| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 03.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 14.03.00 |
| PATENT TITLE |
Process for isolating and purifying viruses, soluble proteins and peptides from plant sources |
| PATENT ABSTRACT |
The present invention features a method for isolating and purifying viruses, proteins and peptides of interest from a plant host which is applicable on a large scale. Moreover, the present invention provides a more efficient method for isolating viruses, proteins and peptides of interest than those methods described in the prior art. In general, the present method of isolating viruses, proteins and peptides of interest comprises the steps of homogenizing a plant to produce a green juice, adjusting the pH of and heating the green juice, separating the target species, either virus or protein/peptide, from other components of the green juice by one or more cycles of centrifugation, resuspenion, and ultrafiltration, and finally purifying virus particles by such procedure as PEG-precipitation or purifying proteins and peptides by such procedures as chromatography and/or salt precipitation. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 10.03.98 |
| PATENT REFERENCES CITED |
Brakke, M., "Density Gradient Centrifugation and its Application to Plant Viruses", Adv. Virus. Res. 7:193-224 (1960) no month found. Gooding et al., "A Simple Technique for Purification of Tobacco Mosaic Virus in Large Quantities", Phytopathological Notes 57:1285 (Nov. 1967). Khan et al., "Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.)", Transgenic Res. 5:178-185 (1996) no month found. Mason et al., "Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice", Proc. Natl. Acad. Sci. USA 93:5335-5340 (May 1996). Turpen et al., "Malarial Epitopes Expressed on the Surface of Recombinant Tobacco Mosaic Virus", BioTechnology 13:53-57 (Jan. 1995). Ma et al., Science 268:716-719 (May 1995). |
| PATENT CLAIMS |
We claim: 1. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of: (a) homogenizing a plant to produce a green juice homogenate; (b) adjusting the pH of the green juice homogenate to less than or equal to about 5.2; (c) heating the green juice homogenate to a minimum temperature of about 45.degree. C.; (d) centrifuging the green juice homogenate to produce a supernatant; and (e) purifying the protein or peptide from the supernatant. 2. The method of claim 1 wherein the pH of the green juice homogenate is adjusted to between about 4.0 and 5.2. 3. The method of claim 1 wherein the pH of the green juice homogenate is adjusted to about 5.0. 4. The method of claim 1 wherein the green juice homogenate is heated to a temperature of between about 45 and 50.degree. C. 5. The method according to claim 1 wherein the supernatant produced in step (d) is further subjected to ultrafiltration. 6. The method according to claim 5 further comprising the step of subjecting a permeate produced by the said ultrafiltration to a second ultrafiltration. 7. The method according to claim 6 further comprising the step of purifying a concentrate resulting from the second ultrafiltration. 8. The method of claim 7 wherein said purifying is performed by chromatography, affinity-based method of purification, or salt precipitation. 9. The method of any one of claim 1 through 8 wherein the soluble protein or peptide is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, Il-5, IL-6, IL-7, Il-8, IL-9, IL-10, IL-11, IL-12, EPO, G-CSF, GM-CSF, M-CSF, Factor VIII, Factor IX, tPA, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone. 10. The method of any one of claims 1 through 8 wherein the soluble protein or peptide is an antimicrobial peptide or protein and is selected from the group consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensions, .beta.-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins. 11. The method of any one of claims 1 through 8 wherein the said protein or peptide is a recombinant protein or peptide. 12. The method according to claim 5 wherein said ultrafiltration produces a permeate comprising one or more molecules selected from the group consisting of sugars, polysaccharides, vitamins, alkaloids, flavor compounds and peptides. 13. The method according to claim 7 wherein said second ultrafiltration produces a permeate containing molecules selected from the group consisting of sugars, polysaccharides, vitamins, alkaloids, flavor compounds and peptides. 14. The method according to any one of the claims 1-8 wherein said protein or peptide is non-native in the plant. 15. A method for obtaining a fusion peptide or fusion protein from a plant comprising the sequential steps of: (a) homogenizing a plant to produce a green juice homogenate; (b) adjusting the pH of the green juice homogenate to less than or equal to about 5.2; (c) heating the green juice homogenate to a minimum temperature of about 45.degree. C.; (d) centrifuging the green juice homogenate to produce a pellet; (e) resuspending the pellet in a liquid solution; (f) adjusting the pH of the liquid solution containing the resuspended pellet to about 2.0 to 4.0; (g) centrifuging the liquid solution of step (f) containing the resuspended pellet to about 2.0 to 4.0; (h) purifying the fusion protein or fusion peptide. 16. The method according to claim 15 wherein the purifying is performed by at least one method selected from the group consisting of chromatography, ultrafiltration, and salt precipitation. 17. The method of claim 15 or claim 16 wherein said fusion protein or fusion peptide comprises a peptide or protein selected from the group consisting of IL-1, IL-2, IL-3, IL-4, Il-5, IL-6, IL-7, Il-8, IL-9, IL-10, IL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIII, Factor IX, tPA, hGH, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, and calcitonin. 18. The method of claim 15 or claim 16 wherein said fusion protein or fusion peptide comprises an antimicrobial peptide or antimicrobial protein selected from the group consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, .beta.-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins. 19. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of: (a) harvesting a plant; (b) homogenizing the plant to produce a green juice homogenate; (c) adjusting the pH of the green juice homogenate to less than or equal to about 5.2. (d) heating the green juice homogenate to a minimum temperature of about 45.degree. C.; (e) centrifuging the green juice homogenate to produce a supernatant; and (f) purifying the protein or peptide from the supernatant. 20. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of: (a) inserting a virus into a plant; (b) harvesting the plant; (c) homogenizing the plant to produce a green juice homogenate; (d) adjusting the pH of the green juice homogenate to less than or equal to about 2.5. (e) heating the green juice homogenate to a minimum temperature of about 45.degree. C. (f) centrifuging the green juice homogenate to produce a supernatant; and (g) purifying the protein or peptide from the supernatant. 21. The method according to claim 20 wherein the virus is a recombinant virus. 22. The method according to claim 21 wherein the virus is a viral vector capable of carrying a heterologous nucleic acid sequence. 23. The method according to any one of the claims 19-22 wherein said protein or peptide is non-native in the plant. 24. The method according to any one of the claims 19-22 wherein said protein or peptide is a recombinant protein or peptide. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to a process for isolating and purifying viruses, soluble proteins and peptides produced in plants. More specifically, the present invention is applicable on a large scale. BACKGROUND OF THE INVENTION Plant proteins and enzymes have long been exploited for many purposes, from viable food sources to biocatalytic reagents, or therapeutic agents. During the past decade, the development of transgenic and transfected plants and improvements in genetic analysis have brought renewed scientific significance and economical incentives to these applications. The concepts of molecular plant breeding and molecular plant farming, wherein a plant system is used as a bioreactor to produce recombinant bioactive materials, have received great attention. Many examples in the literature have demonstrated the utilization of plants or cultured plant cells to produce active mammalian proteins, enzymes, vaccines, antibodies, peptides, and other bioactive species. Ma et al. (Science 268: 716-719 (1995)) were the first to described the production of a functional secretory immunoglobulin in transgenic tobacco. Genes encoding the heavy and light chains of murine antibody, a murine joining chain, and a rabbit secretory component were introduced into separate transgenic plants. Through cross-pollination, plants were obtained to co-express all components and produce a functionally active secretory antibody. In another study, a method for producing antiviral vaccines by expressing a viral protein in transgenic plants was described (Mason et al., Proc. Natl. Acad. Sci. U.S.A. 93: 5335-5340 (1996)). The capsid protein of Norwalk virus, a virus causing epidemic acute gastroenteritis in humans was shown to self-assemble into virus-like particles when expressed in transgenic tobacco and potato. Both purified virus-like particles and transgenic potato tubers when fed to mice stimulated the production of antibodies against the Norwalk virus capsid protein. Alternatively, the production and purification of a vaccine may be facilitated by engineering a plant virus that carries a mammalian pathogen epitope. By using a plant virus, the accidental shedding of virulent virus with the vaccine is abolished, and the same plant virus may be used to vaccinate several hosts. For example, malarial epitopes have been presented on the surface of recombinant tobacco mosiac virus (TMV) (Turpen et al., BioTechnology 13:53-57 (1995)). Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused into the C-terminus. Tobacco plants after infection contain high titers of the recombinant virus, which may be developed as vaccine subunits and readily scaled up. In another study aimed at improving the nutritional status of pasture legumes, a sulfur-rich seed albumin from sunflower was expressed in the leaves of transgenic subterranean clover (Khan et al. Transgenic Res. 5:178-185 (1996)). By targeting the recombinant protein to the endoplasmic reticulum of the transgenic plant leaf cells, an accumulation of transgenic sunflower seed albumin up to 1.3% of the total extractable protein could be achieved. Work has also been conducted in the area of developing suitable vectors for expressing foreign genetic material in plant hosts. Ahlquist, U.S. Pat. No. 4,885,248 and U.S. Pat. No. 5,173,410 described preliminary work done in devising transfer vectors which might be useful in transferring foreign genetic material into plant host cells for the purpose of expression therein. Additional aspects of hybrid RNA viruses and RNA transformation vectors are described by Ahlquist et al. in U.S. Pat. Nos. 5,466,788, 5,602,242, 5,627,060 and 5,500,360 all of which are herein incorporated by reference. Donson et al., U.S. Pat. No. 5,316,931 and U.S. Pat. No. 5,589,367, herein incorporated by reference, demonstrate for the first time plant viral vectors suitable for the systemic expression of foreign genetic material in plants. Donson et al. describe plant viral vectors having heterologous subsenomic promoters for the systemic expression of foreign genes. The availability of such recombinant plant viral vectors makes it feasible to produce proteins and peptides of interest recombinantly in plant hosts. Elaborate methods of plant genetics are being developed at a rapid rate and hold the promise of allowing the transformation of virtually every plant species and the expression of a large variety of genes. However, in order for plant-based molecular breeding and farming to gain widespread acceptance in commercial areas, it is necessary to develop a cost-effective and large-scale purification system for the bioactive species produced in the plants, either proteins or peptides, especially recombinant proteins or peptides, or virus particles, especially genetically engineered viruses. Some processes for isolating proteins, peptides and viruses from plants have been described in the literature (Johal, U.S. Pat. No. 4,400,471, Johal, U.S. Pat. No. 4,334,024, Wildman et al., U.S. Pat. No. 4,268,632, Wildman et al., U.S. Pat. No. 4,289,147, Wildman et al., U.S. Pat. No. 4,347,324, Hollo et al., U.S. Pat. No. 3,637,396, Koch, U.S. Pat. 4,233,210, and Koch, U.S. Pat. No. 4,250,197, the disclosure of which are herein incorporated by reference). The succulent leaves of plants, such as tobacco, spinach, soybeam, and alfalfa, are typically composed of 10-20% solids, the remaining fraction being water. The solid portion is composed of a water soluble and a water insoluble portion, the latter being predominantly composed of the fibrous structural material of the leaf. The water soluble portion includes compounds of relatively low molecular weight (MW), such as sugars, vitamins, alkaloids, flavors, amino acids, and other compounds of relatively high MW, such as nature and recombinant proteins. Proteins in the soluble portion of the plant bombast can be further divided into two fractions. One fraction comprises predominantly a photosynthetic protein, ribulose 1,5-diphosphate carboxylase (or RuBisCO), whose subunit molecular weight is about 550 kD. This fraction is commonly referred to as "Fraction 1 protein." RuBioCO is abundant, comprising up to 25% of the total protein content of a leaf and up to 10% of the solid matter of a leaf. The other fraction contains a mixture of proteins and peptides whose subunit molecular weights typically range from about 3 kD to 100 kD and other compounds including sugars, vitamins, alkaloids, flavors, amino acids. This fraction is collectively referred to as "Fraction 2 proteins." Fraction 2 proteins can be native host materials or recombinant materials including proteins and peptides produced via transaction or transgenic transformation. Transfected plants may also contain virus particles having a molecular size greater than 1,000 kD. The basic process for isolating pant proteins generally begins with disintegrating leaf bombast and pressing the resulting pulp to produce "green juice". The process is typically performed in the presence of a reducing agent or antioxidant to suppress unwanted oxidation. The green juice, which contains various protein components and finely particulate green pigmented material, is pH adjusted and heated. The typical pH range for the green juice after adjustment is between 5.3 and 6.0 This range has been optimized for the isolation of Fraction 1 protein (or ribulose 1,5-diphosphate carboxylase). Heating, which causes the coagulation of green pigmented material, is typically controlled near 50.degree. C. The coagulated green pigmented material can then be removed by moderate centrifugation to yield "brown juice." The brown juice is subsequently cooled and stored at a temperature at or below room temperature. After an extended period of time, e.g. 24 hours, ribulose 1,5-diphosphate carboxylase is crystallized from the brown juice. The crystallized Fraction 1 protein can subsequently be separated from the liquid by centrifugation. Fraction 2 proteins remain in the liquid, and they can be purified upon further acidification to a pH near 4.5. Alternatively, the crystal formation of ribulose 1,5-diphosphate carboxylase from brown juice can be effected by adding sufficient quantities of polyethylene glycol (PEG) in lieu of cooling. The basic process for isolating virus particles is described in Gooding et al. (Phytophathological Notes 57:1285 (1967), the teaching of which are herein incorporated by reference). To purify Tobacco Mosaic Virus (TMV) from plant sources in large quantities, infected leaves are homogenized and n-butanol is then added. The mixture is then centrifuged, and the virus is retained in the supernatant. Polyethylene glycol (PEG) is then added to the supernatant followed by centrifugation. The virus can be recovered from the resultant PEG pellet. The virus can be further purified by another cycle of resuspension, centrifugation and PEG-precipitation. Existing protocols for isolating and purifying plant viruses and soluble proteins and peptides, however, present many problems. First, protein isolation from plant sources have been designed in large part for the recovery of Fraction 1 protein, not for other biologically active soluble protein components. The prior processes for large-scale extraction of F1 proteins was for production of protein as an additive to animal feed or other nutritional substances. Acid-precipitation to obtain Fraction 2 proteins in the prior art is not effective, since most proteins denature in the pellet form. This is especially troublesome for isolating proteins and peptides produced by recombinant nucleic acid technology, as they may be more sensitive to being denatured upon acid-precipitation. Second, the existing methods of separation rely upon the use of solvents, such as n-butanol, chloroform, or carbon tetrachloride to eliminate chloroplast membrane fragments, pigments and other host related materials. Although useful and effective for small-scale virus purification, using solvents in a large-scale purification is problematic. Such problems as solvent disposal, special equipment designs compatible with flammable liquids, facility venting, and worker exposure protection and monitoring are frequently encountered. There are non-solvent based, small-scale virus purification methods, but these are not practical for large scale commercial operations due to equipment and processing limitations and final product purity (Brakke Adv. Virus Res. 7:193-224 (1960) and Brakke et al. Virology 39: 516-533 (1969)). Finally, the existing protocols do not allow a streamline operation such that the isolation and purification of different viruses, proteins and peptides can be achieved with minimum modification of a general purification procedure. There is a need in the art for an efficient, non-denaturing and solvent-limited large-scale method for virus and soluble protein isolation and purification. This need is especially apparent in cases where proteins and peptides produced recombinantly in plant hosts are to be isolated. The properties of these proteins and peptides are frequently different from those of the native plant proteins. Prior art protocols are not suitable to isolate recombinant proteins and peptides of interest. In addition, the vast diversity of recombinant proteins and peptides from plants and the stringent purity requirement for these proteins and peptides in industrial and medical application requires an efficient and economical procedure for isolating and purifying them. Efficient virus isolation is also of great importance because of the utility of viruses as transaction vectors and vaccines. In some situations, proteins and peptides of interest may be attached to a virus or integrated with native viral proteins (fusion protein), such that isolating the protein or peptide of interest may in fact comprise isolating the virus itself. SUMMARY OF THE INVENTION The present invention features a method for isolating and purifying viruses, proteins and peptides of interest from a plant host which is applicable on a large scale. Moreover, the present invention provides a more efficient method for isolating viruses, proteins and peptides of interest than those methods described in the prior art. In general, the present method of isolating viruses, proteins and peptides of interest comprises the steps of homogenizing a plant to produce a green juice, adjusting the pH of and heating the green juice, separating the target species, either virus or protein/peptide, from other components of the green juice by one or more cycles of centrifugation, resuspenion, and ultrafiltration, and finally purifying virus particles by such procedure as PEG-precipitation or purifying proteins and peptides by such procedures as chromoatgraphy, including affinity-based methods, and/or salt precipitation. In one embodiment, the green juice is pH adjusted to a value of between about 4.0 and 5.2 and heated at a temperature of between about 45-50.degree. C. for a minimum of about one min. This mixture is then subjected to centrifugation. The supernatant produced thereby contains virus if transfected and Fraction 2 proteins including recombinant products. Fraction 2 proteins may be separated from the pelleted Fraction 1 protein and other host materials by moderate centrifugation. Virus particles and Fraction 2 proteins may then be further purified by a series of ultrafiltration, chromatography, salt precipitation, and other methods, including affinity separation protocols, which are well known in the art. One of the major advantages of the instant invention is that it allows Fraction 2 proteins to be subjected to ultrafiltration whereas prior methods do not. In a second embodiment, after pH and heat treatment, the pellet from centrifugation containing the virus, Fraction 1 protein and other host materials is resuspended in a water or buffer solution and adjusted to a pH of about 5.0-8.0. The mixture is subjected to a second centrifugation. The resuspension allows the majority of virus to remain in the supernatant after the second centrifugation and Fraction 1 protein and other host materials may be found in the resulting pellet. The virus particles may be further purified by PEG-precipitation or ultrafiltration if necessary prior to PEG-precipitation. In a third embodiment, the coat protein of a virus is a fusion protein, wherein the recombinant protein or peptide of interest is integrated with the coat protein of a virus. During virus replication or during the process of virus isolation and purification, its coat protein may become detached from the virus genome itself, or accumulate as unassembled virus coat protein or the coat fusion may never be incorporated. After centrifugation of the pH adjusted and heated green juice, the pellet may contain the virus, unassembled fusion proteins, Fraction 1 protein, and other host materials. The pellet is then resuspended in water or a buffer solution and adjusted to a pH about 2.0-4.0 followed by a second centrifugation. The protein will remain in the resulting supernatant. The unassembled protein may be further purified according to conventional methods including ultrafiltration, salt precipitation, affinity separation and chromatography. The peptide or protein of interest may be obtained by chemical cleavage of the fusion protein. Such procedures are well known to those skilled in the art. In a fourth embodiment, sugars, vitamins, alkaloids, flavors, and amino acids from a plant may also be conveniently isolated and purified. After centrifugation of the pH adjusted and heated green juice, the supernatant contains the Fraction 2 proteins, viruses and other materials, such as sugars, vitamins, alkaloids, and flavors. The supernatant produced thereby may be separated from the pellected Fraction 1 protein and other host materials by moderate centrifugation. Sugars, vitamins, alkaloids, and flavors may then be further purified by a series of methods including ultrafiltration and other methods, which are well known in the art. In a fifth embodiment, the present invention features viruses, proteins, peptides, sugars, vitamins, alkaloids, and flavors of interest obtained by the procedures described herein. BRIEF DESCRIPTION OF THE FIGURE FIG. 1 represents a flow chart which demonstrates the present method for isolating and purifying viruses and soluble proteins and peptides from plant sources. DETAILED DESCRIPTION OF THE INVENTION The present invention features a novel method for isolating and purifying viruses, proteins and peptides of interest from a plant host. Moreover, the present invention provides a more efficient method for isolating viruses, proteins and peptides of interest than those methods described in the prior art. In addition, the present method is applicable on a large production scale. In general, the present method of isolating viruses, proteins and peptides of interest comprises the steps of homogenizing a plant to produce a green juice, adjusting the pH of and heating the green juice, separating the target species, either virus or protein/peptide, from other components of the green juice by one or more cycles of centrifugation, resuspension, and ultrafiltration, and finally purifying virus particles by such procedure as PEG-precipitation or purifying proteins and peptides by such procedures as chromatography, including affinity separation, and/or salt precipitation. An illustration of the instant invention is presented in FIG. 1. However, this figure is intended merely to visualize the present invention and is not to be construed as being limiting to the procedures or orders of their appearances depicted therein. Any modifications of the instant invention which are functionally equivalent to the procedures and conditions disclosed herein are within the scope of the instant invention. The initial step of the present method features homogenizing the subject plant. Plant leaves may be disintegrated using any appropriate machinery or process available. For instance, a Waring blender for a small scale or a Reitz disintegrator for a large scale has been successfully used in some embodiments of the instant invention. The homogenized mixture may then be pressed using any appropriate machinery or process available. For example, a screw press for a large scale or a cheesecloth for a small scale has been successfully employed in some embodiments of the instant invention. The homogenizing step may be performed in the presence of a suitable reducing agent or oxidizing agent to suppress unwanted oxidation. Sodium metabisulfite (Na.sub.2 S.sub.2 O.sub.5) is successfully used in some embodiments of the instant invention. The subsequent steps to isolate and purify viruses and soluble proteins peptides may be performed generally according to the following procedures. pH Adjustment and Heat Treatment of Green Juice According to the present invention, the pH of the initial green juice is adjusted to a value less than or equal to 5.2 and then heated at a minimum temperature of about 45.degree. C. In preferred embodiments of the instant invention, the green juice is pH adjusted to between about 4.0 and 5.2 and is then heated to a temperature of between about 45-50.degree. C. for a minimum of one minute. In some embodiments of the instant invention, heat treatment between 10 to 15 minutes has been used successfully. Those skilled in the art will readily appreciate that the time allocated for heat treatment will vary depending on the recovery of the described species. Therefore, following pH adjustment, the heating time may vary from about one minute to over 15 minutes. Heat may be applied in any suitable manner, and the invention is not intended to be limiting in this regard. Those skilled in the art will appreciate that pH may be adjusted using many suitable acids or bases well known in the art. In some embodiments of the present invention, phosphoric acid has proven effective. The pH of green juice influences for distribution of virus, proteins and peptides in the supernatant or pellet during subsequent centrifugations. An optimal value for the target species may be obtained by testing the isolation and purification of the virus and or protein or peptide of interest on a small scale. Methods previously described in the literature for non-virus purification adjust the pH of the green juice to a value between 5.3 and 6.0 and use heat treatment of at a temperature of about 48-52.degree. C. The heat-treated and pH adjusted green juice is quite unique in that the pH of green juice influences the distribution of virus, proteins and peptides in the supernatant or pellet during subsequent centrifugations. Depending on the species of interest, the pH of green juice may be readily controlled to facilitate the isolation and purification of the desirable product, either virus particles or proteins and peptides. It thus provides a streamlined operation such that the isolation and purification of different viruses and proteins and peptides can be optimized with small modifications of a general purification procedure. Such modifications are within the routine skill of skilled artisans and do not require undue experimentation. The unique characteristic of green juice has enabled it to be processed in a variety of purification steps described below. Centrifugation of Green Juice The pH- and heat-treated green juice may then be subjected to centrifugation. Those of skill in the art may readily determine suitable conditions for centrifugation, including time interval and G-force. It is generally contemplated that centrifugation should be of sufficient G-force and time to pellet substantially all of Fraction 1 protein, chloroplast and other host materials, while retaining the desired target species in the supernatant fraction or at a sufficient speed and time to pellet the target species with Fraction 1 protein, chloroplast and other host materials. For example, centrifugation at 3000.times.G for two minutes or at 6000.times.G for three minutes have been effectively applied to the green juice in some embodiments of the instant invention. According to the present invention, a majority of Fraction 1 protein, unassembled fusion proteins and peptides, chloroplast and other host materials are pelleted (P1) by centrifugation, while Fraction 2 proteins including recombinant proteins and peptides may generally remain in the supernatant (S1) after this centrifugation (see FIG. 1). The virus, however, may partition between pellet and supernatant after centrifugation, depending upon the pH of the green juice the virus species, virus nucleic acid construct, plant species, plant age, and source of plant tissue, among other factors. At a low pH, preferably below a pH of about 5.0, the virus is predominantly retained in the pellet (P1). At a pH of between about 5.0 and 5.2, virus is present in the supernatant (S1) as well. Depending on the species of interest, the pH of green juice and subsequent centrifugation conditions may be readily controlled to facilitate the isolation and purification of the desirable product, either virus particles or proteins and peptides. Thus, the instant process provides a streamlined operation such that the isolation and purification of different viruses and proteins and peptides can be achieved with small modifications of a general purification procedure, which modifications require no undue experimentation for those of ordinary skill in the art. Resuspension of Pellet in a pH Controlled Buffer The pellet obtained by centrifugation of the pH-adjusted and heated green juice typically contains Fraction 1 protein, unassembled fusion proteins and peptides, viruses, and other host materials. It may be resuspended in water or in a buffer solution having the desired pH range, or pH adjusted to that range. The optimal pH is determined by the final species of interest. In some preferred embodiments, the pH range of resuspension is about 5.0 to 8.0 for isolating and purifying virus particles (see FIG. 1). In other embodiments, the pH range of resuspension is about 2.0 to 4.0 if the desired product is a fusion proteins/peptide (see FIG. 1). Those skilled in the art may readily choose appropriate buffer solution or acids or bases to reach the designed pH range without undue experimentation. Depending upon the percentage of solids of the pellet formed as a result of the first centrifugation procedure, a resuspension volume can be adjusted to a fraction of the starting green juice volume, typically in amounts of 10 to 100-fold of the original green juice volume. Isolation and Purification of Virus Viruses can be recovered from either the pellet (P1) alone, the supernatant (S1), or both the supernatant (S1) and pellet (P1) after centrifugation of the green juice depending upon the pH and degree of virus partitioning. When the pH of green juice is adjusted to a low value, for example, about 4.0, the virus is in general quantitatively retained in the pellet along with Fraction 1 protein chloroplast and other host material after centrifugation of the green juice (see FIG. 1). After resuspension in a solution having a pH of about 5.0 to 8.0, the mixture may be subjected to another centrifugation step. Virus particles are predominantly retained in the supernatant (S2) and may be separated from Fraction 1 protein, chloroplast fragments and other host materials in the pellets. Usually only about 5-10% of the starting green juice protein remains in S2. The virus containing supernatant may then be ultrafiltered, if necessary, using a molecular weight cut-off (MWCO) in the range of about 1-500 kD membrane according to any one of the ultrafiltration techniques known to those of skill in the art. For example, a 100 kD MWCO membrane has been successfully used in some embodiments of the instant invention to retain virus particles in the concentrates, while smaller protein components filter through. The ultrafiltration step results in a substantial further reduction in the process volume. In some embodiments, further reductions in the process volume of 1- to 30-fold or greater are attainable. From ultrafiltration or centrifugation, a final purification of virus may be accomplished by prior art methods such as PEG-precipitation, centrifugation, resuspension, and clarification. In some embodiments of the instant invention, virus particles may also be obtained from the supernatant (S1) after the centrifugation of the green juice. This supernatant fraction normally contains Fraction 2 proteins and peptides (see FIG. 1). In some embodiments of the instant invention, the pH of green juice may be adjusted to a value between about 5.0 and 5.2, preferably around pH 5.0. A significant portion of virus particles may then be recovered from the supernatant (S1) in addition to the pellet (P1) after centrifugation of the green juice. The virus containing supernatant may be ultrafiltered including, if necessary, diafiltration using a molecular weight cut-off membrane in the range of about 1-500 kD according to any one of the ultrafiltration and diafiltration techniques known to those skilled in the art. For example, a 100 kD MWCO membrane has been successfully used in some embodiments of the instant invention to retain virus particles in the concentrates, while smaller protein components, e.g. Fraction 2 proteins filter through. The ultrafiltration step results in a substantial further reduction in the process volume. From ultrafiltration or centrifugation, a final purification of virus may be accomplished by prior art methods such as PEG-precipitation, centrifugation, resuspenion, and clarification. An isolation and purification procedure according to the methods described herein has been used to isolate TMV-based viruses from three tobacco varieties (Ky8959, Tn86 and MD609) and Nicotiana benthamiana. A number of TMV-based viruses have been obtained Figure including, TMV204 (wild type, SEQ ID NO:1:), TMV261 (coat protein read-throughs, SEQ ID. NO:2:), TMV291 (coat protein loop fusion, SEQ ID NO.:3:), TMV811 (SEQ ID NO:4:), and TMV861 (coat protein read throughs, SEQ ID NO.:5:). TMV 261 and TMV291 have been shown to be unstable during some isolation procedures, yet remain intact during the present procedure. These viral vectors are used merely as examples of viruses that can be recovered by the instant invention and are not intended to limit the scope of the invention. A person of ordinary skill in the art will be able to use the instant invention to recover other viruses. The virus of interest may be a potyvirus, a tobamovirus, a bromovirus, a armovirus, a luteovirus, a marafivirus, the MCDV group, a necrovirus, the PYFV group, a sobemovirus, a tombusvirus, a tymovirus, a capillovirus, a closterovirus, a carlavirus, a potexvirus, a comovirus, a dianthovirus, a fabavirus, a repovirus, a PEMV, a furovirus, a tobravirus, an AMV, a tenuivirus, a rice necrosis virus, caulimovirus, a geminivirus, a reovirus, the commelina yellow mottle virus group and a cryptovirus, a Rhabovirus, or a Bunyavirus. The present methods of isolating and purifying virus particles represent significant advantages over the prior art methods. They allow the ultrafiltration of virus-containing supernatant (S1 and/or S2), which significantly reduces the processing volume and removes plant components, such as, sugars, alkaloids, flavors, and pigments and Fraction 1 and 2 proteins. Desired virus particles can be enriched as particulate. The concentration and purification of virus particles is thus rapid and effective. Isolation and Purification of Soluble Proteins and Peptides The Fraction 2 proteins including recombinant proteins and peptides remain soluble after pH adjustment and heat treatment and centrifugation of green juice (see FIG. 1). The Fraction 2 protein-containing supernatant has removed sufficient Fraction 1 proteins, chloroplast and other host materials, to enable an efficient isolation and purification of Fraction 2 proteins, especially recombinant proteins and peptides, using size fractionation by ultrafiltration, concentration and diafiltration. Ultrafiltration is typically performed using a MWCO membrane in the range of about 1 to 500 kD according to methods well known in the art. In some embodiments of the instant invention, a large MWCO membrane is first used to filter out the residual virus and other host materials. Large molecular weight components may remain in the concentrates. Filtrates containing the proteins/peptides of interest may be optionally passed through another ultrafiltration membrane, typically of a smaller MWCO, such that the target compound can be collected in the concentrates. Additionally cycles of ultrafiltration may be conducted, if necessary, to improve the purity of the target compound. The choice of MWCO size and ultrafiltration conditions depends on the size of the target compound and is an obvious variation to those skilled in the art. The ultrafiltration step generally results in a reduction in process volume of about 10- to 30-fold or more and allows diafiltration to further remove undesired molecular species. Finally, proteins or peptides of interest may be purified using standard procedures such as chromatography, salt precipitation, solvent extractions including super critical fluids such as CO.sub.2 and other methods known to those of skill in the art. The present isolation procedure has been used to successfully isolate and concentrate secretory IgA antibody and .alpha.-trichosanthin. The invention is also specifically intended to encompass embodiments wherein the peptide or protein of interest is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, -IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIII, Factor IX, tPA, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone. In yet other embodiments, the soluble protein or peptide of interest may be an antimicrobial peptide or protein consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, .beta.-defensins, cryptdins, clavainins, plant defensins, nicin and bacterecins. These and other proteins and peptides of interest may be naturally produced or produced by recombinant methodologies in a plant. The present method of isolating and purifying Fraction 2 proteins represents significant advantages from the prior art methods. First, it does not require acid-precipitation of F2 proteins. Acid-precipitation in the prior art may not be described since many proteins may be denatured or lose enzymatic or biological activity. Fraction 2 proteins including recombinant proteins and peptides in the instant invention are not retained in a pellet form, thereby minimizing the risk of protein denaturation. The present method thereby minimizes denaturation of proteins and peptides of interests. Second, because the more abundant component, Fraction 1 protein, is eliminated during the early stages of purification, the downstream process allows the ultrafiltration of Fraction 2 proteins. Ultrafiltration of Fraction 2 proteins permits significant reduction of processing volume and allows rapid concentration and purification of proteins and peptides. Desirable proteins and peptides can be enriched by molecular weight. Rapid concentratin and purification also reduces or eliminates the degradation or denaturation due to endogenous protease activities. Ultrafiltration of Fraction 2 proteins is not applicable with methods in the prior art. Finally, the concentration of Fraction 2 proteins including recombinant proteins and peptides requires no solvents and no additional chemicals. Plant protein and peptide isolation procedures in the prior art frequently use solvents such as n-butanol, chloroform, and carbon tetrachloride to eliminate chloroplast membrane fragments, pigments and other host related materials. Such methods are not easily practiced on a large and commercially valuable scale since these methods present the problems of safety and solvent disposal, which often require designing special equipment compatible with flammable fluids, and hence require facility venting and providing protective equipment to workers. Isolation and Purification of Unassembled Fusion Proteins and Fusion Peptides During virus replication or during the process of isolating and purifying a virus, its coat protein may become detached from the virus genome itself, or accumulate as unassembled virus coat protein, or the coat protein may never be incorporated. One of ordinary skill in the art can invision that the coat protein can be designed through established recombinant nucleic acid protocols to intentionally be unassembled for commercial recovery of proteins having a plurality of biochemical features. This coat protein may contain a recombinant component integrated with the native coat protein, or fusion proteins. These unassembled fusion proteins typically co-segregate in the pellet (P1) with Fraction 1 protein after centrifugation of pH adjusted and heated green juice (see FIG. 1). The pellet may then be resuspended in water or in a buffer with a pH value within the range of about 2.0 to 4.0 followed by another centrifugation. The unassembled protein may be further purified according to conventional methods including a series of ultrafiltration, centrifugation and chromatography steps. The fusion peptide may be obtained followed by chemical cleavage of the desired peptide or protein from the fusion peptide (fusion proteins). Such procedures are well known to those skill in the art. The present invention procedure has been used to successfully isolate and concentrate .alpha.-amylase-indolicidin fusion protein. The invention is also specifically intended to encompass embodiments wherein the fusion protein or peptide may contain a peptide or protein selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, I1-8, IL-9, IL-10, IL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, MCSF, Factor VIII, Factor IX, tPA, receptors receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone. In yet other embodiments, the protein or peptide present in the fusion protein or peptide may be an antimicrobial peptide or protein consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, .beta.-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins. Isolation and Purification of Sugars, Vitamins, Alkaloids, and Flavors Sugars, vitamins, alkaloids, flavors, amino acids from a plant may also be conveniently isolated and purified using the method of the instant invention. After centrifugation of the pH adjusted and heated green juice, the supernatant contains the Fraction 2 proteins, viruses and other materials, including sugars, vitamins, alkaloids, and flavors. The supernatant produced thereby may be separated from the pelleted Fraction 1 protein and other host materials by centrifugation. Sugars, vitamins, alkaloids, flavors may then be further purified by a series of low molecular weight cutoff ultrafiltration and other methods, which are well known in the art. Definitions In order to provide an even clearer and more consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided: A "virus" is defined herein to include the group consisting of a virion wherein said virion comprises an infectious nucleic acid sequence in combination with one or more viral structural proteins; a non-infectious virion wherein said non-infectious virion comprises a non-infectious nucleic acid in combination with one or more viral structural proteins; and aggregates of viral structural proteins wherein there is no nucleic acid sequence present or in combination with said aggregate and wherein said aggregate may include virus-like particles (VLPs). Said viruses may be either naturally occurring or derived from recombinant nucleic acid techniques and include any viral-derived nucleic acids that can be adopted whether by design or selection, for replication in whole plant, plant tissues or plant cells. A "virus population" is defined herein to include one or more viruses as defined above wherein said virus population consists of a homogeneous selection of viruses or wherein said virus population consists of a heterogenous selection comprising any combination and proportion of said viruses. "Virus-like particles" (VPLs) are defined herein as self-assembling structural proteins wherein said structural proteins are encoded by one or more nucleic acid sequences wherein said nucleic acid sequence(s) is inserted into the genome of a host viral vector. "Protein and peptides" are defined as being either naturally-occurring proteins and peptides or recombinant proteins and peptides produced via transfection or transgenic transformation. |
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