The present invention relates to the production of proteins such as thioredoxin and thioredoxin reductase on oil bodies.
Many very diverse methods have been tested for the production of recombinant molecules of interest and commercial value. Different organisms that have been considered as hosts for foreign protein expression include single celled organisms such as bacteria and yeasts, cells and cell cultures of animals, fungi and plants and whole organisms such as plants, insects and transgenic animals.
The use of fermentation techniques for large-scale production of bacteria, yeasts and higher organism cell cultures is well established. The capital costs associated with establishment of the facility and the costs of maintenance are negative economic factors. Although the expression levels of proteins that can be achieved are high, energy inputs and protein purification costs can greatly increase the cost of recombinant protein production.
The production of a variety of proteins of therapeutic interest has been described in transgenic animals, however the cost of establishing substantial manufacturing is prohibitive for all but high value proteins. Numerous foreign proteins have been expressed in whole plants and selected plant organs. Methods of stably inserting recombinant DNA into plants have become routine and the number of species that are now accessible to these methods has increased greatly.
Plants represent a highly effective and economical means to produce recombinant proteins as they can be grown on a large scale with modest cost inputs and most commercially important species can now be transformed. Although the expression of foreign proteins has been clearly demonstrated, the development of systems with commercially viable levels of expression coupled with cost effective separation techniques has been limited.
The production of recombinant proteins and peptides in plants has been investigated using a variety of approaches including transcriptional fusions using a strong constitutive plant promoter (e.g., from cauliflower mosaic virus (Sijmons et al., 1990, Bio/Technology, 8:217-221); transcriptional fusions with organ specific promoter sequences (Radke etal., 1988, Theoret. Appl. Genet., 75:685-694); and translational fusions which require subsequent cleavage of a recombinant protein (Vanderkerckove et al., 1989, Bio/Technology, 7:929-932).
Foreign proteins that have been successfully expressed in plant cells include proteins from bacteria (Fraley et al., 1983, Proc. Natl. Acad. Sci. USA, 80:4803-4807), animals (Misra and Gedamu, 1989, Theor. Appl. Genet., 78:161-168), fungi and other plant species (Fraley et al., 1983, Proc. Natl. Acad. Sci. USA, 80:4803-4807). Some proteins, predominantly markers of DNA integration, have been expressed in specific cells and tissues including seeds (Sen Gupta-Gopalan et al., 1985, Proc. Natl. Acad. Sci. USA, 82:3320-3324); Radke et al., 1988, Theor. Appl. Genet., 75:685-694). Seed specific research has been focused on the use of seed-storage protein promoters as a means of deriving seed-specific expression. Using such a system, Vanderkerckove et al., (1989, Bio/Technol., 7:929-932) expressed the peptide leu-enkephalin in seeds of Arabidopsis thaliana and Brassica napus. The level of expression of this peptide was quite low and it appeared that expression of this peptide was limited to endosperm tissue.
It has been generally shown that the construction of chimeric genes which contain the promoter from a given regulated gene and a coding sequence of a reporter protein not normally associated with that promoter gives rise to regulated expression of the reporter. The use of promoters from seed-specific genes for the expression of recombinant sequences in seed that are not normally expressed in a seed-specific manner have been described.
Sengupta-Gopalan et al., (1985, Proc. Natl. Acad. Sci. USA, 82:3320-3324) reported expression of a major storage protein of french bean, called xcex2-phaseolin, in tobacco plants. The gene expressed correctly in the seeds and only at very low levels elsewhere in the plant. However, the constructs used by Sengupta-Gopalan were not chimeric. The entire xcex2-phaseolin gene including the native 5xe2x80x2-flanking sequences were used. Subsequent experiments with other species (Radke et al., 1988, Theor. App. Genet. 75:685-694) or other genes (Perez-Grau, L., Goldberg, R. B., 1989, Plant Cell, 1:1095-1109) showed the fidelity of expression in a seed-specific manner in both Arabidopsis and Brassica. Radke et al., (1988, vide supra), used a xe2x80x9ctaggedxe2x80x9d gene i.e., one containing the entire napin gene plus a non-translated xe2x80x9ctagxe2x80x9d.
The role of the storage proteins is to serve as a reserve of nitrogen during seed germination and growth. Although storage protein genes can be expressed at high levels, they represent a class of protein whose complete three-dimensional structure appears important for proper packaging and storage. The storage proteins generally assemble into multimeric units which are arranged in specific bodies in endosperm tissue. Perturbation of the structure by the addition of foreign peptide sequences leads to storage proteins unable to be packaged properly in the seed.
In addition to nitrogen, the seed also stores lipids. The storage of lipids occurs in oil or lipid bodies. Analysis of the contents of lipid bodies has demonstrated that in addition to triglyceride and membrane lipids, there are also several polypeptides/proteins associated with the surface or lumen of the oil body (Bowman-Vance and Huang, 1987, J. Biol. Chem., 262:11275-11279, Murphy et al., 1989, Biochem. J., 258:285-293, Taylor et al., 1990, Planta, 181:18-26). Oil-body proteins have been identified in a wide range of taxonomically diverse species (Moreau et al., 1980, Plant Physiol., 65:1176-1180; Qu et al., 1986, Biochem. J., 235:57-65) and have been shown to be uniquely localized in oil-bodies and not found in organelles of vegetative tissues. In Brassica napus (rapeseed, canola) there are at least three polypeptides associated with the oil-bodies of developing seeds (Taylor et al., 1990, Planta, 181:18-26).
The oil bodies that are produced in seeds are of a similar size (Huang A. H. C., 1985, in Modern Meths. Plant Analysis, Vol. 1:145-151 Springer-Verlag, Berlin). Electron microscopic observations have shown that the oil-bodies are surrounded by a membrane and are not freely suspended in the cytoplasm. These oil-bodies have been variously named by electron microscopists as oleosomes, lipid bodies and spherosomes (Gurr MI., 1980, in The Biochemistry of Plants, 4:205-248, Acad. Press, Orlando, Fla.). The oil-bodies of the species that have been studied are encapsulated by an unusual xe2x80x9chalf-unitxe2x80x9d membrane comprising, not a classical lipid bilayer, but rather a single amphophilic layer with hydrophobic groups on the inside and hydrophillic groups on the outside (Huang A. H. C., 1985, in Modern Meths. Plant Analysis, Vol. 1:145-151 Springer-Verlag, Berlin).
The numbers and sizes of oil-body associated proteins may vary from species to species. In corn, for example, there are two immunologically distinct polypeptide classes found in oil-bodies (Bowman-Vance and Huang, 1988, J. Biol. Chem., 263:1476-1481). Oleosins have been shown to comprise alternate hydrophillic and hydrophobic regions (Bowman-Vance and Huang, 1987, J. Biol. Chem., 262:11275-11279). The amino acid sequences of oleosins from corn, rapeseed, and carrot have been obtained. See Qu and Huang, 1990, J. Biol. Chem., 265:2238-2243, Hatzopoulos et al., 1990, Plant Cell, 2:457-467, respectively. In an oilseed such as rapeseed, oleosin may comprise between 8% (Taylor et al., 1990, Planta, 181:18-26) and 20% (Murphy et al., 1989, Biochem. J., 258:285-293) of total seed protein. Such a level is comparable to that found for many seed storage proteins.
Genomic clones encoding oil-body proteins with their associated upstream regions have been reported for several species, including maize (Zea mays, Bowman-Vance and Huang, 1987, J. Biol. Chem., 262:11275-11279; and Qu Huang, 1990, J. Biol. Chem., 265:2238-2243) and carrot (Hatzopoulos et al., 1990, Plant Cell, 2:457-467). cDNAs and genomic clones have also been reported for cultivated oilseeds, Brassica napus (Murphy, et al., 1991, Biochem. Biophys. Acta, 1088:86-94; and Lee and Huang, 1991, Plant Physiol 96:1395-1397), sunflower (Cummins and Murphy, 1992, Plant Molec. Biol. 19:873-878) soybean (Kalinski et al., 1991, Plant Molec. Biol. 17: 1095-1098), and cotton (Hughes et al., 1993, Plant Physiol 101:697-698). Reports on the expression of these oil-body protein genes in developing seeds have varied. In the case of Zea mays, transcription of genes encoding oil-body protein isoforms began quite early in seed development and were easily detected 18 days after pollination. In non-endospermic seeds such as the dicotyledonous plant Brassica napus (canola, rapeseed), expression of oil-body protein genes seems to occur later in seed development (Murphy, et al., 1989, Biochem. J., 258:285-293) compared to corn.
A maize oleosin has been expressed in seed oil bodies in Brassica napus transformed with a Zea mays oleosin gene. The gene was expressed under the control of regulatory elements from a Brassica gene encoding napin, a major seed storage protein. The temporal regulation and tissue specificity of expression was reported to be correct for a napin gene promoter/terminator (Lee et al., 1991, Proc. Natl. Acad. Sci. USA, 88:6181-6185).
Thus the above demonstrates that oil body proteins (or oleosins) from various plant sources share a number of similarities in both structure and expression. However, at the time of the above references it was generally believed that modifications to oleosins or oil body proteins would likely lead to abherant targeting and instability of the protein product. (Vande Kerckhove et al., 1989. Bio/Technology, 7:929-932; Radke et al., 1988. Theor. and Applied Genetics, 75:685-694; and Hoffman et al., 1988. Plant Mol. Biol. 11:717-729).
Of particular relevance to the present invention are the redox proteins thioredoxin and its reductant thioredoxin reductase. Thioredoxins are relatively small proteins (typically approximately 12 kDa) that belong to the family of thioltransferases which catalyze oxido-reductions via the formation or hydrolysis of disulfide bonds and are widely, if not universally, distributed throughout the animal, plant and bacterial kingdom. In order to reduce the oxidized thioredoxin two cellular reductants provide the reduction equivalents, reduced ferredoxin and NADPH. These reduction equivalents are supplied via different thioredoxin reductases including the NADPH thioredoxin reductase and ferredoxin thioredoxin reductase. The latter thioredoxin reductase is involved in the reduction of plant thioredoxins designated as TRx and TRm, both of which are involved in the regulation of photosynthetic processes in the chloroplast. The NADPH/thioredoxin active in plant seeds is designated TRh and is capable of the reduction of a wide range of proteins thereby functioning as an important cellular redox buffer.
Thioredoxins have been obtained from several organisms including Arabidopsis thaliana (Riveira Madrid et al. (1995) Proc. Natl. Acad. Sci. 92: 5620-5624), wheat (Gautier et al. (1998) Eur. J. Biochem. 252: 314-324); Escherichia coli (Hoeoeg et al (1984) Biosci. Rep. 4: 917-923) and thermophylic microorganisms such as Methanococcus jannaschii and Archaeoglobus fulgidus (PCT Patent Application 00/36126). Thioredoxins have also been recombinantly expressed in several host systems including bacteria (Gautier et al. (1998) Eur J. Biochem. 252: 314-324) and plants (PCT Patent Application WO 00/58453) Commercial preparations of E. coli sourced thioredoxin are readily available from for example: Sigma Cat No. T 0910 Thioredoxin (E. coli, recombinant; expressed in E. coli).
NADPH-thioredoxin reductase is a cytosolic homodimeric enzyme comprising typically 300-500 amino acids. Crystal structures of both E. coli and plant NADPH-thioredoxin reductase have been obtained (Waksman et al. (1994) J. Mol. Biol. 236: 800-816; Dai et al. (1996) J. Allergy Clin. Immunol. 103: 690-697). NADPH-thioredoxin reductases have been expressed in heterologous hosts, for example the Arabidopsis NADPH-thioredoxin reductase has been expressed in E. coli (Jacquot et al. (1994) J. Mol. Biol. 235: 1357-1363) and wheat (PCT Patent Application 00/58453).
There is a need in the art to further improve the methods for the recombinant expression of thioredoxin and thioredoxin reductase in association with oil bodies.
The present invention describes the use of an oil body protein gene to target the expression of a heterologous polypeptide, to an oil body in a host cell. The unique features of both the oil body protein and the expression patterns are used in this invention to provide a means of synthesizing commercially important proteins on a scale that is difficult if not impossible to achieve using conventional systems of protein production. In a preferred embodiment of the present invention, the heterologous peptide is a thioredoxin or thioredoxin reductase.
In particular, the present invention provides a method for the expression of a thioredoxin or thioredoxin reductase by a host cell said method comprising:
a) introducing into a host cell a chimeric nucleic acid sequence comprising:
1) a first nucleic acid sequence capable of regulating the transcription in said host cell of
2) a second nucleic acid sequence, wherein said second sequence encodes a fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein gene to provide targeting of the fusion polypeptide to a lipid phase linked in reading frame to (ii) a nucleic acid sequence encoding thioredoxin or thioredoxin reductase; and
3) a third nucleic acid sequence encoding a termination region functional in the host cell; and
b) growing said host cell to produce the fusion polypeptide.
In a preferred embodiment the oil body protein is an oleosin. Preferred host cells are plant cells, bacterial cells and yeast cells.
The present invention also provides a chimeric nucleic acid sequence encoding a fusion polypeptide, capable of being expressed in association with an oil body of a host cell comprising:
1) a first nucleic acid sequence capable of regulating the transcription in said host cell of
2) a second nucleic acid sequence, wherein said second sequence encodes a fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein gene to provide targeting of the fusion polypeptide to a lipid phase linked in reading frame to (ii) a nucleic acid sequence encoding a thioredoxin or thioredoxin reductase; and
3) a third nucleic acid sequence encoding a termination region functional in the host cell.
In a preferred embodiment the oil body protein is an oleosin. Preferred host cells are plant cells, bacterial cells and yeast cells.
The present invention also includes a fusion polypeptides encoded by a chimeric nucleic acid sequence comprising (i) a nucleic acid sequence encoding a sufficient portion of an oil body protein to provide targeting of the fusion polypeptide to an oil body linked in reading frame to (ii) a nucleic acid sequence encoding a thioredoxin or thioredoxin reductase.
The invention further provides methods for the separation of a thioredoxin or thioredoxin reductase from host cell components by partitioning of the oil body fraction and subsequent release of the thioredoxin or thioredoxin reductase via specific cleavage of the thioredoxin or thioredoxin reductase-oil body protein fusion. Optionally a cleavage site may be located prior to the N-terminus and after the C-terminus of the thioredoxin or thioredoxin reductase protein allowing the fusion polypeptide to be cleaved and separated by phase separation into its component peptides. This production system finds utility in the production of many proteins and peptides such as those with pharmaceutical, enzymic, rheological and adhesive properties.
The methods described above are not limited to thioredoxin or thioredoxin reductase produced in plant seeds as oil body proteins may also be found in association with oil bodies in other cells and tissues. Additionally the methods are not limited to the recovery of thioredoxin or thioredoxin reductase produced in plants because the extraction and release methods can be adapted to accommodate oil body protein-thioredoxin/thioredoxin reductase protein fusions produced in any cell type or organism. An extract containing the fusion protein is mixed with additional oleosins and appropriate tri-glycerides and physical conditions are manipulated to reconstitute the oil-bodies. The reconstituted oil-bodies are separated by floatation and the recombinant thioredoxin or thioredoxin reductase released by the cleavage of the junction with the oil body protein.