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WO1993018181A1 - Sequence genique synthetique pour un peptide-signal - Google Patents

Sequence genique synthetique pour un peptide-signal Download PDF

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Publication number
WO1993018181A1
WO1993018181A1 PCT/US1993/001494 US9301494W WO9318181A1 WO 1993018181 A1 WO1993018181 A1 WO 1993018181A1 US 9301494 W US9301494 W US 9301494W WO 9318181 A1 WO9318181 A1 WO 9318181A1
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WIPO (PCT)
Prior art keywords
protein
signal peptide
dna molecule
hybrid
glucuronidase
Prior art date
Application number
PCT/US1993/001494
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English (en)
Inventor
Sheng-Zhi Pang
John C. Sanford
Original Assignee
Cornell Research Foundation, Inc.
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Publication date
Application filed by Cornell Research Foundation, Inc. filed Critical Cornell Research Foundation, Inc.
Publication of WO1993018181A1 publication Critical patent/WO1993018181A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates generally to recombinantly produced proteins in plant cells and a process of transporting such proteins into the endoplasmic reticulum of plant cells, where they can subsequently be transported outside the cell or deposited in specific intracellular compartments.
  • the endoplasmic reticulum plays a number of important functions.
  • the smooth endoplasmic reticulum which lacks ribosomes, provides a site for synthesis and metabolism of fatty acids and phospholipids.
  • the rough endoplasmic reticulum is studded with ribosomes which participate in the synthesis of cytoplasmic, membrane, and organelle proteins as well as in the synthesis of proteins to be secreted from the cell.
  • RNA polymerase activates a control region on the gene which transcribes the DNA (i.e. encoded information) into messenger RNA. Transcription is ended by one or more "stop" codons on the gene.
  • the messenger RNA is then translated into the encoded protein at the ribosomes bound to the rough endoplasmic reticulum.
  • a growing polypeptide emerges from the ribosome, passes through the membrane of the rough endoplasmic reticulum, and accumulates in its lumen or central cavity.
  • Proteins destined for direct transport include a lead signal peptide section, which is what directs the protein to enter the endoplasmic reticulum, and is then cleaved from the remainder of the protein after such entrance.
  • proteins and membrane constituents are directed to their appropriate destinations. This is achieved by enzymatically modifying the proteins with a "tag" (e.g. , a carbohydrate or phosphate residue) to direct them to vesicles which transport the modified proteins outside the cell (or to intracellular compartments) .
  • a "tag” e.g. , a carbohydrate or phosphate residue
  • vicilins the major storage protein of developing legume seeds.
  • Vicilins are synthesized as prepro-vi ⁇ ilins on membrane-bound ribosomes of the rough endoplasmic reticulum.
  • prepro- vicilins pass into the lumen of the rough endoplasmic reticulum, their signal peptides are removed, as recognized by T.J.V. Higgins and D. Spencer, "Precursor forms of Pea Gene Encoding Vicilin Subunits," 67 Plant Phvsiol 205-11 (1981) , which is hereby incorporated by reference.
  • the resulting peptides may then undergo various post-translational modifications.
  • the protein in the lumen of the endoplasmic reticulum or in the Golgi complex, the protein may undergo glycosylation so that it is ready for intracellular transport, as recognized by B.S.L. Beevers, "Glycoprotein Metabolism in the Cotyledons of Pisum Sativum During Development and Germination," 57 Plant Ph ⁇ siol. 93-97 (1976) and R.A. Davey and .F. Dudman, "The Carbohydrate of Storage Glycoproteins from Seeds of Pisum Sativum: Characterization and Distribution of Component Polypeptides," 6 Aust. J. Plant Physiol.
  • Boulter "Characterization of the Storage Protein Subunits Synthesized in vitro by Polyribosomes and RNA from Developing Pea (Pisum sativum L.), I.” 148 Le umin. Planta 49- 56 (1980); R.R.D. Croy, J.A. Gatehouse, I.M. Evans, and D. Boulter, "Characterization of the Storage Protein Subunits Synthesized in vitro by Polyribosomes and RNA from Developing Pea (Pisum sativum L.), II.” 148 Vicilin Planta 57-63 (1980); J.A. Gatehouse, R.R.D. Croy, H. Morton, M. Tyler, and D.
  • heterologous genes i.e. DNA that encodes proteins not ordinarily produced by the host organism containing that vehicle.
  • a heterologous gene is first inserted into a closing vector, such as a plasmid.
  • the vector is then transfected into the host cells, which are thereafter transformed.
  • the tranformed cells then multiply to increase the population containing copies of the gene to be expressed in the host cell for production of a protein foreign to that cell.
  • the signal peptides were derived from gene fragments encoding for the pathogenesis-related protein 16(sPRI) , a protein secreted in tobacco mosaic virus-infected tobacco leaves-, and the prepro-peptide of cecropin B(sCEC), a peptide secreted into the hemolymph of the insect Hyalophora cecropia. It was found that the total level of enzyme expression was lower in secretion constructs (with the signal peptide sequence) than for unmodified genes which encoded for the enzyme alone.
  • genes encoding for a hybrid protein consisting of a signal peptide sequence fused to another protein for the purpose of secretion is thus well known in plant cells.
  • these signal peptides are not satisfactory, because they do not achieve high expression levels and, in fact, decrease the expression level of the desired proteins. Accordingly, the need exists for effective signal peptide sequences which can maintain or enhance high expression levels of desired proteins in plant cells.
  • the present invention relates to a process of transporting a desired protein to the endoplasmic reticulum of plant cells for secretion.
  • This process utilizes a DNA molecule encoding for a hybrid protein which includes a first protein containing a signal peptide, as described below, which was derived from the putative published sequence for the vicilin signal peptide, or its homologs, or any fragments thereof fused to a second protein which confers an extracellular protective or other desired effect in plant cells.
  • the second protein may be a protein or fragment thereof which imparts bacterial, fungal, or freeze resistance outside the cell walls of plant cells transformed with an expression system in which that DNA molecule is cloned.
  • the DNA molecule encoding the hybrid protein of the present invention may be cloned into an expression vector (e.g., a plasmid) to form a recombinant DNA expression system. This system is then transfected into plant cells. The plant cells of the present invention are then transformed and grown in a suitable medium to increase the number of such transformed cells. The transformed cells thus express the DNA encoding the hybrid protein of the present invention.
  • the hybrid protein includes the signal peptide described below, its homologs, or any fragment thereof which directs transport of the second protein into the endoplasmic reticulum. The signal peptide is cleaved off upon entry into the rough endoplasmic reticulum.
  • the second protein which is the protein of interest, is further modified in the endoplasmic reticulum lumen or in the Golgi complex and then secreted outside the plant cell or otherwise transported within the cell. Any plant cell transformed with this recombinant DNA expression system may then secrete the desired protein.
  • This system can be used to secrete proteins which will condition resistance to bacteria, fungi, or frost, or will affect plant cell walls or function as markers or tags or color factors.
  • Figure IB shows the 5' end of the hybrid gene encoding for the signal peptide and j8-glucuronidase, and the corresponding amino acids.
  • Figure 2A is a graph showing the 0-glucuronidase activity for transformed leaf cells of transgenic tobacco plants.
  • Figure 2B is a graph showing the ⁇ -glucuronidase activity for transformed root cells of transgenic tobacco plants.
  • Figure 3A shows a chart of the distribution of transient
  • Figure 3B shows a chart of the distribution of transient j3-glucuronidase activity in transformed tobacco protoplasts in the presence of tunicamycin.
  • Figure 4 is a chart showing the total transient ⁇ - glucuronidase activity (pooled from all fractions) in transformed protoplasts in the presence of tunicamycin.
  • the present invention relates to a process of transporting proteins to the endoplasmic reticulum of plant cells for secretion or other targeting within the cell.
  • This process utilizes a DNA molecule encoding a hybrid protein.
  • the hybrid protein includes a first part of the protein which contains a signal peptide derived from a putative sequence for the vicilin protein, its homologs, or any fragments thereof.
  • This first protein is fused to a second protein which confers an extracellular protective effect in plant calls or any other desired effect such as for marking, color, cell wall modification, etc.
  • the signal peptide, its homologs, or fragments thereof is present to enable the hybrid protein to enter the cell's transport pathway via the endoplasmic reticulum.
  • the signal peptide Upon entry into the lumen, the signal peptide is cleaved from the second protein in the same fashion as the vicilin signal peptide would be separated from the vicilin protein to which it is fused in nature.
  • the second protein can then be processed in the Golgi complex or in the endoplasmic reticulum lumen for extracellular secretion or transport within the cell.
  • the DNA molecule of the present invention encodes for an amino acid signal sequence substantially corresponding to the sequence:
  • the second protein is a protein or fragment thereof which imparts a resistance to bacteria, fungi, or freezing or any other desired effect.
  • the synthetic sequence derived from the Pisum vicilin signal peptide is effective in producing a hybrid protein which includes and carries a second protein into the cell transport path. As a result the resistance provided by the second protein is imparted to plants extracellularly (i.e. outside the cell wall, particularly to extracellular surfaces of the plant cell walls) .
  • proteins which may impart extracellular bacterial resistance to plants include lytic proteins. See, e.g., L. Destefano-Beltran, "Enhancing Bacterial and Fungal Disease Resistance in Plants: Application to Potato," Molecular Biology of the Potato, p. 205-21 (1990) , which is hereby incorporated by reference.
  • proteins which may impart extracellular fungal resistance to plants include chitinases and glucanases. See, e.g., C. Castresana, "Tissue-Specific and Pathogen-Induced Regulation of a Nicotiana plumbaginifolia ⁇ - 1,3-Glucanase Gene," 2 The Plant Cell. 1131-43 (1990); J. Neuhaus, "High-level Expression of a Tobacco Chitanase Gene in Nicotiana sylvestris . Susceptibility of Transgenic Plants to Cercospora Nicotianae Infection," 16 Plant Molec. Biol. 141-51 (1991) ; and L. Destefano-Beltran, "Enhancing Bacterial and Fungal Disease Resistance in Plants: Application to Potato,” Molecular Biology of the Potato, p. 205-21 (1990), all of which are hereby incorporated by reference.
  • proteins which may impart extracellular antifreezing capability in plants include numerous fish antifreeze peptides. See, e.g., P.L. Davies, "Antifreeze Protein Genes of the Winter Flounder," 259 J. Biol. Chem. 9241-47 (1984) , which is hereby incorporated by reference.
  • the second protein may include polygalacturonase, /3-glucuronidase, other marker proteins, and their homologs, fragments, or portions thereof.
  • jS-glucuronidase primarily serves as an example.
  • the gene encoding for jS-glucuronidase is a widely used marker gene having a well known sequence and known to be useful in forming N-terminal fusions. See, e.g., R.A. Jefferson, "Assaying Chimeric Genes in Plants: The GUS Gene Fusion System," 5 Plant Molecular Biology Reporter. 387-405 (1987) and R.A. Jefferson, "j ⁇ -Glucuronidase from Escherichia coli as a Gene-fusion Marker," 83 Proc.
  • the polypeptide formed from the fusion of the first protein to the second protein is a hybrid in that the first and second proteins are not normally joined in nature. Likewise, the corresponding portions of the DNA molecule encoding each of these proteins makes the DNA molecule a hybrid.
  • the portion of the DNA molecule encoding the first protein is fused in proper orientation and correct reading frame to that part of the DNA molecule encoding the second protein.
  • a linker sequence connects these portions of the DNA molecule.
  • the expression vector into which this fusion DNA molecule is inserted includes a promoter sequence which functions to regulate polypeptide expression.
  • a wide variety of promoters may be utilized, such as the 35S promoter of cauliflower mosaic virus.
  • the DNA molecule encoding for the hybrid protein of the present invention is inserted into an expression system to which the DNA molecule is heterologous (i.e., not normally present) .
  • the heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame.
  • plasmids include pBI426 and pMON316, preferably pMON316.
  • the expression system of the present invention can be used to transform virtually any crop plant cell under suitable conditions. Such cells are transformed with the DNA molecule of the present invention by conventional procedures. Cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to produce the desired second protein. This protein can then be harvested or recovered by conventional purification techniques. Alternatively, transformed cells can be regenerated into whole plants such that secreted proteins impart desired characteristics to the intact plants.
  • DNA molecule of the present invention Regardless of whether the DNA molecule of the present invention is expressed in intact plants or in culture, expression of the desired protein follows essentially the same basic mechanism. Specifically, transcription of the DNA molecule is initiated by the binding of RNA polymerase to the DNA molecule's promoter. During transcription, movement of the RNA polymerase along the DNA molecule forms messenger RNA. As a result, the DNA molecule that encodes for the hybrid protein of the present invention is transcribed into the corresponding messenger RNA. This messenger RNA then moves to the ribosomes of the rough endoplasmic reticulum which, with transfer RNA, translates the messenger RNA into the hybrid protein of the present invention.
  • the resulting hybrid protein passes into the lumen of the rough endoplasmic reticulum.
  • the signal sequence i.e., the first protein, its homolog, or a fragment thereof
  • the second protein having the ability to confer desired effects will continue to advance into the lumen in an unfused state.
  • This protein is then modified by glycosylation in the lumen or in Golgi complex.
  • the modified protein is then secreted by the Golgi complex to a location outside the host cell or is transported within the cell to a specific site.
  • Complementary oligodeoxyribonucleotides of each segment were then annealed by combining 0.2 nmole of each, and incubating the mixture at 90°C for 10 minutes, at 65°C for 2 hours, and at room temperature (i.e. 32°C) overnight.
  • the resulting DNA fragment was then purified from a polyacrylamide gel and used for construction of plant j8-glucuronidase-secretion vector through a multiple-step procedure.
  • the gene encoding jS-glucuronidase was excised from the plas id pBI101.3 (Clontech Laboratories, Inc.) with Hindlll+SstI , and ligated into the vector p ⁇ Cll ⁇ . See, J. Vieira and J. Messing, "Production of Single-stranded Plasmid DNA," 153 Methods Enzymol 3-11 (1987) , which is hereby incorporated by , reference, that had been cut with Hindlll+SstI .
  • a Kpnl linker ( ⁇ '-GGGTACCC) and a Hindlll linker (5'CCAAGCTTGG) were inserted into the unique Hindlll and Smal sites upstream of the gene encoding ⁇ -glucuronidase in the resulting p ⁇ C118- ⁇ -glucuronidase in the resulting p ⁇ C118- ⁇ -glucuronidase plasmid.
  • the purified signal peptide fragment was then fused in-frame to the N-terminus of the gene encoding ⁇ -glucuronidase to form pUC118- ⁇ -glucuronidasel, by replacing the Kpnl-Hindlll multi-cloning fragment of the modified p ⁇ cll8- ⁇ -glucuronidase with the assembled signal peptide-encoding fragment.
  • the fusion region of the hybrid gene in pUC118- ⁇ -glucuronidasel ( Figure 1) was sequenced by the dideoxy chain-termination method of F. Sanger, S. Nicklen, and A.R. Coulson, "DNA Sequencing with Chain-terminating Inhibitors," 74 Proc. Natl. Acad. Sci. USA 5463-67 (1977), the disclosure of which is hereby incorporated by reference, using the universal M13 primer.
  • the KpnI-EcoRI fragment coding for the signal peptide//3-glucuronidase fusion was isolated from pUcll8- ⁇ -glucuronidasel and cloned into the plant expression vector pMON316. See, S.G. Rogers, H.J.
  • Plasmid pM0N316- ⁇ -glucuronidasel-2 was similarly constructed, according to the above-described procedures in an independent cloning experiment.
  • the 3-glucuronidase-encoding sequence was closed into pMON316 in both orientations to generate positive (pMON316- ⁇ -glucuronidase (+) ) and negative (p ⁇ fON31t5-3-grIu ⁇ uronidase (-) ) controls for expression of the gene encoding for
  • the fusion gene was then placed under the 35S promoter of cauliflower mosaic virus in a plant expression vector, and introduced into tobacco protoplasts for transient expression, and into tobacco leaf discs/NTl cells for stable transformation, using conventional recombinant technology procedures.
  • Leaf discs from in vitro grown plantlets were transformed using an improved biolistic device of G.N. Ye, M. Daniell, and J.C. Sanford, "Optimization of Delivery of Foreign Gene into Higher Plant Chloroplasts," 15 Plant Mol. Biol. 809-19 (1990) , which is hereby incorporated by reference, and transformants were selected on kanamycin medium following two days of incubation on non-selective medium.
  • Transgenic plants from leaf discs were regenerated from shoots after transfer to a hormone-free tobacco medium. 20 ⁇ g tunicamycin per ml was added to the culture medium to inhibit glycosylation of the fusion -glucuronidase.
  • Leaf or root extracts were prepared by grinding in the extraction buffer (50 mM Tris-HCl, pH 7.5/0.15 mg phenylmethylsulfonyl fluoride/0.3 mg dithiothreitol/0.3 mg bovine serum albumin, all per ml) and removing the cell debris by centrifugation.
  • the /3-glucuronidase fluorometric assay described by J.H. Gould and R.M. Smith "A Non-destructive Assay for 0-glucuronidase in the Media of Plant Tissue Cultures," 7 Plant Mol. Biol. Rep.
  • GUS(-) and GUS(+) represent the plants transformed with pMON316- ⁇ -glucuronidase (-) and pMON316- ⁇ -glucuronidase (+) , respectively, while GUSl-1 and GUS1-2 represent plants transformed with the similar vicilin(s) signal peptide/3-glucuronidase-encoding constructs pMON316- ⁇ -glucuronidasel-l and pMON316- ⁇ -glucuronidase 1-2.
  • pMON316- ⁇ -glucuronidase (-) Since the fusion region of the hybrid gene encoding for jS-glucuronidase was sequenced, and confirmed to be correct, the absence of 8-glucuronidase activity in the plants containing the fusion gene must be attributed to the interference of gene expression by the addition of the signal peptide sequence. Alternatively, the gene fusion or post-translational modification of the fusion protein in the transport pathway could cause loss of /3-glucuronidase activity.
  • Protoplasts were prepared from leaves derived from seedlings of N. tabacum var Havana cv 423 grown in vitro at low light intensity, generally as described by J.I. Negy and P. Maliga, "Callus Induction and Plant Regeneration from Mesophyll Protoplasts of Nicotiana Sylvestris.” 78 Z. convincedvsiol 453-55 (1976) , which disclosure is hereby incorporated by reference, with some modifications. Briefly, the sliced leaves were digested in an isolation medium containing 1.25% Cellulase RIO and 0.4% Macerozyme RIO (Karlan Chemical Corporation, Torrance, CA) at 26°C overnight.
  • the protoplasts at the interface were transferred to new tubes and washed with W5 solution.
  • the isolated protoplasts were transformed with the plasmid constructs described above using the polyethylene glycol method of Negrutiu.
  • the transformed protoplasts were then cultured at the final density of lxlO 6 protoplasts/ml in the culture medium (containing 20 ⁇ g tunicamycin/ml where indicated) at 26°C in the dark.
  • the transformed tobacco protoplasts were fractionated into extracellular, cytoplasmic and membrane-associated fractions about 50 hours after the polyethylene glycol-mediated transformation.
  • the extracellular fraction (the culture medium) was collected as supernatant from the protoplast cultures following centrifugation at lOOxg for 5 minutes.
  • the pelleted protoplasts were washed once in W5 solution and lysed by osmotic shock in an appropriate volume of extraction buffer
  • the transport system appears to become saturated or partially blocked, resulting in accumulation of 3-glucuronidase in the cytoplasm.
  • the protoplasts transiently expressing the hybrid gene produced 10-30 fold more j8-glucuronidase protein (Fig. 4) than the transgenic plants containing the same construct (Fig. 2) .

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Abstract

L'invention se rapporte à un procédé de transport d'une protéine désirée dans le réticulum endoplasmique de cellules végétales à l'intérieur ou à l'extérieur de la cellule. Dans ce procédé, on utilise une molécule d'ADN codant une protéine hybride. Ladite protéine hybride codée se compose d'une première protéine comprenant un peptide-signal dérivé d'un présumé peptide comprenant un peptide-signal de vicilline, de son homologue, ou d'un morceau de celui-ci, et d'une seconde protéine qui produit un effet protecteur extracellulaire ou un autre effet désiré dans les cellules végétales. Cette molécule d'ADN peut être insérée dans un système d'expression qui peut ensuite être utilisé pour transformer les cellules qui dirigeront la seconde protéine désirée dans son système de transport.
PCT/US1993/001494 1992-03-13 1993-02-19 Sequence genique synthetique pour un peptide-signal WO1993018181A1 (fr)

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US07/851,429 1992-03-13

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006501818A (ja) * 2002-07-23 2006-01-19 デルタ バイオテクノロジー リミテッド 遺伝子およびポリペプチド配列
WO2019241315A1 (fr) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Constructions régulatrices dérivées de pde5 et procédés d'utilisation en immunothérapie
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
WO2020185632A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procédés de régulation accordable
WO2020252405A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation ajustable
WO2020252404A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation accordable
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr

Citations (1)

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US4940840A (en) * 1984-03-26 1990-07-10 Dna Plant Technology Corporation Novel chitinase-producing bacteria and plants

Patent Citations (1)

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US4940840A (en) * 1984-03-26 1990-07-10 Dna Plant Technology Corporation Novel chitinase-producing bacteria and plants

Non-Patent Citations (5)

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Title
GENE, Volume 99, No. 1, issued 01 March 1991, M. DE LOOSE et al., "The Extensin Signal Peptide Allows Secretion of a Heterologous Protein form Protoplasts", pages 95-100. *
NUCLEIC ACIDS RESEARCH, Volume 11, No. 8, issued 1983, "The Vicilin Gene Family of Pea (Pisum Sativum L.): A Complete cDNA Coding Sequence for Preprovicilin", pages 2367-2380. *
PLANT MOLECULAR BIOLOGY, Volume 11, issued 1988, T.J.V. HIGGINS et al., "The Sequence of a Pea Vicilin Gene and its Expression in Transgenic Tobacco Plants", pages 683-695. *
PLANT PHYSIOLOGY, Volume 91, issued 1989, P. LUND et al., "Bacterial Chitanse is Modified and Secreting in Transgenic Tobacco", pages 130-135. *
THE PLANT CELL, Volume 1, issued March 1989, G. ITURRIAGA et al., "Endoplasmic Reticulum Targeting and Glycosylation of Hybrid Proteins in Transgenic Tobacco", pages 381-390. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006501818A (ja) * 2002-07-23 2006-01-19 デルタ バイオテクノロジー リミテッド 遺伝子およびポリペプチド配列
EP2390333A1 (fr) * 2002-07-23 2011-11-30 Novozymes Biopharma DK A/S Polypeptides avec une pre-séquence de sécrétion de la sérum albumine humaine modifiée avec rendement de sécrétion amelioré
US8258102B2 (en) 2002-07-23 2012-09-04 Novozymes Biopharma Dk A/S Gene and polypeptide sequences
US9133265B2 (en) 2002-07-23 2015-09-15 Novozymes Biopharma Dk A/S Gene and polypeptide sequences
WO2019241315A1 (fr) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Constructions régulatrices dérivées de pde5 et procédés d'utilisation en immunothérapie
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
WO2020185632A1 (fr) 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procédés de régulation accordable
WO2020252405A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation ajustable
WO2020252404A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation accordable
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr

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