector) and the chimeric aprotinin HA-SP-3F-FX-APRO. pAU76 allowed the same targeted integration of the recombinant genes into the C. reinhardtii chloroplast genome as pLE63.
[0241] The nucleic acid sequence encoding ELP4 were amplified by PCR from pLA00 using the primers O5′Gibs01BE (SEQ ID N.sup.o 74) and O3′Gibs01BE (SEQ ID N.sup.o 75). The PCR fragment FPCR-AP-FELP4 (SEQ ID N.sup.o 76) of 359 pb were cloned using the Gibson Assembly Master Mix into the pLE63 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 containing ELP4 linked at its N-terminus to the chimeric aprotinin HA-SP-3F-FX-APRO followed by the 1×Flag Tag (
[0242] The chloroplast transformation vector pLA02 was obtained by cloning by the Gibson Assembly method into pLE63 (linearized by BamHI and PmeI), the PCR fragment FPCR-FELP4-HA (SEQ ID N.sup.o 77) (359 pb) amplified from pLA00 with primers O5′Gibs02BE (SEQ ID N.sup.o 78) and O3′Gibs02BE (SEQ ID N.sup.o 79). 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 (
[0243] 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 N.sup.o 88).
[0244] 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 (SEQ ID N.sup.o 24) present in the 1×Flag Tag just upstream ELP4.
[0245] The elastin like polypeptide named ELPE4 consisted of a repeat of the VGVAPGE (SEQ ID N.sup.o 9), a derivative of the peptide VGVAPG, more particularly of a 4-fold repeat of this peptide (SEQ ID N.sup.o 80, 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.
[0246] In order to separate in vitro by the ELPE4 from the carrier, a flexible linker LGM (SEQ ID N.sup.o 50: RSGGGGSSGGGGGGSSRS) followed by a cleavage site for TEV protease (TV; SEQ ID N.sup.o 52: ENLYFQG) or enterokinase (EK; SEQ ID N.sup.o 38: DDDDK) were added.
[0247] Two types of fusion proteins have been produced from two different chloroplast expression vectors: HA-SP-3F-FX-APRO-LGM-TV-ELPE4 (SEQ ID N.sup.o 89) or HA-SP-3F-FX-APRO-LGM-EK-ELPE4 (SEQ ID N.sup.o 90).
[0248] 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 N.sup.o 40) and Igm-ek-elpe4 (SEQ ID N.sup.o 48) were synthetized by Eurofins. These optimized genes were cloned by the Gibson assembly method downstream the gene encoding the carrier into an expression cassette (SEQ N.sup.o 82) present in the chloroplast transformation vector pAU76 linearized by PmeI to give respectively, pLA03 and pLA04.
Transformation of Algae
[0249] The transformation vectors pAL01, pLA02, pLA03 and pLA04 were bombarded in C. reinhardtii cell (137c and CW15) as described in the Example 1.
[0250] 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 ID N.sup.o 67) and O3′SUTRpsbD (SEQ ID N.sup.o 54) annealing, respectively, in the atpA 3′UTR and psbD 5′UTR.
Analyses and Results
[0251] 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 N.sup.o 87), HA-SP-3F-FX-APRO-LGM-TV-ELPE4 (SEQ ID N.sup.o 89) and HA-SP-3F-FX-APRO-LGM-EK-ELPE4 (SEQ ID N.sup.o 91) were produced in the C. reinhardtii chloroplast.
[0252] As shown in the
[0253] In the LA02 transformants, no recombinant protein were detected (
[0254] Biomass of one transformant CW-LA01 was produced. Cell pellet was resuspended in sonication buffer.
[0255] 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.
[0256] 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.
[0257] In the case of LA03 transformants and after the TEV protease digestion, the released polypeptide was GVGVAPGEVGVAPGEVGVAPGEVGVAPGE (SEQ ID N.sup.o 53).
[0258] 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.
[0259] 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.