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WO2006005362A1 - Production de proteines mammiferes dans des cellules vegetales - Google Patents

Production de proteines mammiferes dans des cellules vegetales Download PDF

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Publication number
WO2006005362A1
WO2006005362A1 PCT/EP2004/007727 EP2004007727W WO2006005362A1 WO 2006005362 A1 WO2006005362 A1 WO 2006005362A1 EP 2004007727 W EP2004007727 W EP 2004007727W WO 2006005362 A1 WO2006005362 A1 WO 2006005362A1
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Prior art keywords
solulin
protein
dspa
dspaαi
recombinant
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Inventor
Stefan Schillberg
Helga Schinkel
Andreas Schiermeyer
Aachim Schüttler
Wolfgang Söhngen
Rainer Fischer
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Paion Deutschland GmbH
Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Paion Deutschland GmbH
Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon

Definitions

  • the invention relates to the field of production of mammalian proteins or peptides in plant cells ("molecular farming”).
  • Thrombomodulin is an endothelial, cell-surface glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III (Esmon et al., 1982).
  • Thrombomodulin consists of a lectin-like domain, six epidermal growth factor-like domains, an O-linked glycosylation site, a transmembrane region and a cytosolic domain (Suzuki et al., 1987).
  • Solulin a soluble derivative of human thrombomodulin, lacks the transmembrane and cytosolic domains and has six mutations that improve protein stability (Weisel et al., 1996).
  • Solulin is a glycoprotein con ⁇ taining an 18-amino-acid N-terminal signal sequence, which promotes co- translational transport into the secretory pathway in mammalian cells (Glaser et al., 1991).
  • the mature glycosylated protein is 487 amino acids in length has an apparent molecular mass of -75 kDa as determined by SDS-PAGE under non-reducing conditions.
  • Thrombotic disorders are caused by clot for ⁇ mation in response to a stimulus like a damaged vessel wall. This triggers the coagulation cascade generating thrombin, which is converting fibrinogen to fibrin, the matrix of the clot.
  • soluble, oxida ⁇ tion-resistant thrombomodulin analogs such as Solulin inhibits the proco- agulant activity of thrombin by building a thrombin-thrombomodulin complex and acting as an anticoagulant by activation of protein C, which in turn at ⁇ tenuates the clotting cascade by inactivation of several activated cofactors (Esmon et al., 1989).
  • This mechanism can be used to treat diseases in which thrombus formation plays a significant etiological role, e.g.
  • recombinant thrombomodulin is produced in mammalian cell cul ⁇ ture such as Chinese hamster ovary (CHO) cells (Lin et al., 1994).
  • mammalian cell cul ⁇ ture such as Chinese hamster ovary (CHO) cells
  • CHO Chinese hamster ovary
  • a number of expression platforms based on plants have been developed recently, and these offer several advantages in terms of production scales, safety and economy for the production of pharmaceutical proteins (Twyman et al., 2003). We therefore assessed the feasibility of producing recombinant solu- ble thrombomodulin in tobacco plants and BY2 cell suspension cultures.
  • the recombinant protein was fused to apoplast, protein body and vacuolar targeting sequences.
  • the highest yields in transgenic plants were achieved by apoplast targeting, whereas the highest yields in BY2 cells were achieved using a targeting sequence for protein bodies.
  • Active Solulin was produced in plant cells showing correct processing of the signal sequence demonstrating the potential of plants as an alternative production platform.
  • Escherichia coli strain DH5 ⁇ (Ausubel et al., 1994) was used for general cloning.
  • Agrobacterium tumefaciens strain GV3101 (pMp90RK, GmR KmR), RifR (Koncz & Schell, 1986) was used for agroinfiltration and stable trans ⁇ formation.
  • Tobacco plants Nicotiana tabac ⁇ m cv. Petite Havana SR1 and cv. Maryland Mammoth were used for agroinfiltration, and cv. Maryland Mam ⁇ moth was also used for stable transformation.
  • Suspension cells originated from N. tabacum L. cv. bright yellow 2 (BY2).
  • the cDNA of the thrombomodulin derivative ( Solulin) including the original signal sequence (ORI) was amplified by PCR from the source plasmid pTHR525 (provided by PAION GmbH, Aachen, Germany) using the primer set Solulin-for (signal) 5'-TTT ATA AAA AAA AAA AAA ACA ATG CTT GGG GTC CTG GTC-3' and Solulin-back (stop) 5'- GCG CGA AGC TTC TCG AGT TTA CGG AGG AGT CAA GGT AGA CCC-3'.
  • the primary PCR prod ⁇ uct was then used as the template in a second PCR with primers Solulin-for (5'UT) 5'-CGC GAA TTC ACA ACA CAA ATC AGA TTT ATA GAG AGA TTT ATA AAA AAA AAA AC-3' and Solulin-back (stop) to fuse the Pet- roselinum crispum chalcone synthase 5' untranslated region to the 5' end of the cDNA.
  • the product was ligated into the vector pTRAkc (a derivative of pPAM, GenBank accession number AY027531 , with an Xba ⁇ site omitted), using the EcoRI and Xho ⁇ sites.
  • This vector contained the double-enhanced CaMV 35S promoter (Kay et al., 1987) and the CaMV termination sequence, preceded by a polyadenylation site ( Figure 1).
  • the resulting construct was named Solulin-ORI.
  • the cDNA was fused to an apoplast targeting signal, which was based on the N-terminal signal sequence of a murine antibody heavy chain (Voss et al., 1995; Vaquero et al., 1999).
  • the DNA sequence encoding the last three amino acids of this signal was removed since the algorithm SignalP (Nielsen et al., 1997; http://www.cbs.dtu.dk/services/SiqnalP ' ) predicted that such action was nec ⁇ essary to achieve correct processing.
  • the resulting APO "3 sequence was amplified by PCR using the forward primer 35Sseq2 5'-ATC CTT CGC AAG ACC CTT CC-3' and the reverse primer Apo-back 5'-GCC ACC CGG CTG CGG CTC ACC TGC AGT GCC GCT G-3'.
  • the Solulin cDNA was amplified from plasmid pTHR525 using the primers Solulin-Apo 5'-CAG CGG CAC TGC AGG TGA GCC GCA GCC GGG TGG CAG CCA G-3' and SoIu- lin-back.
  • the two PCR fragments were then assembled in a SOE-PCR using the primers 35Sseq2 and Solulin-back.
  • the product of the SOE-PCR was ligated into pTRAkc using EcoRI and Xho ⁇ restriction sites, resulting in the final construct Solulin-APO "3 .
  • Solulin-VTS "4 the Solulin cDNA was combined with the N-ter ⁇ minal vacuolar targeting sequence of Catharanthus roseus strictosidine syn ⁇ thase (VTS; McKnight et al., 1990). The DNA encoding the last four amino acids was omitted to ensure proper signal sequence cleavage, based on predictions by the SignalP algorithm (see above). This truncation was achieved by cutting the VTS sequence from the template vector using Asc ⁇ and Sapl.
  • the Solulin cDNA was amplified by PCR from the source plasmid pTHR525 using primers Solulin-VTS 5'-CTA GCT CTT CAA GCG GAG CCG CAG CCG GGT GGC AGC-3' and Solulin-back.
  • the PCR product was digested with Xho ⁇ and Sap ⁇ , mixed with the Asc ⁇ and Sap ⁇ restricted VTS "4 sequence, and introduced into the pTRAkc vector (prepared by digestion with Asc ⁇ and Xho ⁇ ) in a triple ligation, resulting in the final construct Solulin- VTS "4 .
  • Solulin was fused to the signal sequence of Pisum sativum legumin A2 (PbTS; Rerie et al., 1990).
  • PbTS was amplified from a template vector with the primer 35Sseq2 and PbTS- back 5'-GCC ACC CGG CTG CGG TCG GCA AAG CAG CCT CCC-3'.
  • the Solulin cDNA was amplified from plasmid pTHR525 using the primers SoIuNn-PbTS 5'-GGG AGG CTG CTT TGC CGA GCC GCA GCC GGG TGG-3' and Solulin-back.
  • the two PCR fragments were then assem ⁇ bled by SOE-PCR using the primers 35S SEQ2 and Solulin-back.
  • the prod ⁇ uct of the SOE-PCR was ligated into pTRAkc using EcoRI and Xho ⁇ , result ⁇ ing in the final construct Solulin-PbTS.
  • the plant expression vectors described above were introduced into A. tumefaciens using a Gene Pulser Il electroporation system (BioRad, Hercu ⁇ les, USA) according to the manufacturer's instructions.
  • Transient expression in N. tabacum cv. Petit Havana SR1 or cv. Maryland Mammoth leaves was carried out by vacuum infiltration as previously described (Kapila et al., 1997; Vaquero et al., 1999).
  • Transgenic N. tabacum cv. Maryland Mammoth plants were generated by the leaf disc transformation method and transgenic plants were regenerated from transformed callus (Horsch et al., 1985). Plants were grown in a greenhouse under a 16-h natural daylight photoperiod and a 25°C/day, and 22°C/night temperature regime.
  • BY2 cells were generated by co-cultiva ⁇ tion with recombinant A. tumefaciens (An, 1985). BY2 cells were maintained in Murashige and Skoog (1962) medium supplemented with minimal organ- ics (BY2 medium: 0.47% (w/v) Murashige and Skoog salts, 0.15 ⁇ g ml "1 thiamine, 0.02 ⁇ g ml "1 KH 2 PO 4 and 3% (w/v) sucrose, pH 5.2) in an orbital shaker (New Brunswick Scientific, Edison, USA) at 180 rpm and 26°C in the dark. Cultures were subcultered every week with a 5% (v/v) inoculum.
  • the total soluble protein (TSP) from tobacco leaves was extracted as described by Fischer et al. (1999) using a 1 :2 (w/v) ratio of plant material and extraction buffer I (200 mM Tris-HCI, pH 7.4, 5 mM EDTA, 5 mM DTT, 0.1 % (v/v) Tween 20) or extraction buffer Il (150 mM NaCO 3 , pH 9.5, 500 mM NaCI, 1 % (v/v) DMSO). Protein extraction from cell cultures was carried out 4-6 days after reseeding, when the packed cell volume was 25-40 %.
  • extraction buffer I 200 mM Tris-HCI, pH 7.4, 5 mM EDTA, 5 mM DTT, 0.1 % (v/v) Tween 20
  • extraction buffer Il 150 mM NaCO 3 , pH 9.5, 500 mM NaCI, 1 % (v/v) DMSO.
  • Cells were centrifuged (500 x g, 5 min, room temperature) and the packed cells were sonicated (UW2070 with SH70G, MS-72 probe tip, 9x10%, 20%, 1 min; Bandelin, Berlin, Germany) in a 1 :2 (w/v) ratio of BY2 cells and extraction buffer I. The extracts were clarified by centrifugation prior to immunoblot analysis. Cell culture supematants were used without further treatment.
  • TSP concentrations were determined according to Bradford (1976) (Roti- Quant, Roth, Düsseldorf, Germany) using bovine serum albumin (BSA) as a standard.
  • BSA bovine serum albumin
  • TSP extracted from leaves or cells was resolved by SDS-polyacrylamide gel electrophoresis and blotted onto PVDF membrane.
  • Blotted Solulin was detected using the monoclonal anti- Solulin primary antibody 1043 (kindly provided by PAION GmbH, Aachen, Germany) diluted either 1 :1000 in PBS with 0.05% (v/v) Tween 20 (PBS-T) or 1 :1500 in PBS-T with 1% (w/v) skimmed milk powder.
  • Binding of the primary antibody was detected using a goat-anti-mouse secondary antibody conjugated to alkaline phosphatase (Jackson lmmuno Research Laboratories, West Grove, USA) diluted 1 :5000 in PBS-T, or a goat-anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson lmmuno Research Laborato ⁇ ries) diluted 1 :8000 in PBS-T with 1 % (w/v) skimmed milk powder.
  • alka ⁇ line phosphatase the signal was developed with nitroblue tetrazolium and 5- bromo-4-chloro-3-indolyl-phosphate solution (NBT/BCIP, Roth).
  • Recombinant soluble thrombomodulin was purified from suspension cell cultures by ammonium sulfate precipitation and anion exchange chromatog ⁇ raphy. The supernatant from a 300- ⁇ ml culture was retrieved by vacuum filtra ⁇ tion and was buffered with 8 mM Tris-HCI (final pH 8.5). MgSO 4 was added to a final concentration of 2 mM. To remove DNA, 500 units of Benzonase (Merck, Darmstadt, Germany) were added and the solution was incubated for 1.5 h at room temperature, followed by centrifugation (27138 x g, 20 min, 4°C).
  • a 50% ammonium sulfate precipitation was performed, followed by a centrifugation as above to collect the precipitate.
  • the pellet was redissolved in dialysis buffer (30 mM ammonium acetate, pH 6) and dialysed against the same buffer.
  • the desalted sample was processed by anion exchange chromatography using a 1-ml Sepharose Q column (Amersham Biosciences) equilibrated with dialysis buffer. The column was then washed with dialysis buffer containing 100 mM NaCI, and proteins were eluted with dialysis buffer containing 300 mM NaCI. The elution fraction was concentrated in centrifugal concentration devices (Pall Filtron, East Hills, USA) and separated by SDS-PAGE (Lammli 1970) prior to transfer onto PVDF membrane using CAPS-buffer (10 mM CAPS, pH 11 , 10% (v/v) methanol) in a semi-dry blotting apparatus (BioRad). The blot was stained with amido black, and the band correspond ⁇ ing to the recombinant thrombomodulin was cut out and used for N-terminal protein sequencing performed by TopLab (Martinsried, Germany). Thrombomodulin activity test
  • hirudin (Roche, Mannheim, Germany) was then added, bringing the total volume to 100 ml, followed by incubation at 37 0 C for 15 min. Subsequently, 100 ⁇ l of the chro- mogenic substrate S-2366 (1.4 mM; Haemochrom) was added. The reaction was incubated at room temperature for two minutes and measured for 15 min at 405 nm using a kinetic protocol. Solulin standards (0, 2, 4, 6, 10 and 20 ng) derived from CHO cells were included with each test. The standards were kindly provided by PAION GmbH.
  • Solulin yields in transformed BY2 cell cultures Recombinant Solulin levels in BY2 cell extracts and culture medium were determined by immunoblot analysis using a Solulin-specific monoclonal antibody and an alkaline phos- phatase-conjugated secondary antibody followed by NBT/BCIP staining ( a ) or a horseradish peroxidase-conjugated secondary antibody followed by chemiluminescence based detection ( b ).
  • M culture medium
  • C cell fresh weight.
  • 35SS CaMV 35S promoter with duplicated 35S enhancer region
  • CHS 5'UT Chalcone syn ⁇ thase 5' untranslated region
  • SS signal sequence
  • Solulin coding sequence for Solulin
  • T35S CaMV 35S terminator sequence.
  • Figure 2 lmmunoblot analysis of Solulin constructs transiently expressed in N. taba- cum cv. Petite Havana SR1. Total soluble proteins were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. Solulin was detected using a monoclonal mouse anti- Solulin antibody as primary antibody and a goat-anti mouse secondary antibody conjugated to alkaline phosphatase, followed by NBT/BCIP staining. Size standards are indicated to the right. 1: Solulin-ORI, 2: Solulin-APO '3 , 3: Solulin-PbTS, 4: Solulin-VTS "4 , S: 200 ng purified Solulin standard from CHO cells.
  • Solulin-PbTS SDS-PAGE analysis of partially purified Solulin-PbTS.
  • Solulin -PbTS was purified from 300 ml of BY2 suspension culture (4 d after seeding) by pre ⁇ cipitation and anion exchange chromatography. The elution fraction was concentrated 20-fold and 16 ⁇ l was separated by 10% SDS-PAGE. After electrophoresis, the gel was stained with Coomassie brilliant blue. Size stan ⁇ dards are indicated to the left. Lane 1 : 800 ng purified Solulin standard from CHO cells. Lane 2: partially purified Solulin-PbTS.
  • Nucleic acid and protein sequence of Solulin The entire coding region of the nucleic acid fragment used in the expression of Solulin is shown.
  • the deducted amino acid sequence is shown below the nucleic acid sequence using the single letter amino acid code.
  • the one letter code symbol is printed below the first nucleotide of the corresponding amino acid code.
  • the one letter symbol is printed below the first nucleotide of the corresponding codon.
  • the arrow indicates the first amino acid of the mature secreted protein.
  • the cDNA of the mature Solulin was fused to different N-terminal tar ⁇ geting sequences to enable transport of the Solulin to the apoplast ( Solulin- APO "3 , Solulin-ORI), the vacuole ( Solulin-VTS '4 ) or protein bodies ( Solulin- PbTS) ( Figure 1).
  • the plant-derived Solulin should be identical in terms of amino acid sequence to the Solulin produced in CHO cells.
  • the nucleotide sequences of Solulin joined to the three non-innate signal sequences were analysed by the SignalP server, which predicts cleavage sites using a com ⁇ bination of several artificial neural networks (Nielsen et al., 1997).
  • the analy ⁇ sis showed that the P. sativum legumin A2 signal sequence PbTS (Rerie et al., 1990) could be used without any changes.
  • PbTS P. sativum legumin A2 signal sequence
  • VTS '4 roseus strictosidine synthase
  • APO '3 Vaquero et al., 1999
  • Solulin accumulation levels were similar whether the protein was targeted to the apoplast or protein bodies, but variations were seen between different infiltrations. However, higher yields of the protein were achieved using the Maryland Mammoth cultivar compared to Petite Havana SR1 (data not shown), so the former was chosen for stable transformation experiments.
  • Solulin-APO "3 , Solulin-PbTS and Solulin-ORI constructs were introduced into tobacco leaf discs. Transgenic plants were recovered and tested for recombinant Solulin accumulation by immunoblot (Table 1). The maximum yields of Solulin differed according to which signal sequence was used with Solulin-APO "3 showing the highest accumulation (115 ⁇ g g "1 fresh weight), followed by Solulin-PbTS (38 ⁇ g g '1 fresh weight) and Solulin-ORI (27 ⁇ g g "1 fresh weight).
  • the culture medium contains 50-100 times less total soluble protein than the total cell extract (40-75 ⁇ g ml "1 ), resulting in a favourable recovery of active Solulin when expressed as percentage TSP (0.8-3.3%). This is a good start ⁇ ing point for the purification of Solulin, since the amount of contaminating proteins is low.
  • Solulin was purified from the BY2 culture medium.
  • the crucial steps prior to anion exchange chromatography were DNAse treatment and ammonium sulfate precipitation with subsequent dialysis to remove DNA and other com ⁇ pounds interfering with the chromatography process.
  • this procedure resulted in only partial purification of Solulin-APO "3 and Solulin-PbTS ( Figure 3), the yield and purity was sufficient to perform N-terminal protein sequencing.
  • the results of these experiments revealed that the five N-terminal amino acids (Glu-Pro-Gln-Pro-Gly) were identical to those of the mature protein derived from CHO cells, demonstrating that the two targeting sequences are processed correctly in plant cells. Discussion of the results
  • plant cells can produce intact and functional Solulin, with a yield of 27 ⁇ g g "1 fresh weight in BY2 cells and 115 ⁇ g g "1 in transgenic tobacco leaves (corresponding to 0.4% and 1.5% TSP, respec ⁇ tively).
  • the levels obtained in transgenic plants were in a comparable order of magnitude as those obtained in CHO cells.
  • the overall space time yield of Solulin in mammalian cell systems is much higher since produc ⁇ tion levels of up to several hundred ⁇ g per ml culture medium can be achieved and harvested daily. Nevertheless, the yield of recombinant soluble thrombomodulin in plants is high when compared to other thrombolytic or blood proteins.
  • hirudin was produced in trans ⁇ genic canola seeds (Brassica napus cv. Westar) as a fusion protein with oleosin.
  • the expression was restricted to seeds and accumulation levels of approx. 1% of total soluble seed protein were reported (Parmenter et al., 1995).
  • human protein C reached only 0.002% of TSP (Cramer et al., 1996).
  • the Solulin produced in transgenic plants and suspension cells had a molecular mass of ⁇ 75 kDa, which is the same size as the Solulin produced in CHO cells but 25 kDa larger than expected from the amino acid sequence. This deviation can be attributed to glycosylation. It is well known that N-gly- can structures slightly differ between mammalian and plant cells (Bardor et al., 1999). However, functional tests showed that thrombomodulin activity was not impaired by the presence of plant-derived N-glycans. Active Solulin was recovered from the BY2 culture supernatant but not from total BY2 cell extracts.
  • the method according to the invention does not only provide a way how to produce Solulin but is also applicable for the production of native thrombo ⁇ modulin or other structural and functional derivatives of thrombomodulin or analogs thereof. It can be used for producing derivatives of thrombomodulin and solulin which are encoded by nucleic acid sequences which have at least an identity of 80 %, preferably 90% identity to the sequences of Solulin as shown in figure 4.
  • thrombomodulin analgos suitable for the method according to 'the invention are disclosed in US patents 5,256,770, 5,466,668, 5,827,824, 5,863,760 and 6,063,763, incorporated herein by references.
  • DSPA ⁇ i is one of four plasminogen activators that have been isolated from the saliva of the common vampire bat Desmodus rotundus (Kratzschmar et al. 1991). Among these proteins, DSPA ⁇ i is the largest with a molecular weight of 50 kDa. DSPA ⁇ i is a serine protease which specifically cleaves serum plasminogen converting it into its active form, plasmin. Activated plasmin dissolves blood clots through the degradation of fibrin fibers. Its unique fibrin specificity (Schleuning et al. 1992) has led to the development of DSPA ⁇ i as a drug for the treatment of arterial thrombosis (Witt et al. 1994). Currently, recombinant DSPA ⁇ i is undergoing clinical trials for the treatment of acute ischemic stroke (Liberatore et al. 2003).
  • recombinant DSPA ⁇ i is produced in transformed Chi ⁇ nese hamster ovary (CHO) cells (Gohlke et al. 1997; Petri et al. 1995). How ⁇ ever, future applications of this recombinant protein, including the treatment of stroke patients, may require a more efficient production system to provide sufficient amounts at low costs. Therefore, we explored the use of transgenic tobacco plants and suspension cells as alternative production platforms.
  • Transgenic plants are now becoming established as hosts for the production of valuable pharmaceutical proteins (Hood et al. 2002; Ma et al. 2003; Twy- man et al. 2003).
  • Several plant species have been utilized, with maize and tobacco being the most common systems.
  • plant suspension cells have been used for protein expression under controlled conditions in biore- actors (Fischer et al. 1999; James and Lee 2001).
  • Many molecular medi ⁇ cines have been synthesized in plants, including plasma proteins, enzymes, growth factors, vaccines and recombinant antibodies.
  • recombinant proteins can be expressed in transgenic plants on an agricultural scale, pro ⁇ viding a technology that can produce huge quantities of recombinant prod ⁇ ucts for use as diagnostics and therapeutics in modern health care and the life sciences (Gruber et al. 2001 ; Ma et al. 1998).
  • the coding sequence of the mature DSPA protein was amplified from genomic DNA isolated from CHO cell line CD16.4 (provided by PAION GmbH, Aachen, Germany) using the following primers: DSPA-for (Nde ⁇ ), 5 ' GGT GTT CAC TCC GCA TAT GGT GTG GCC TGC AAA G 3 ' and DSPA- back (stop), 5 ' G CGA AGC TTC TCG AGT TTA CAG GTG CAT GTT GTC TCG AAT CC 3 ' . This generated a 1340-bp product.
  • LPH the plant codon-optimized, 19-amino-acid leader peptide from the heavy chain of murine monoclonal antibody 24 (Vaquero et al. 1999), was amplified from the source vector pTRAkc-LPH (provided by Thomas Rade- macher, RWTH Aachen).
  • the 240-bp product was fused to the 1340-bp DSPA sequence by splice overlap extension (SOE) PCR (Horton et al. 1990).
  • the fragment was inserted at EcoRI and Xho ⁇ sites in the plant expression vector pTRAkc, containing the T-DNA border sequences, the double enhanced CaMV 35S promoter, the 5 ' untranslated region (UTR) of the chalcone synthetase (CHS) gene from Petroselinum crispum, the 35S polyadenylation signal, scaffold attachment regions and the nptW gene as a selectable marker, resulting in the final construct DSPA-APO.
  • pTRAkc containing the T-DNA border sequences, the double enhanced CaMV 35S promoter, the 5 ' untranslated region (UTR) of the chalcone synthetase (CHS) gene from Petroselinum crispum, the 35S polyadenylation signal, scaffold attachment regions and the nptW gene as a selectable marker, resulting in the final construct DSPA-APO.
  • the PbTS leader peptide sequence (22 amino acids) is derived from legu- minA2 of Pisum sativum (GenBank ® accession X17193) and targets native leguminA2 to protein bodies in pea seeds (Hinz et al. 1995).
  • the PbTS cod ⁇ ing sequence was assembled by two consecutive rounds of the PCR, using pTRAkc-LPH as a template.
  • the forward primer pSS-LPH forw (5 ' ACC ACG TCT TCA AAG CAA GTG G 3') was used for both rounds of amplification.
  • the primers PbTS-back 1 (5 ' GCA GAA AGA AAG AGA AAG TGC AAG GAG TTT AGT AGC CAT TGT TTT TTT TTT TTT TAT A 3 ' ) and PbTS-back 2 (5 ' GGC CAC ACC ATA TGC GGC AAA GCA GCC TCC CAA AAG GAG AAA GCA GAA AGA AAG AGA AAG 3 ' ) were used successively.
  • the final product was cleaved with EcoRI and Nde ⁇ and ligated with the Nde ⁇ IXho ⁇ fragment derived from DSPA-APO into the EcoRI- and X/?ol-digested vector backbone of pTRAkc, resulting in the final vector DSPA-PbTS.
  • the VTS "4 leader sequence is derived from the strictosidine synthase gene of Catharanthus roseus (GenBank ® accession X61932) and comprises 28 amino acids. The C-terminal four serine residues from the native sequence were omitted since they would lead to incorrect cleavage as predicted by the CBS SignalP prediction server (http://www.cbs.dtu.dk/services/SignalP-2.0/) (Nielsen et al. 1997). The native enzyme is localized in the vacuoles of leaf tissue in C. roseus as well as in transgenic tobacco plants (McKnight et al. 1991).
  • the leader sequence was assembled through three consecutive rounds of PCR using the forward primer pSS-LPH forw in all reactions in combination with one of the backward primers: VTS-back 1 , 5 ' CAT CAT GGA CTT AGA TTC AGA GAA GTT TGC CAT TGT TTT TTT TTT TAT A 3 ' ; VTS-back 2, 5 ' AAG GAG AAG AAG GAA AAA CAT GAA GAA GAA AAC TGC CAT CAT GGA CTT AG ATT C 3 ' ; or VTS-back 3, 5 ' GGC CAC ACC ATA TGC GCT TGA AGA GCT AGA TGA TGA TGA TGA TGA TGA TGA TGA TGA AAG GAG AAG GAA AAA 3 ' .
  • the final expression construct, DSPA-VTS "4 was generated by assem ⁇ bling the amplified VTS "4 and DSPA sequence with the pTRAkc backbone in a triple ligation as described above.
  • the fourth construct contained the native DSPA ⁇ i sequence including the original N-terminal signal peptide of 21 amino acids and a prosequence of 15 amino acids.
  • the coding sequence was amplified from genomic DNA derived from CHO cell line CD16.4 in two consecutive PCRs.
  • the two forward prim ⁇ ers were designed to introduce the CHS 5 ' UTR: DSPAorM-for, 5 ' GAT TTA TAG AGA GAT TTA TAA AAA AAA AAA AAA AAA CAA TGG TGA ATA CAA TGA AGA 3 ' , and DSPAori2-for, 5 ' GGA ATT CAC AAC ACA AAT CAG ATT TAT AGA GAG ATT TAT 3 ' .
  • DSPA( ⁇ /col)-back primer were used together with the DSPA( ⁇ /col)-back primer.
  • the final product of 788 bp was digested with EcoRI and ⁇ /col and replaced with the corresponding fragment of DSPA- APO in pTRAkc, resulting in the
  • DSPA-coAPO, DSPA-coVTS '4 , DSPA-coPbTS, DSPA-coORI To insert a C-terminal His ⁇ tag, a Not ⁇ restriction site was placed at the 3' end of the DSPAaI cDNA by PCR using the primer pair DSPA-for (Nde ⁇ ) and DSPA-back ( ⁇ fofl) (5'-CGC GTA CTC GAG AGC GGC CGC CAG GTG CAT GTT GTC TCG-3'). Genomic DNA from CHO cells containing the DSPAaI cDNA was used as the template.
  • DSPA-coAPO the ⁇ /col- and ⁇ /ofl-digested PCR product, the EcoR ⁇ INot ⁇ pTRAkc-LPH vec ⁇ tor backbone and the EcoRI/ ⁇ /col fragment of the vector DSPA-APO were assembled in a triple ligation.
  • the remaining constructs DSPA-coVTS "4 , DSPA-coPbTS and DSPA-coORI were generated by cloning the corre ⁇ sponding targeting sequences plus the 5' end of DSPAaI via EcoRI and Nco ⁇ into the vector backbone of the EcoRI/ ⁇ /col-digested DSPA-coAPO construct.
  • BY-2 cells were maintained in Murashige and Skoog (1962) basal salt with minimal organics (BY-2 medium: 0.47 % (w/v) Murashige and Skoog salts, 0.15 ⁇ g/ml thiamine, 0.02 ⁇ g/ml KH 2 PO 4 and 3 % (w/v) sucrose, pH 5.8) on an orbital shaker (New Brunswick Scientific, Edison, USA) at 180 rpm and 26°C in the dark. Cells were passed into fresh medium every week with a 5 % (v/v) inoculum.
  • Total RNA was prepared from 200 mg of infiltrated leaves using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Total RNA (10 ⁇ g) was loaded onto denaturing formaldehyde agarose gels and then capillary blotted onto nylon membranes (Hybond N + , Amersham Biosciences, Freiburg, Germany) using 10 ⁇ SSC. The membrane was probed with a 770-bp Nco ⁇ /Xba ⁇ DSPA ⁇ i fragment radiolabeled with [ ⁇ 32 ]P-dATP (Amersham Biosciences) using the DecaLabel DNA labeling kit (Fermentas GmbH, St. Leon-Rot, Germany) according to the manufacturer's instructions.
  • the membrane was hybridized with Church solution (1 % (w/v) BSA, 1 mM EDTA, 7 % (w/v) SDS, 0.5 M NaH 2 PO 4 , pH 7.0) for two hours at 65°C. After prehybridization the labeled probe was added and incubated over night at 65°C. The membrane was washed twice using 2 x SSC, 0.1 % (w/v) SDS at 65 0 C and once with 0.2 x SSC, 0.1 % (w/v) SDS at 65°C for 30 minutes each. The membrane was exposed to an X-ray film for six hours using an intensifier screen.
  • BY-2 extraction buffer 50 mM Tris/HCI, 100 mM NaCI, 5 mM EDTA, 0.1 % (v/v) Tween 20, 10 mM DTT, pH 8.0
  • sonication UW2070 with SH70G, MS-72 probe tip, 9 x 10%, 20%, 1 min; Bandelin, Berlin, Germany.
  • the total soluble protein from leaf material was obtained by extraction in buffer I (200 mM Tris-HCI, 5 mM EDTA, 5 mM DTT, 0.1 % (v/v) Tween 20, pH 7.4) or buffer Il (150 mM citrate buffer, pH 4.8). Protein extracts were separated on a 12 % SDS-polyacrylamide gel using the Mini- protean system (Biorad, Hercules, CA, USA). The gels were run with a con ⁇ stant current of 20 mA per gel.
  • the secondary antibody was either an alkaline phos ⁇ phatase- or peroxidase-labeled goat anti-rabbit antibody (Dianova GmbH, Hamburg, Germany). Blots were developed with NBT/BCIP or the ECLTM Western Blotting Analysis System (Amersham Biosciences) according to the manufacturer's instructions.
  • the quantity of recombinant DSPA ⁇ i was calculated using the LAS-1000 luminescence image analyzer (Fuji, Duesseldorf, Germany) and Aida pro ⁇ gram version 2.31 (Raytest GmbH, Straubenhardt, Germany) using CHO- derived DSPA ⁇ i as a standard.
  • PVDF polyvinylidene fluo ⁇ ride
  • the activity assay was carried out according to a standard operating proce ⁇ dure developed by Berlex Biosciences (Richmond, CA, USA). The assay is based on the liberation of a chromogenic moiety (p-nitroaniline, pNA) from the small peptide S2288TM (Ile-Pro-Arg-pNA, Haemochrom Diagnostica, Essen, Germany).
  • pNA chromogenic moiety
  • a standard curve was included with the following concentrations of CHO-derived DSPA ⁇ i (kindly provided by PAION GmbH, Aachen, Germany): 20 ⁇ g/ml; 10 ⁇ g/ml; 5 ⁇ g/ml; 2.5 ⁇ g/ml; 1.25 ⁇ g/ml; 0.625 ⁇ g/ml and 0.312 ⁇ g/ml in assay buffer (50 mM Na 2 CO 3 , 0.1 % (v/v) Tween 80, pH 9.5). A 10 mM stock solution of S2288TM was diluted 1 :5 with the assay buffer immediately before use.
  • Plant-derived and purified recombinant DSPA ⁇ i containing a C-terminal His 6 -tag was desalted by gel filtration using a PD 10 column (Amersham Biosciences). The samples were diluted in assay buffer to a final volume of 100 ⁇ l and the reaction was started by the addition of 100 ⁇ l of the substrate solution in 96- well microtiter plates (Greiner Bio-One, Frickenhausen, Germany). The reac ⁇ tions were incubated for two minutes at room temperature and DSPA ⁇ i activity was measured at OD 4 os for 5 min at 9-s intervals.
  • gelatin As a substrate for proteolysis, gelatin (Merck, Darmstadt, Germany) was co- polymerized with a 12.5% SDS polyacrylamide gel at a final concentration of 0.1 % (w/v). Gels were run at 20 mA and, after electrophoresis, washed once with 2.5 % (v/v) Triton ® X-100 for 20 min at room temperature and twice in protease buffer (5O mM Tris, 5 mM CaCI 2 , 100 ⁇ M ZnCI 2 , pH 7.6) for 15 minutes each. The gels were incubated overnight in protease buffer at room temperature with gentle agitation. Subsequently, gels were stained with Coomassie Brillant Blue followed by destaining to reveal areas of proteolytic gelatin degradation.
  • Results Figure 5 lmmunoblot analysis of recombinant DSPA ⁇ i transiently expressed in N. tabacum cv Petite Havana SR1.
  • M pre-stained broad range marker
  • Figure 6 lmmunoblot and northern blot analysis of transiently transformed tobacco leaves using different tobacco cultivars.
  • TSP total soluble proteins
  • RNA (10 ⁇ g per lane) was extracted from leaves vacuum infiltrated with DSPA-coPbTS (1 , N. tabacum cv Petite Havana SR1 ; 2, N. tabacum cv Maryland Mammoth; 3, N. tabacum cv NFT 51) or from a non-infiltrated leaves (4, N. tabacum cv Petite Havana SR1 ; 5, N. tabacum cv Maryland Mammoth; 6, N. tabacum cv NFT 51).
  • DSPA ⁇ i Determination of recombinant DSPA ⁇ i enzyme activity.
  • Standard DSPA ⁇ i purified from CHO cells was used in the following con ⁇ centrations as references: 20 ⁇ g/ml; 10 ⁇ g/ml; 5 ⁇ g/ml; 2.5 ⁇ g/ml; 1.25 ⁇ g/ml; 0.675 ⁇ g/ml and 0.312 ⁇ g/ml.
  • the plant-derived and purified DSPA-coAPO sample was diluted 1 :5 and 1 :10 in a final volume of 100 ⁇ l.
  • Figure 8 lmmunoblot analysis of recombinant DSPA ⁇ i in BY-2 cells and cell culture medium.
  • M Pre-stained broad range marker (NEB); 1 , DSPA-APO extracted from BY- 2 cells; 2, DSPA-APO from cell culture medium; arrow indicates the full-size DSPA ⁇ i product.
  • NEB Pre-stained broad range marker
  • BY-2 culture medium 15 ⁇ l of four-day-old cultures; wild type, DSPA-coAPO, DSPA-PbTS or DSPA-VTS "4 ) was separated on a 10% SDS polyacrylamide gel (without 2-mercaptoethanol) containing 0.1% (w/v) gelatin. After electro ⁇ phoresis, the gel was washed twice in 2.5% (v/v) Triton ® X-100 and twice in protease buffer (50 mM Tris, 5 mM CaCI 2 , 100 ⁇ M ZnCI 2 , pH 7.6). Protease digestion was carried out for 17 h in protease buffer before staining the gel with Coomassie Brilliant Blue. Clear bands on the dark background indicate protease activity.
  • DSPA ⁇ i N-terminal sequencing of plant-derived recombinant DSPA ⁇ i .
  • Recombinant DSPA ⁇ i proteins were purified via IMAC from transiently transformed tobacco leaves. The purified proteins were separated by SDS- PAGE and blotted onto PVDF membranes. The highest molecular weight band was cut out and used for Edman sequencing.
  • FIG. 7 shows as an example the enzymatic activity of DSPA- coAPO. According to this activity, we calculated an accumulation level of 5.5 ⁇ g DSPA-coAPO per gram of transiently transformed tobacco leaf tissue. The levels for the remaining proteins - DSPA-coVTS '4 , DSPA-coPbTS and DSPA-coORI - were 3.1 ⁇ g, 5.6 ⁇ g and 0.4 ⁇ g per gram of leaf material, respectively. This agreed with the levels determined by immunoblot (data not shown) indicating that the purified proteins are predominantly functional. The low value for DSPAcoORI is at least partially due to a less efficient Ni-NTA purification. However, in general, the accumulation of DSPAcoORI and DSPA-ORI in plant tissue were low. Therefore, both constructs were excluded from further characterization.
  • DSPA-coAPO transiently expressed and purified His 6 -tagged proteins
  • DSPA-coVTS '4 and DSPA-coPbTS were analyzed by N-terminal protein sequencing.
  • the plant-derived DSPA-coAPO were present in two different forms (Table 4). One form started with an alanine residue as expected, but the second started with a valine, missing the first three amino acids of the mature DSPA ⁇ i protein. The ratio of the two forms was 0.7: 1.
  • DSPA-coVTS "4 was processed uniformly but the initial alanine residue was missing. The mature recombinant protein starts with a tyrosine residue. Sequencing of DSPA- coPbTS failed, probably due to a blocked N-terminus.
  • DSPA ⁇ i expression in tobacco BY-2 cells is the facilitated purification of the recombinant protein when targeted for secretion into the culture medium (Sijmons et al. 1990).
  • DSPA-APO, DSPA-VTS "4 and DSPA- PbTS constructs were used for stable transformation of tobacco BY-2 cells, and approximately 35 kanamycin resistant calli were tested for DSPA ⁇ i accumulation by immunoblotting.
  • Transgenic BY-2 calli showing the highest level of recombinant protein accumulation were selected and used to estab ⁇ lish cell cultures. These were tested for recombinant DSPA ⁇ i accumulation by activity assays and immunoblotting.
  • the lack of intact recombinant DSPA ⁇ i in the BY-2 culture medium might be due to rapid degradation of the full-size molecule by extracellular plant pro ⁇ teases or the retention of the full-size molecule by the plant cell wall.
  • Spiking experiments using DSPA ⁇ i from CHO cells revealed that instability was not caused by components in the Murashige and Skoog plant cell culture medium.
  • the DSPA ⁇ i standard was stable in fresh culture medium for at least 20 hours without any degradation (data not shown).
  • the DSPA ⁇ i standard was rapidly degraded in medium taken from a wild type BY-2 cell suspension culture ( Figure 9, lanes 1 and 4).
  • DSPA ⁇ i The rapid proteolysis of DSPA ⁇ i could be reduced significantly by the use of a protease inhibitor blend for the simultaneous inhibition of proteases from different classes.
  • a 10 x concentrated CompleteTM proteinase inhibitor mix (Roche) was the most effective in reducing the degradation of DSPA ⁇ i ( Figure 9, lanes 2 and 5; Figure 10). It was therefore assumed that proteases in the cell culture super ⁇ natant were responsible for the observed degradation of DSPA ⁇ i . This pro- teolysis-inhibiting effect could also be achieved by the addition of EDTA at a concentration of 5 mM ( Figure 9, lanes 3 and 6).
  • Plant-derived DSPA ⁇ i is enzymatically active but inhibited by components present in total soluble protein extracts from intact plants. No major influence of the targeting signal on enzyme activity or accumulation levels were observed with the exception of the native signal sequence (ORI) from Des- modus rotundus wh ⁇ ch reduced yields approximately ten-fold.
  • the N-terminal ORI signal peptide consists of a 21-amino-acid signal sequence and a 15- amino-acid propeptide (Kratzschmar et al. 1991 ). It is possible that incorrect processing of the ORI signal resulted in reduced secretion efficiency and low product yield.
  • Proteolytic degradation was most severe in conditioned BY-2 cell culture medium.
  • Several groups have reported the instability of recombinant proteins secreted into the plant cell culture medium (Bateman et al. 1997; LaCount et al. 1997; Lee et al. 2002; Tsoi and Doran 2002).
  • recombinant proteins were stabilized by addition of polymers such as PVP or gelatin leading to increased levels of intact product (LaCount et al. 1997; Lee et al. 2002).
  • these polymers did not stabilize recombinant DSPA ⁇ i in the plant cell culture medium.
  • DSPA ⁇ i full-size DSPA ⁇ i was detectable after the addition of a protease inhibitor mix or 5 mM EDTA indicating that proteases present in the culture medium are responsible for DSPA ⁇ i degra ⁇ dation. Indeed we showed the presence of several active proteases in condi ⁇ tioned culture medium (see Figure 11 ). Our ability to inhibit this protease activity through the addition of EDTA indicates that metalloproteases are responsible for the degradation of DSPA ⁇ i . In contrast to the aspartate, serine, threonine and cysteine proteases, metalloproteases require a divalent metal ion cofactor for their activity (Mayne and Robinson 1996).
  • cofactors Zn 2+ but other divalent cations including cobalt or nickel have also been described as cofactors. Therefore, metalloproteases can be inhibited effectively by chelating agents such as EDTA or 1 ,10-phe- nanthroline (Belozersky et al. 1990).
  • the method according to the invention does not only provide a way how produced DSPA ⁇ 1 but is also applicable for the production of other Des- moteplase isoforms as disclosed in US 6,008,019 and US 5,830,849 incor ⁇ porated herein by references or for plasminogen activators with a structural identity to DSPA ⁇ 1 of at least 75 %, preferably 80 through 90 % (compared with the amino acid sequence as shown in figure 12.
  • TL-DNA gene 5 controls the tissue specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Molecular and General Genetics 204: 383- 396.
  • Maize (Zea mays)-derived bovine trypsin characterization of the first large-scale, commercial pro ⁇ tein product from transgenic plants. Biotechnology and Applied Bio ⁇ chemistry 38:123-30. Yang XY, Mackins JY, Li QJ, Antony AC. 1996. Isolation and characterization of a folate receptor-directed metalloprotease from human placenta. Journal of Biological Chemistry 271 (19): 11493-9.

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Abstract

La présente invention se rapporte à un procédé destiné à produire de la soluline ou de la DSPAa1, qui consiste à faire croître des cellules végétales comportant une séquence intégrée qui contient une cassette de transcription fonctionnelle renfermant un gène structurel codant ladite soluline ou desmoteplase.
PCT/EP2004/007727 2004-07-13 2004-07-13 Production de proteines mammiferes dans des cellules vegetales Ceased WO2006005362A1 (fr)

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CN103074329A (zh) * 2013-02-04 2013-05-01 首都师范大学 一种聚合酶链反应增强方法
WO2016118732A1 (fr) * 2015-01-21 2016-07-28 Northeastern State University Protéines de dissolution de caillots de sang produites dans des graines

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103074329A (zh) * 2013-02-04 2013-05-01 首都师范大学 一种聚合酶链反应增强方法
WO2016118732A1 (fr) * 2015-01-21 2016-07-28 Northeastern State University Protéines de dissolution de caillots de sang produites dans des graines
CN107427562A (zh) * 2015-01-21 2017-12-01 东北州立大学 种子中产生的血液凝块溶解蛋白

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