WO2001019969A1

WO2001019969A1 - A process for isolating and purifying viruses, soluble proteins and peptides from plant sources

Info

Publication number: WO2001019969A1
Application number: PCT/US2000/013680
Authority: WO
Inventors: Stephen J. Garger; Barry R. Holtz; Michael J. Mcculloch; Thomas H. Turpen
Original assignee: Large Scale Biology Corporation
Priority date: 1999-09-16
Filing date: 2000-05-19
Publication date: 2001-03-22
Also published as: AU5142000A

Abstract

The present invention features a method for isolating and purifying viruses, proteins and peptides of interest from a plant host that is applicable on a large scale. In order to isolate the bioactive species from the undesirable photosynthetic proteins in photosynthetic plants, the present invention extends the periods during which the plants are in an environment substantially without light. Moreover, plant material is cut from the photosynthetic plants during a cutting period when the quantity of ribulose 1,5-diphosphate carboxylase in the tobacco plant is at a minimum.

Description

A PROCESS FOR ISOLATING AND PURIFYING VIRUSES, SOLUBLE PROTEINS AND PEPTIDES FROM PLANT SOURCES

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application number 09/037,751, filed March 10, 1998, the entirety of which is incorporated herein by reference.

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 improvement 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 describe 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. USA 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 liters 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. Patent 4,885,248 and U.S. Patent 5,173,410 describe 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. Patents 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. Patent 5,316,931 and U.S. Patent 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 subgenomic 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. Patent, 4,400,471, Johal, U.S. Patent, 4,334,024, Wildman et al., U.S. Patent 4,268,632, Wildman et al., U.S. Patent 4,289, 147, Wildman et al, U.S. Patent 4,347,324, Hollo et al., U.S. Patent 3,637,396, Koch, U.S. Patent 4,233,210, and Koch, U.S. Patent 4,250,197, the disclosure of which are herein incorporated by reference). The succulent leaves of plants, such as tobacco, spinach, soybean, 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 native and recombinant proteins. Proteins in the soluble portion of plant biomass can be further divided into two fractions. One fraction comprises predominantly a photosynthetic protein, ribulose 1,5- diphosphate carboxylase (hereinafter referred to as "RuBisCo"), whose subunit molecular weight is about 550 kD. This fraction is commonly referred to as "Fraction 1 protein." RuBisCo 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 transfection or transgenic transformation. Transfected plants may also contain virus particles having a molecular size greater than 1,000 kD.

The basic process for isolating plant proteins generally begins with disintegrating leaf biomass 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 (ribulose 1,5-diphosphate carboxylase). Heating, which causes the coagulation of green pigmented material, is typically controlled near 50°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.

(Phytopathological 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 has 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 FI proteins was for production of protein as an additive to animal feed or other nutritional substances. For example, the processes described in U.S. Patent Nos. 4,268,632 and 4,347,324 to Wildman et al. are specifically directed to the isolation of RuBisCo in high yield and high purity. These processes are focused on improving the yield and purity with which the RuBisCo can be obtained so that this protein can be used for other purposes. As such, the foregoing patents and processes are particularly inapposite for processes where RuBisCo is an undesirable, waste protein, and other bioactive species are desired to be isolated.

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 non-native proteins and peptides, such as those 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 non-native proteins and peptides or 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 transfection 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. There is a further need in the art for a method by which undesirable photosynthetic proteins can be minimized, to thereby improve the efficiency of the isolation of other bioactive species of interest. There is a particular need in the art for a method by which the presence of RuBisCo in photosynthetic plants can be minimized.

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 photosynthetic plants, the bioactive species of interest must be isolated from undesirable photosynthetic proteins, particularly RuBisCo. The present invention provides novel methods by which the photosynthetic plants are grown and processed to minimize the presence of RuBisCo.

In one aspect of the present invention, a method of processing photosynthetic plants to obtain a plant product suitable for isolation of one or more bioactive species from RuBisCo present in the plant is provided. The method comprises cutting plant material from a plant during a cutting period. The cutting period is a period of a light/dark cycle during which a quantity of RuBisCo is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle. Preferably, the quantity of RuBisCo is substantially at a minimum during the cutting period. The method may also comprise, prior to the cutting step, inoculating the photosynthetic plant with a virus. The method may then further comprise the step of isolating the virus from the plant material, using one or more of the isolating and purifying methods of the present invention.

In a further aspect of the present invention, a method of processing photosynthetic plants is provided. The method comprises: maintaining a plant in an environment substantially without light for a selected period during which a quantity of RuBisCo is reduced, wherein the selected period occurs during daylight; cutting plant material from the plant; and isolating a bioactive species from the RuBisCo in the plant material. The cutting step may be carried out during a cutting period during which a quantity of RuBisCo is substantially at a minimum. The method may also comprise, prior to the cutting step, inoculating the photosynthetic plant with a virus.

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 homogenate, adjusting the pH of and heating the green juice homogenate, separating the target species, either virus or protein/peptide, from other components of the green juice homogenate 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-based methods, and/or salt precipitation. The various aspects of the present invention may be combined into a method for obtaining a soluble protein or peptide from a plant. Such a method comprises the sequential steps of: (a) cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of RuBisCo in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle; (b) homogenizing the plant material 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°C;

(e) centrifuging the green juice homogenate to produce a supernatant; and (f) purifying the protein or peptide from the supernatant.

The various aspects of the present invention may be combined into a method for obtaining a fusion protein or fusion peptide from a plant. Such a method comprises the sequential steps of:

(a) cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of RuBisCo in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle;

(b) homogenizing the plant material to produce a green juice homogenate;

(d) heating the green juice homogenate to a minimum temperature of about 45°C;

(e) centrifuging the green juice homogenate to produce a pellet;

(f) resuspending the pellet in a liquid solution;

(g) adjusting the pH of the liquid solution containing the resuspended pellet to about 2.0 to 4.0;

(h) centrifuging the liquid solution of step (g) containing the resuspended pellet; and

(i) purifying the fusion protein or fusion peptide.

In another aspect of the present invention, a method for obtaining a soluble protein or peptide from a plant is provided. Such a method comprises the sequential steps of:

(a) homogenizing the plant material to produce a green juice homogenate; (b) simultaneously adjusting the pH of the green juice homogenate to less than or equal to about 5.2 and heating the green juice homogenate to a minimum temperature of about 45°C;

(c) centrifuging the green juice homogenate to produce a supernatant; and (d) purifying the protein or peptide from the supernatant.

In still another aspect of the present invention, a method for obtaining a soluble protein or peptide comprises the sequential steps of:

(a) homogenizing the plant material to produce a green juice homogenate;

(b) heating the green juice homogenate to a minimum temperature of about 45°C; (c) adjusting the pH of the green juice homogenate to less than or equal to about

5.2;

(d) centrifuging the green juice homogenate to produce a supernatant; and

(e) purifying the protein or peptide from the supernatant.

In another aspect of the present invention, the green juice homogenate is pH adjusted to a value of between about 4.0 and 5.2 and heated at a temperature of between about 45-

50°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 another aspect, 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 still a further aspect of the present invention, 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 still a further aspect of the present invention, 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 pelleted 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 still a further aspect, the present invention features viruses, proteins, peptides, sugars, vitamins, alkaloids, and flavors of interest obtained by the procedures described herein.

In yet another aspect of the present invention, a method of increasing the number of harvests in a growing season is provided. The method comprises: a) growing a plant to a desirable height; b) harvesting biomass from the plant; c) allowing the plant to generate new biomass; d) harvesting the new biomass; and e) repeating steps c) and d).

In various aspects of the invention, the plant is grown to a desirable height of no more than 4 feet, 3 feet, 2 feet, 1 foot, or 6 inches.

In still a further aspect of the invention, a method of increasing the yield of biomass in a growing season is provided. The method comprises: a) harvesting the younger biomass from a plant; b) allowing the plant to regenerate new biomass; c) harvesting the new biomass; and d) repeating steps b) and c).

BRIEF DESCRIPTION OF THE FIGURE Figure 1 represents a flow chart that illustrates methods for isolating and purifying viruses and soluble proteins and peptides from plant sources in accordance with the present invention.

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. The present invention also provides novel methods by which the presence of RuBisCo in photosynthetic plants can be reduced or minimized, thereby improving the efficiency with which other bioactive species of interest can be isolated from this undesirable photosynthetic protein.

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 homogenate, adjusting the pH of and heating the green juice homogenate, separating the target species, either virus or protein/peptide, from other components of the green juice homogenate 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 Figure 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-S₂O_s) 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°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°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 desired 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 the 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°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 x G for two minutes or at 6000 x 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 (PI) by centrifugation, while Fraction 2 proteins including recombinant proteins and peptides may generally remain in the supernatant (SI) after this centrifugation (see Figure 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 (PI). At a pH of between about 5.0 and 5.2, virus is present in the supernatant (SI) 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 Figure 1). In other embodiments, the pH range of resuspension is about 2.0 to 4.0 if the desired product is a fusion protein/peptide (see Figure 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 (PI) alone, the supernatant (SI), or both the supernatant (SI) and pellet (PI) 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 chroloplast and other host material after centrifugation of the green juice (see Figure 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, choloroplast 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 (SI) after the centrifugation of the green juice. This supernatant fraction normally contains Fraction 2 proteins and peptides (see Figure 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 (SI) in addition to the pellet (PI) 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, resuspension, 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 LD NO:l:), 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 carmovirus, 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 nepovirus, 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 (SI 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 Figure 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. Additional 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 CO2 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 α-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, LL-3, LL-4, JL-5, LL-6, LL-7, IL-8, IL-9, IL-10, JL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIU, Factor LX, 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, β- defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins. 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 desired 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 interest. 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 concentration 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 (PI) with Fraction 1 protein after centrifugation of pH adjusted and heated green juice (see Figure 1). The pellet may then be resuspened 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 skilled in the art.

The present isolation procedure has been used to successfully isolate and concentrate -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, 11-5, IL-6, IL-7, 11-8, JL- 9, IL-10, IL-11, EL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VTJL Factor LX, 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, β-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 plants, 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 homogenous selection of viruses or wherein said virus population consists of a heterogeneous 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.

EXAMPLES The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. The examples are intended specifically to illustrate recoveries of virus, protein and peptide of interest which may be attained using the process within the scope of the present invention.

EXAMPLE 1 Fraction 1 Protein Pelleted From Green Juice at Low pH A tobacco plant of variety MD609 was inoculated 27 days after sowing with TMV

811. Forty days after inoculation, the plant was harvested. Leaf and stalk tissue (150 g) were combined with 0.04% sodium metabisulfite solution (150 ml) in a 1-L Waring blender. The plant tissue was ground on high speed for a period of two minutes. The resulting homogenate was pressed through four layers of cheesecloth, and the pressed fiber was discarded. The volume of juice collected was 240 ml and its pH was 5.57.

With constant stirring, the pH was slowly adjusted downward with dilute phosphoric acid (H₃PO₄). A juice sample (35 ml) was removed at each of the following pH values: pH 5.4, pH 5.3, pH 5.2, pH 5.1, and pH 5.0. Subsequently, all samples were heated to 45°C in a water bath and maintained at this temperature for ten minutes. Samples were then cooled to 25°C in a cold water bath. The cooled samples were centrifuged at 10,000 x G for 15 minutes.

The supernatants (SI in Figure 1) were decanted and analyzed for Fraction 1 protein level by the Bradford assay and SDS-PAGE. The virus was PEG-precipitated and isolated from a portion of each supernatant (25 ml) by the method of Gooding, supra. Virus concentrations were determined by spectrophotometric analysis at 260 nm.

Table 1. Total protein concentrations and virus yields in SI portion after green juices are adjusted to low pH and heated at 45°C for 10 minutes.

Results:

The total protein as determined by the method of Bradford retained in the soluble portion (SI) as determined by the method of Bradford after centrifugation is gradually reduced when the pH of the green juice is adjusted downwards from 5.4 to 5.0. In particular, at pH 5.0 of green juice followed by heat-treatment at 45°C for 10 minutes (referred to as "pH 5.0/45°C process"), the amount of Fraction 1 protein left in SI shows more than a fivefold reduction compared to the pH 5.5/45°C process. More Faction 1 protein is pelleted at low pH value of green juice. The solubility of virus in S 1, however, remains unaffected.

Subsequent examples also demonstrate that while Fraction 1 protein is pelleted at this pH range, the majority of Fraction 2 proteins remains in the supernatant. A conventional method of isolating soluble plant proteins adjusts the pH of green juice within the range of 5.3-6.0, which directs Fraction 1 protein to the supernatant after the centrifugation. The pH adjustment of green juice to a value below 5.2 followed by moderate heating in the instant procedure thus allows the separation of Fraction 1 and Fraction 2 protein upon the centrifugation of green juice. Eliminating the abundant Fraction 1 protein from the soluble portion simplifies the subsequent isolation and purification of Fraction 2 proteins. An ultrafiltration method can now be successfully applied to the purification of Fraction 2 proteins. This is an appreciable advantage over the prior art, where Fraction 1 protein is preferably retained in the soluble portion until the final crystallization or precipitation. Ultrafiltration in the presence of a large amount of Fraction 1 protein and other host materials is not efficient.

EXAMPLE 2 Distribution of Virus From Green Juice At Different pH Values Nicotiana tabacum (KY8959) grown in a greenhouse was inoculated with a TMV derivative (coat protein loop fusion), TMV291, seven weeks post seed germination. Plants were harvested two and half weeks post inoculation after systemic spread of the virus. Leaf and stalk tissue (150 g) was macerated in a 1 -liter Waring blender for two minutes at the high setting with 0.04% Na₂S₂O₅ (150 ml). The macerated material was strained through four layers of cheesecloth to remove fibrous material. The remaining green juice was adjusted to the pHs of 5.0, 4.8, 4.6, 4.4, 4.2, and 4.0 with H₃PO . Green juice aliquots of 30 ml were removed at each pH for further processing. All pH adjusted green juice samples were heat- treated at 45°C for 15 minutes in a water bath and then cooled to 15°C. Samples were centrifuged in a JS-13.1 rotor at 10,000 RPM for 15 minutes resulting in two fractions, supernatant (SI) and pellet (PI) (see Figure 1). Pellets were resuspended in 15 ml of 50 mM phosphate buffer, pH 7.2 and centrifuged in a JS-13.1 rotor at 10,000 RPM for 15 minutes resulting in two fractions, supernatant (S2) and pellet (P2), see Figure 1. Virus was recovered from both supernatant fractions by PEG-precipitation (8,000 MW PEG) as described by Gooding, supra and quantified by spectrophotometric analysis at 260 nm.

Table 2. Distribution of Virus in SI and S2 at Different Green Juice pHs

Results: This example examines the relative distribution of virus in supernatant, SI and S2, during the first and second centrifugation, respectively. S 1 is obtained after pH adjustment of green juice, from 5.0 to 4.0, followed by heat treatment and centrifugation. The pellet (PI) is resuspended in a buffer (pH 7.2) and subsequently subjected to a second centrifugation, which produces supernatant (S2). The amount of virus recovered from SI and S2 portion is similar at pH 5.0 of green juice in Table 2. Upon lowering the pH, however, virus gradually migrates from the supernatant portion (SI) to the pellet portion (PI) and reappears in S2. At pH 4.0 in Table 2, the amount of virus isolated from S2 portion is more than 100-fold higher than in the S 1 portion. The pH of green juice and the pH of the resuspension buffer are shown to have a great effect on the relative distribution of virus in the supernatant or pellet during centrifugation. At a low pH, e.g. pH 4.0/45°C process and pH 7.2 suspension buffer, the virus can be quantitatively recovered from the S2 portion alone. This process concentrates the virus into one fraction. This results in a fraction that can be ultrafiltered thereby significantly reducing the process volume and overall efficiency of virus purification. Adjusting the value of the green juice and suspension buffer offers a method for controlling the distribution of virus and thus facilitates the isolation of virus with large recovery yields.

EXAMPLE 3

Small-Scale Isolation of Virus from S2 Using the pH 4.2/45°C process A tobacco plant of variety MD609 was inoculated with TMV 811. Eleven weeks after sowing, the plant was harvested. Leaf and stalk tissue (250 g) were combined with 0.04% sodium metabisulfite solution (250 ml) in a 1 -liter Waring blender. The plant tissue was ground on high speed for a period of two minutes. The resulting homogenate was pressed through four layers of cheesecloth and the pressed fiber discarded. The volume of juice collected was 408 ml and its pH was 5.4. With constant stirring, the pH was adjusted to 4.2 with dilute phosphoric acid.

A portion of the juice (285 ml) was heated to 45°C in a water bath and maintained at this temperature for 10 minutes. Without cooling, the juice was centrifuged at 10,000 x G for 15 minutes. The supernatant was decanted and discarded, and the pellet was resuspended in double distilled deionized water (142 ml). The pH of the resuspended pellet was adjusted to pH 8.0 with dilute sodium hydroxide.

The resuspended and pH-adjusted pellet was divided into eight aliquots (15 ml each). These aliquots were centrifuged at different RPMs in a JA-20 rotor in a Beckman J2-21 centrifuge. The second supernatants (S2) were decanted and analyzed by SDS-PAGE. The virus was PEG-precipitated and isolated from the remaining supernatant (S2) portion according to the method of Gooding, supra. Supernatant clarity was also gauged visually. Table 3. Virus and Protein Yields of S2 under Different Centrifugation Conditions.

Results:

Example 2 demonstrates that a low pH of green juice and a neutral pH of suspension buffer directs most of virus into the soluble portion of the second centrifugation (S2). Example 3 further tests the optimal condition for the second centrifugation. If the target species is a virus, one prefers that the supernatant S2 contains as little protein as possible. Such a condition can be generally achieved with a high speed centrifugation for a long time interval, as shown in Aliquot 1 in Table 3. Such a condition, although effective, confers a larger cost and a longer process. An optimal condition provides a lower RPM rate for a shorter period of time without greatly compromising the yield and purity is desirable.

Although Aliquots 2-5 operate at a much lower centrifugation speed and for a shorter period, the exclusion of protein is, however, poor, as evidenced by a larger soluble protein concentration and a cloudy solution (an indication of large protein content). Aliquots 6-8 leave much protein out of supernatant (an almost clear solution), the amount of virus recovered in the S2 portion is comparable to that of Aliquot 1, but confers only moderate centrifugation speed and shorter time interval comparing to aliquot 1.

Although it can be seen from the instant example that there is no danger of over centrifuging (Aliquot 1), for a cost-effective virus purification process, centrifugation at a moderate speed and reasonable time interval, sufficient to eliminate the interfering proteins, is preferred. Those skilled in the art can readily determine the optimal condition of centrifugation that is suitable for isolation of virus of interest. EXAMPLE 4

Effect of Host Components and Suspension Volume on Virus Recovery from S2 Using the pH 4.2/45°C Process

Nicotiana tabacwn MD609 grown in a greenhouse was inoculated with a TMV derivative (coat protein leaky-stop), TMV811, six weeks post seed germination. Plants were harvested five weeks post inoculation after systemic spread of the virus. Leaf and stalk tissue

(150 g) was macerated in a 1 -liter Waring blender for two minutes at the high setting with

0.04% Na S O₅ (150 ml). The macerated material was strained through four layers of cheesecloth to remove fibrous material. The remaining green juice was adjusted to a pH of 4.2 with H₃PO . The pH-adjusted green juice was heated to 45°C under hot tap water and incubated for 10 minutes in a 45°C water bath. The heat-treated green juice was separated into 30 ml aliquots and then centrifuged in a JS-13.1 rotor at 10,000 RPM for 15 minutes. The pelleted material was adjusted to either 10 or 20% of the starting 30 ml volume by the addition of supernatant and then further adjusted to 1/4, 1/2 or 1 volume of the starting 30 ml volume by the addition of deionized H₂O. The average pellet volume from 30 ml of green juice was 1.7 ml.

All pellets were completely resuspended in the added supernatant and deionized FLO and then adjusted to a pH of 7.5-7.7 by the addition of NaOH. The resuspended samples were centrifuged in a JS 13.1 rotor at 10,000 RPM for 15 minutes. Virus was recovered from the supernatants by PEG-precipitation (8,000 MW PEG) as described by Gooding, supra.

Table 4. Virus Yield under Different Resuspension Volume.

Results:

When pellets are obtained from centrifugation, they are frequently contaminated with residual supernatant, which may or may not affect the subsequent recovery of the target species. In addition, the resuspension volume may also exert an effect on the recovery of target species. This example is designed to test the virus recovery under the condition where a defined volume of supernatant is added back to the pellet and the resuspension volume is systematically varied in order to assess its effect on virus recovery.

Table 4 demonstrates the inverse relationship of resuspension volume to virus yield. When resuspension volume increases from V* to Vι and Vz to 1 equivalent of the starting volume (30 ml), the recovery of virus is increased (compare 1 through 3 and 4 through 6). Thus, as the percentage of pellet volume increases, the resuspension volume should also increase to maximize the recovery of virus. For the effect of residual supernatant, the yield of virus recovery is higher when less supernatant is added back to the pellet (compare 1 and 4, 2 and 5, 3 and 6). Host component(s) in the supernatant may affect the ability to resuspend dissociate virions from the pellet. Thus, a smaller pellet volume with less residual supernatnant after centrifugation is desirable. In summary, factors such as the resuspension volume and dryness of the pellet may be optimized to maximize the yield and purity of target species.

EXAMPLE 5 Effect of Feed Rate on Large Scale Virus Isolation Using pH 5.0/47°C Process

Field grown tobacco of variety KY8959 was inoculated with TMV 291 and harvested ten weeks after setting. The plant tissue (8,093 lbs.) was ground in a Reitz^® disintegrator and the fiber removed using a screw press. Water was added to the disintegrator at the rate of 120 gallons per ton of tobacco. The juice from the press was collected in a stirred tank where the pH was adjusted to 5.0 with phosphoric acid. The pH-adjusted juice was pumped through a heat exchanger in a continuous manner so that the temperature of the juice reached 47°C. The heated juice was then pumped through holding tubes, which ensures that this temperature was maintained for at least ten minutes.

The treated juice was then fed to a Westfalia^® SAMR 15037 disk stack-type centrifuge at a feed rate of five gallons per minute to twenty gallons per minute. Samples of the concentrate were taken at each feed rate and analyzed for virus concentration. Table 5. Virus Yield Versus Feed Rate.

Results:

The virus recovery yield was examined using different feed rates. Table 5 shows that virus recovery was lowered with a low feed rate of green juice to the centrifuge. Since the feed rate is inversely proportional to the retention time of green juice in the centrifuge, these data demonstrate virus is lost if it is subjected to too much centrifugation (low feed rate). Thus, feed rate may also be optimized to maximize the yield and purity of target species in a large scale isolation and purification.

EXAMPLE 6 Isolation of Recombinant Protein α-Trichosanthin Using the pH 5.0/45°C Process

Nicotiana benthamiana grown in a greenhouse was inoculated with TMV containing the gene coding for α-trichosanthin. Plants were harvested ten days post inoculation after systemic spread of the virus. Leaf and stalk tissue (150 g) was macerated in a 1 -liter Waring blender for two minutes at the high setting with 0.04% Na₂S O₅(150 ml). The macerated material was strained through four layers of cheesecloth to remove fibrous material. The remaining green juice was adjusted to pH 5.0 with HC1. The pH adjusted green juice was heat-treated at 45°C for ten minutes in a water bath and then cooled to 28°C. Heat treated juice was centrifuged in a KA-12 rotor (Kompspin, Sunnyvale, CA) at 10,000 RPM (15,600 x G) for 15 minutes. The supernatant (SI) (50 ml aliquots) was subjected to ultrafiltration using 100 and 10 kD MWCO regenerated cellulose membranes in an Amicon^® stirred-cell at 50 PSI. The 100 kD permeate fraction was then concentrated via filtration through a 10 kD membrane and diafiltered three times. The α-trichosanthin is collected from the 10 kD concentrate. The 10 kD permeate contains the sugars, alkaloids, flavors, vitamins and peptides below 10 kD MW. The relative quantity of α-trichosanthin in green juice, supernatant, 100 kD and 10 kD concentrates and the 100 to 10 kD fraction was determined by Western analysis using α-trichosanthin antibody.

Table 6. α-trichosanthin Yield in a pH 5.0/45°C process.

Results:

This example demonstrates the ability to extract and purify a soluble F2 protein, α- trichosanthin, using the pH 5.0/45°C process and ultrafiltration. The α-trichosanthin was quantitatively retained in the supernatant (SI) fraction, relative to amounts present in the green juice, (based upon Western analysis). In addition, α-trichosanthin present in the S 1 was purified 6-fold relative to green juice (based on Bradford protein and Western analysis). α-Trichosanthin present in the S 1 fraction was quantitatively retained and concentrated 4-fold, by ultrafiltration using a 10 kD MWCO membrane (50 ml of SI was concentrated to 13.5 ml and 96% of the α-trichosanthin was present in the 10 kD concentrate, based upon Western analysis). α-Trichosanthin was also purified away from large molecular weight proteins and viruses via ultrafiltration with a 100 kD MWCO membrane. The 100 kD concentrate fraction was diafiltered three times to allow recovery of additional α-Trichosanthin. After 100 kD concentration and diafiltration, only 40.8% of the α-Trichosanthin remained in the 100 kD concentrate, indicating that 59.2% of the α-Trichosanthin would be present in the 100 kD permeate fraction. The 100 kD permeate fraction was concentrated using a lOkD MWCO membrane. The resultant 10 kD concentrate (derived from the 100 kD permeate), contained 34% of α-Trichosanthin, relative to the amount of αTrichosanthin present in 50 ml of the starting SI fraction. The α-trichosanthin present in the 100-lOkD fraction was determined to be purified 8-fold relative to Green juice (based on Bradford protein and Western analysis) and concentrated 12.5-fold (50 ml of SI was concentrated to 4.0 ml of 100-10 kD fraction).

EXAMPLE 7

Isolation of Secretory IgA Antibody From Transgenic Plants Using the pH5.0/47°C Process

Leaf and stalk tissue (50 g fresh weight) of greenhouse grown transgenic tobacco, which expresses four secretory IgA (SlgA) protein components, was macerated in a Virtis blender for two minutes at the high setting with 0.04% Na₂S₂O₅ (75 ml). The macerated material was strained through four layers of cheesecloth to remove fibrous material. The remaining green juice was adjusted to pH 5.0 with H₃PO₄. The pH-adjusted green juice was heat-treated at 47°C for ten minutes in a water bath and then cooled to 28°C. Heat treated juice was centrifuged in a JA-13.1 rotor at 3,000 RPM for three minutes. The supernatant fraction was subjected to ultrafiltration using 10 kD MWCO, regenerated cellulose membrane (Amicon^®, Centriprep^®). The relative quantity of SlgA in green juice, supernatant and the 10 kD concentrate was determined by Western analysis using an antibody reactive with the heavy chain.

Table 7. Secretory IgA and Other Proteins Recovered from the pH 5.0/47°C Process.

Results:

Secretory IgA antibody, recombinantly produced in transgenic plants, was successfully recovered in this example. Following pH adjustment and heat treatment, centrifugation reduced the total protein in the supernatant by 85%. The SlgA in the supernatant was recovered and ultrafiltered resulting in a 12-fold concentration of the total protein and the SlgA components.

EXAMPLE 8 Small Scale Isolation of Virus Using pH5.0/45°C Process and Ultrafiltration Field-grown tobacco of variety MD609 and infected with TMV 261 was harvested and frozen at -20°C until use. The frozen tissue was ground in four batches in a 4-liter Waring blender. In each batch, plant tissue (1500 g) was ground for three minutes at high speed in 0.04% sodium metabisulfite solution (1500 ml). The homogenates were strained through four layers of cheesecloth and the juices combined to give a volume of approximately 10 liters.

The pH of the juice was adjusted from a starting value of 5.8 to 5.0 using concentrated phosphoric acid (H₃PO₄). The juice was then heated to 45°C using a stainless steel coil heated by hot tap water. After maintaining the juice at 45°C for ten minutes, it was cooled to 25°C using the coil with chilled water. The heat-treated juice was centrifuged at 12,000 x G for five minutes and the resulting supernatant was decanted through Miracloth^®. This supernatant was processed using a one square foot, 100 kD MWCO regenerated cellulose, spiral ultrafiltration membrane. With an inlet pressure of 50 psi and a recirculation rate of five liters per minute, the supernatant was concentrated to about 5% of the starting volume. The final concentrate was drained from the ultrafiltration apparatus and the system was rinsed with a small volume of water. Samples of the starting supernatant, the final concentrate, the water rinse, and the combined permeate were assayed for protein by Bradford analysis. They were also PEG-precipitated according to the method of Gooding, supra, to isolate any virus present. Virus concentrations were determined spectrophotometrically .

Table 8. Protein Concentration and Virus Yield in Supernatant (S 1) and Subsequent Ultrafiltration.

Results:

In this example, a small scale virus isolation was successfully carried out. Green juice was pH adjusted to 5.0 and heat-treated followed by centrifugation. The supernatant containing virus (1.94 g) was passed through a 100 kD MWCO membrane. The virus (1.64 g) was quantitatively recovered from the concentrate. Proteins of smaller size were collected in the permeate. Only a small amount of virus is lost by ultrafiltration using a 100 kD membrane.

EXAMPLE 9 Large Scale Virus Isolation Using pH4.0/47°C Process

Field grown tobacco of variety KY8959 was inoculated with TMV 291 and harvested ten weeks after setting. The plant tissue (8,382 lbs.) was ground in a Reitz® disintegrator and the fiber removed using a screw press. Water was added to the disintegrator at the rate of 120 gallons per ton of tobacco. The juice from the press was collected in a stirred tank where the pH was adjusted to 4.0 with phosphoric acid. The pH adjusted juice was pumped through a heat exchanger in a continuous manner so that the temperature of the juice reached 47°C. The heated juice was then pumped through holding tubes which ensures that this temperature was maintained for at least ten minutes.

The treated juice was then fed to a Westfalia SAMR 15037 disk stack type centrifuge at a feed rate of 10 gallons per minute. A total of 1120 gallons of supernatant and 200 gallons of pellet were produced during centrifugation. A volume of 380 gallons of water was added to the pellet, and the resuspended pellet pH was adjusted to 7.12 by the addition of KOH. The pH adjusted, resuspended pellet was then fed to a Westfalia SAMR 15037 disk stack type centrifuge at a feed rate of 5 gallons per minute resulting in the recovery of 435 gallons of supernatant (S2). Supernatant (435 gallons) was concentrated to 24.8 gallons by ultrafiltration through 1,000 square feet of 100 kD MWCO, cellulose acetate, spiral membrane (SETEC, Livermore, CA). After removal of the concentrate, the membranes were washed with 31.5 gallons of water. Virus (158 g) was purified from the 100 kD MWCO concentrate and then further concentrated and washed by PEG -precipitation (8,000 MW PEG) as described by Gooding, supra. This quantity of virus recovered is two orders of magnitude greater than ever isolated before.

This example demonstrates an efficient large scale virus isolation using the pH4.0/47°C process. Example 2, supra, demonstrates that the pH 4.0/47°C process allows the concentration of virus in the supernatant, S2 on a small scale. The virus can be further concentrated using ultrafiltration by passing the supernatant (S2) through a 100 kD MWCO membrane. The virus particles can be recovered at high yield as shown in this example.

EXAMPLE 10 Large Scale Virus and Fraction 2 Protein Isolation Using pH5.0/47°C Process

Field-grown tobacco of variety KY8959 was inoculated with TMV 291 and harvested ten weeks after setting. The plant tissue (8,093 lbs.) was ground in a Reitz® disintegrator and the fiber removed using a screw press. Water was added to the disintegrator at the rate of 120 gallons per ton of tobacco. The juice from the press was collected in a stirred tank where the pH was adjusted to 5.0 with phosphoric acid. The pH-adjusted juice was pumped through a heat exchanger in a continuous manner so that the temperature of the juice reached 47°C. The heated juice was then pumped through holding tubes which ensures that this temperature was maintained for at least 10 minutes. The treated juice was then fed to a Westfalia® SAMR 15037 disk stack type centrifuge at a feed rate of ten gallons per minute. A total of 760 gallons of the 990 gallons of supernatant produced during centrifugation was concentrated to 32 gallons by ultrafiltration through 1,000 square feet of 100 kD MWCO, cellulose acetate, spiral membrane. Virus (213 g) was purified from the 100 kD concentrate fraction by PEG (8,000 MW) precipitation as described by Gooding, supra. The soluble Fraction 2 proteins (<100 kD) located in the 100-kD filtration permeate, were concentrated by ultrafiltration through 40 square feet of 10 kD MWCO, regenerated cellulose, spiral membrane. A total of 60 gallons of 100 kD permeate was concentrated to 3.5 gallons, yielding 1.69 g of soluble Fraction 2 proteins.

This example successfully demonstrates that a large-scale process for isolating and purifying Fraction 2 proteins and virus using pH 5.0/47°C process. The first centrifugation produces a supernatant fraction that contains both virus and other soluble proteins. It is possible to use ultrafiltration to concentrate and separate the virus and soluble Fraction 2 proteins, where virus remains in the concentrate of a large MW MWCO membrane and Fraction 2 proteins in the permeate. Fraction 2 proteins can be further purified and concentrated by passing through a smaller MW MWCO membrane, where different sizes of Fraction 2 proteins can be individually obtained. Fraction 2 protein and virus can be recovered with high yields using the instant method at a large scale.

EXAMPLE 11

Physiochemical Properties of the Purified Virus Particles Produced bv the pH5.0/47°C or the pH4.0/47°C Process

Wild type tobacco mosaic virus (TMV204, sample 960808) was extracted from field grown tobacco (variety KY8959, 11,884 lbs.) using the large-scale pH4.0/47°C process as described in Example 9. Recombinant TMV291 (sample 960829) was extracted from field grown tobacco (variety KY8959, 14,898 lbs.) using the pH5.0/47°C extraction procedure as described in Example 10. The virion, after PEG precipitation, were subjected to various analyses to ascertain biochemical and purity profiles. Table 9. Virion Purity Profiles after Large Scale Isolation using pH4.0/47°C and pH5.0/47°C Processes.

* Matrix Assisted Laser Desorption Ionization-Time of Flight, Mass Spectrometry.

Table 10. Elemental Analysis of Virions after Large Scale Isolation Using pH4.0/47°C and pH5.0/47°C Processes.

Table 11. Amino Acid Analysis of Virions after Large Scale Isolation Using pH4.0/47°C and pH5.0/47°C Processes.

*** Quantity of sample analyzed, wet weight (960808: 537.47 mg, 960829: 554.28 mg).

Results:

The analysis of PEG purified virion preparations produced via the large-scale pH5.0/47°C and pH4.0/47°C processes, indicate a high degree of purity and no detectable TMV coat protein degradation. Absorbance ratios of 1.20 at 260/280 nm (Table 9) are indicative of highly purified TMV. In addition, the MALDI-TOF mass of both virus preparations (Table 9) are within experimental ranges for the predicted coat protein molecular weight. Both virus preparations contained low levels of lipids, nicotine and endotoxin, again demonstrating the utility of these methods in the isolation and purification of virions and virus fusion coat protein. The elemental analyses of the virus extracts (Table 10) are indicative of highly purified proteins as determined by the relative ratios of the various elements. The amino acid profiles of the virus samples (Table 11) reflect the relative abundance of each predicated amino acid and also reflects the predicted differences in amino acids between the two test samples.

Both virus samples were shown to be infective when passed onto host plants, indicating that the described methods resulted in the recovery of biologically active virions. RT-PCR analysis of the virus extracts produced the predicated nucleic acid fragments, indicative of intact RNA genomes.

As demonstrated by the examples described above, one aspect of the present invention is the isolation of one or more bioactive species from RuBisCo present in photosynthetic plants. By "bioactive species" is meant any protein, peptide, nucleic acid, vitamin, membrane, cell wall, sugar, alkaloid, flavor, fusion peptide, or virus that can be isolated from the biomass of a plant. Such a bioactive species may include, for example, but is not limited to, a virus, such as a tobacco mosaic virus (TMV) or a TMV derivative, a recombinant virus, or a viral vector capable of carrying a heterologous nucleic acid sequence. The virus may contain a gene that encodes for a protein of interest. The virus and/or the protein of interest can be isolated using the methods of the present invention. The bioactive species may also be a soluble protein or peptide, a non-native protein or peptide, a recombinant protein or peptide, or a fusion protein or fusion peptide. Examples of such proteins or peptides include EL-1, IL-2, LL-3, IL-4, 11-5, EL-6, IL-7, 11-8, LL-9, LL-10, LL- 11, LL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIH, Factor LX, tPA, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, human growth hormone, and an antimicrobial peptide or protein, such as protegrins, magainins, cecropins, melittins, indolicidins, defensins, β-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins. To facilitate the isolation of such bioactive species from RuBisCo, the present invention includes methods for reducing or minimizing the presence of RuBisCo in photosynthetic plants, and to cut the plant material from the plants when RuBisCo is substantially at a minimum. The inventors have discovered that, because the quantity of RuBisCo drops during dark periods, extending the dark periods to which the plants are exposed, and cutting plant material during dark periods, the quantity of RuBisCo in the plant material can be reduced from the maximum quantity of RuBisCo that is present in the plants during the light portion of a light/dark cycle. This is particularly advantageous since the leaves with the highest titer of bioactive species are the leaves that are the most photosynthetically active. A light/dark cycle can be any period of hours that are desirable for crop yield and production. In the field, the light/dark cycle is 24 hours. However, the cycle may be lengthened by covering the plants with an opaque material to extend the time during which the plant is deprived of sunlight. For plants subjected to artificial light, the lights may be left on for an extended period or left off for an extended period as desired.

The quantity of RuBisCo in the chloroplasts of photosynthetic plants increases to a maximum during the light portion of a light/dark cycle to which the plants are exposed. The quantity of RuBisCo decreases during the dark portion of the light/dark cycle. In order to reduce or minimize the amount of RuBisCo that needs to be eliminated as waste during the isolation and purification process, the plants are harvested when the quantity of RuBisCo is reduced from the maximum quantity that occurs during the light portion of the light/dark cycle. Preferably, the plants are harvested when the quantity of RuBisCo is substantially at a minimum. By adjusting the time of harvesting, the efficiency of the isolation and purification process can be increased, and the costs reduced, by reducing the amount of waste material (RuBisCo) that needs to be eliminated during the process.

It may be desirable to extend the time during which a plant is deprived of light to reduce the amount of RuBisCo in the plant prior to harvesting the biomass. In this manner, the isolation and purification of bioactive species may be made more efficient. To determine the optimal reduction in RuBisCo might be the greatest reduction possible. The lowest amount of RuBisCo would provide the highest efficiency of purification of bioactive species available by reduction of RuBisCo. However, optimal reduction of RuBisCo might represent a balance between purification efficiency and other factors.

To determine the lowest content of RuBisCo, one can determine the concentration of RuBisCo during several sample times during darkness. For example, the plants are subjected to darkness and a sample of biomass is harvested and homogenized at time 0 (zero). A sample of the juice is subjected to protein electrophoresis together with a RuBisCo standard. The spot on the gel representing the RuBisCo in the plant sample is subjected to a densitometry reading. Later samples are treated in the same manner as the first. With each sample, the density reading will be reduced until some minimum density is reached. One can conclude from the minimum density that the minimum amount of RuBisCo has been reached. If the optimum reduction of RuBisCo is reached during a dark period that is greater than the dark period natural for that location and that time of year, then the dark period can be increased by maintaining the plants in darkness by artificial means. In one embodiment of the present invention, the plants are maintained in an environment substantially without light for a selected period during which the quantity of RuBisCo in the plant is reduced, the selected period occurring during daylight. In this manner, the photosynthetic plants may be subjected to extended periods of darkness in order to reduce or minimize the presence of RuBisCo in the plants. Plant material is then cut from the plant, and the bioactive species is isolated from the RuBisCo in the plant material.

In a preferred embodiment of the present invention, the tobacco plants are maintained in an environment substantially without light for a selected period during daylight. The length and duration of the selected period are selected to reduce or minimize the presence of RuBisCo in the plants, as could be readily determined by one skilled in the art. The step of maintaining the plants in an environment substantially without light may be carried out in a greenhouse, or outside, such as in a tobacco field. This can be accomplished, for example, by placing a cover over the plants that shields them from natural or artificial light. This can be carried out in either a greenhouse or outside. As would be readily apparent to one skilled in the art, other mechanisms can be used to maintain the plants in an environment substantially without light. For example, depending upon the greenhouse design, shades or panels can be used to substantially eliminate natural light from entering the greenhouse, or artificial light in the greenhouse can be turned on and off at predetermined times.

To further improve the efficiency of isolating bioactive species from RuBisCo, the plant material is preferably cut from the plant during a cutting period. As used herein,

"cutting period" refers to a period of time that occurs during a light/dark cycle. During the cutting period, a quantity of RuBisCo in the photosynthetic plant is reduced from the maximum quantity that occurs in the plant during the light portion of the light/dark cycle. In a particularly preferred embodiment, the quantity of RuBisCo is substantially at a minimum during the cutting period. Harvesting the plants during such a cutting period minimizes the presence of the undesirable RuBisCo protein that will be eliminated as waste during the subsequent isolation and purification. Minimizing the presence of RuBisCo is particularly advantageous because this protein is sticky, thereby making it more difficult to remove during the subsequent isolation and purification. The cutting period preferably occurs during a dark period of the light/dark cycle, more preferably after sunset and prior to sunrise. Cutting of the plant material from the plant during the cutting period when the RuBisCo in the plant is substantially at a minimum can be carried out in a greenhouse or outside, such as in a field. The plant material cut from the plant will comprise leaf tissue, as well as stalk tissue.

In a particularly preferred embodiment of the present invention, the harvesting or cutting operation is optimized in order to minimize damage to the cells and biomass containing the bioactive species of interest. The height above the ground at which the plant is cut is selected to allow the plant to regenerate for a second growth and harvest, and also to obtain that portion of the plant that is most actively producing protein, when desirable. Preferably, plant material is cut from the plant at a height in the range of from about one foot to about three feet (above the ground or soil in which the plants are growing). In a particularly preferred embodiment, the plant material is cut from the plant at a height of about two feet. In this manner, the most photosynthetically active leaves, with the highest levels of protein and virus, are being removed from the plant. In a particularly preferred embodiment, the photosynthetic plant is a tobacco plant. However, it should be understood by one skilled in the art that the present invention is not limited to tobacco plants. During the harvesting or cutting operation, the plant is cut to remove or separate plant material from the plant. The plant material is also preferably cut or chopped into pieces having a particular chop size. As used herein, "chop size" means the size of the biomass that is presented to a disintegrator or homogenizer. The biomass is cut from the plant and if necessary, chopped to a smaller size which will not foul the disintegrator. Preferred chop size is about 4 inches (about 4" x 4" or about 16 square inches). However, a chop size of about 1 or 2 inches reduces the loss of material to about 1%. Chop size is determined by balancing the need to avoid stalling the disintegrator with the desire to minimize the amount of cell damage. Cell damage results in release of proteolytic enzymes, interstitial and intracellular fluid. The net effects include reduced yield and increased microbial activity. In accordance with the present invention, the size of the chop is optimized to obtain a high titer of the bioactive species of interest. The size of the chop is also optimized to minimize compaction of the chopped pieces so that they stay aerated to prevent any anaerobic activity prior to the subsequent isolation and purification process. In a preferred embodiment, the plant material is cut into pieces that have a mean chop size in the range of about two to about eight square inches. In a particularly preferred embodiment, the chop size is approximately four square inches, preferably approximately 2"x2". It has been unexpectedly discovered by the inventors that a mean chop size in the range of about two to about eight square inches also minimizes loss of liquid containing the bioactive species during the isolation and purification process. Cutting the plant material into such a preferred chop size can take place, for example, in a greenhouse using manual or automated cutting techniques known to one skilled in the art. Alternatively, cutting the plant material into such a preferred chop size can take place in a field using a cutting or harvesting machine. To further improve the efficiency of isolating bioactive species from RuBisCo, the present invention optimizes the harvesting process in order to harvest biomass with the highest amount of titer of the bioactive species of interest. To do so, the present invention provides for inoculating the plant with, for example, a virus, when a growth rate of the plant is substantially at a maximum. By introducing the virus into rapidly growing tissue during the fast growth period of the growing cycle of the plant, the quantity of virus in the harvested biomass can be maximized. In a preferred embodiment, the inoculating step is carried out after growth by the plant of primary leaves. In another embodiment, the inoculating step is carried out when the plant is approximately 12 to 18 inches in height.

In another embodiment of the present invention it is desirable to harvest biomass having the highest titre of bioactive species of interest so that yield of protein is increased and the number of harvests per season may be increased. The present invention provides for cutting the portion of the plant that is more actively photosynthesizing. The remaining plant may be allowed to regrow so that another harvest may be achieved from the same crop. For example, a plant in the field with 48 inches of height may be cut to about 24 inches to obtain the more active biomass. A less active biomass or the biomass that is senescent is left behind to regenerate. By this method, a second or even third harvest may be obtained in a single season. However, by allowing a plant to achieve only 24 to 30 inches in height and cutting as low as 6 inches or closer to the ground, an increased number of harvests may be obtained from regrowth. The new growth is more actively photosynthesizing and producing protein than the old growth. Also, a shorter plant will grow faster than a taller plant, because it will apply less energy to transporting water and nutrients throughout the plant, or to maintaining older leaves.

The advantages to this method include but may not be limited to increased titre of bioactive species, increased number of harvests from a single plant in a season, avoidance of flowering which diverts energy from a plant, senescence is reduced, easier cultivation, easier to weed between the plants, greater air movement between the plants helps to prevent mold, and young plants are more resistant to disease. Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims and their equivalents.

Claims

WE CLAIM:

1. A method of processing plants to obtain a plant product suitable for isolation of one or more bioactive species from ribulose 1,5-diphosphate carboxylase present in the plant, comprising: cutting plant material from a plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the lrght/dark cycle.

2. The method of claim 1, wherein the cutting period occurs during a dark period of the light/dark cycle.

3. The method of claim 1 , wherein the cutting period occurs after sunset and prior to sunrise.

4. The method of claim 1 , wherein the cutting step is carried out in a greenhouse.

5. The method of claim 1, wherein the cutting step is carried out outside.

6. The method of claim 1, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about one foot to about three feet.

7. The method of claim 1, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about eight square inches.

8. The method of claim 6, wherein the cutting step is carried out to cut the plant material from the plant at a height of about two feet.

9. The method of claim 1, wherein the plant material comprises leaf tissue.

10. The method of claim 1, further comprising prior to the cutting step: inoculating the plant with a virus.

11. The method of claim 10, wherein the virus is a tobacco mosaic virus (TMV) or a TMV derivative.

12. The method of claim 10, wherein the inoculating step is carried out after growth by the plant of primary leaves.

13. The method of claim 10, wherein the inoculating step is carried out when the plant is approximately 12-18 inches in height.

14. The method of claim 10, wherein the inoculating step is carried out when a growth rate of the plant is substantially at a maximum.

15. The method of claim 10, further comprising after the cutting step: isolating the virus from the plant material.

16. The method of claim 10, wherein the virus contains a gene encoding for a protein of interest.

17. The method of claim 16, further comprising after the cutting step: isolating the protein of interest from the plant material.

18. The method of claim 17, further comprising after the cutting step: isolating the virus from the plant material.

19. A method for obtaining a virus of interest, comprising: inoculating a plant with the virus of interest; cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle; and isolating the virus of interest from the plant material.

20. The method of claim 19, wherein the cutting period occurs during a dark period of the light/dark cycle.

21. The method of claim 19, wherein the cutting period occurs after sunset and prior to sunrise.

22. The method of claim 19, wherein the cutting step is carried out in a greenhouse.

23. The method of claim 19, wherein the cutting step is carried out outside.

24. The method of claim 19, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about one foot to about three feet.

25. The method of claim 19, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about eight square inches.

26. The method of claim 24, wherein the cutting step is carried out to cut the plant material from the plant at a height of about two feet.

27. The method of claim 19, wherein the plant material comprises leaf tissue.

28. The method of claim 19, wherein the virus of interest is a tobacco mosaic virus (TMV) or a TMV derivative.

29. The method of claim 19, wherein the inoculating step is carried out after growth by the plant of primary leaves.

30. The method of claim 19, wherein the inoculating step is carried out when the plant is approximately 12-18 inches in height.

31. The method of claim 19, wherein the inoculating step is carried out when a growth rate of the plant is substantially at a maximum.

32. The method of claim 19, wherein the virus of interest contains a gene encoding for a protein of interest.

33. The method of claim 32, further comprising after the cutting step: isolating the protein of interest from the plant material.

34. A virus obtained according to the method of claim 19.

35. A method of processing plants, comprising: maintaining a plant in an environment substantially without light for a selected period during which a quantity of ribulose 1,5-diphosphate carboxylase is reduced, wherein the selected period occurs during daylight; cutting plant material from the plant; and isolating a bioactive species from the ribulose 1,5-diphosphate carboxylase in the plant material.

36. The method of claim 35, wherein the maintaining step is carried out in a greenhouse.

37. The method of claim 35, wherein the maintaining step is carried out outside.

38. The method of claim 35, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is substantially at a minimum.

39. The method of claim 35, wherein the cutting step is carried out to cut the tobacco plant material from the plant at a height in the range of from about one foot to about three feet.

40. The method of claim 35, wherein the bioactive species is a virus.

41. The method of claim 35, further comprising prior to the cutting step: inoculating the plant with a virus.

42. The method of claim 41, wherein the virus is a tobacco mosaic virus (TMV) or a TMV derivative.

43. The method of claim 41, wherein the isolating step is carried out to isolate the virus.

44. The method of claim 43, wherein the virus contains a gene encoding for a protein of interest.

45. The method of claim 44, further comprising: isolating the protein of interest.

46. The method of claim 35, further comprising prior to the cutting step: inoculating the plant with a virus that contains a gene encoding for a protein of interest.

47. The method of claim 46, wherein the isolating step is carried out to isolate the protein of interest.

48. The method of claim 46, wherein the virus is a tobacco mosaic virus (TMV) or a TMV derivative.

49. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of:

(a) cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle;

(b) homogenizing the plant material to produce a green juice homogenate;

(d) heating the green juice homogenate to a minimum temperature of about 45°C;

(e) centrifuging the green juice homogenate to produce a supernatant; and

(f) purifying the protein or peptide from the supernatant.

50. A method for obtaining a fusion peptide or fusion protein from a plant comprising the sequential steps of:

(a) cutting plant material from the tobacco plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle;

(b) homogenizing the plant material 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°C;

(e) centrifuging the green juice homogenate to produce a pellet;

(f) resuspending the pellet in a liquid solution;

(i) purifying the fusion protein or fusion peptide.

1. The method of claim 49 wherein the pH of the green juice homogenate is adjusted to between about 4.0 and 5.2.

52. The method of claim 49 wherein the pH of the green juice homogenate is adjusted to about 5.0.

53. The method of claim 49 wherein the green juice homogenate is heated to a temperature of between about 45° and 50°C.

54. The method of claim 49 wherein the supernatant produced in step (e) is further subjected to ultrafiltration.

55. The method of claim 54 further comprising the step of subjecting a permeate produced by the said ultrafiltration to a second ultrafiltration.

56. The method of claim 55 further comprising the step of purifying a concentrate resulting from the second ultrafiltration.

57. The method of claim 56 wherein said purifying is performed by chromatography, affinity-based method of purification, or salt precipitation.

58. The method of claim 49 wherein the soluble protein or peptide of interest is selected from the group consisting of IL-1, IL-2, IL-3, LL-4, 11-5, IL-6, IL-7, H-8, IL-9, IL-10, IL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VTJL Factor LX, tPA, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone.

59. The method of claim 49 wherein the soluble protein or peptide of interest is an antimicrobial peptide or protein and is selected from the group consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, β-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins.

60. The method of claim 50 wherein the purifying is performed by at least one method selected from the group consisting of chromatography, ultrafiltration, affinity-based method of purification, and salt precipitation.

61. The method of claim 50 wherein said fusion protein or fusion peptide comprises a peptide or protein selected from the group consisting of IL-1, IL-2, EL-3, LL-4, 11-5, IL-6, JLL- 7, D-8, IL-9, EL- 10, IL-11, IL-12, EPO, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VTLL Factor IX, tPA, hGH, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, and calcitonin.

62. The method of claim 50 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, β-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins.

63. A method according to claim 54 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.

64. A method according to claim 55 wherein said second ultrafiltration produces in a permeate containing molecules selected from the group consisting of sugars, polysaccharides, vitamins, alkaloids, flavor compounds and peptides.

65. The method of claim 10, wherein the virus is a recombinant virus.

66. The method of claim 10, wherein the virus is a viral vector capable of carrying a heterologous nucleic acid sequence.

67. The method of claim 19, wherein the virus of interest is a recombinant virus.

68. The method of claim 19, wherein the virus of interest is a viral vector capable of carrying a heterologous nucleic acid sequence.

69. The method of claim 49, wherein the soluble protein or peptide is recombinant protein or peptide.

70. The method of claim 49, wherein the soluble protein or peptide is a non-native protein or peptide.

71. The method of claim 1 , wherein the quantity of ribulose 1 ,5-diphosphate carboxylase is substantially at a minimum during the cutting period.

72. The method of claim 1 , wherein the plant is a tobacco plant.

73. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of:

(a) homogenizing the plant material to produce a green juice homogenate;

(b) simultaneously adjusting the pH of the green juice homogenate to less than or equal to about 5.2 and heating the green juice homogenate to a minimum temperature of about 45 °C;

(c) centrifuging the green juice homogenate to produce a supernatant; and

(d) purifying the protein or peptide from the supernatant.

74. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of:

(a) homogenizing the plant material to produce a green juice homogenate;

(b) heating the green juice homogenate to a minimum temperature of about 45°C;

(d) centrifuging the green juice homogenate to produce a supernatant; and

(e) purifying the protein or peptide from the supernatant.

75. The method of claim 73, further comprising before step (a): cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle.

76. The method of claim 74, further comprising before step (a): cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle.

77. The method of claim 1, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 4 inches x 4 inches.

78. The method of claim 1, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 2 inches x 2 inches.

79. the method of claim 1, wherein the cutting step is carried out to cut the plant material from the plant at a height of about six inches or less.

80. The method of claim 19, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 4 inches x 4 inches.

81. The method of claim 19, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 2 inches x 2 inches.

82. The method of claim 19, wherein the cutting step is carried out to cut the plant material from the plant at a height of about six inches or less.

83. The method of claim 35, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 4 inches x 4 inches.

84. The method of claim 35, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 2 inches x 2 inches.

85. The method of claim 35, wherein the cutting step is carried out to cut the plant material from the plant at a height of about six inches or less.

86. The method of claim 49, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about six inches to about three feet.

87. The method of claim 49, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about sixteen square inches.

88. The method of claim 50, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about six inches to about three feet.

89. The method of claim 50, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about sixteen square inches.

90. The method of claim 73, further comprising before step (a): cutting plant material from the plant at a height in the range of from about six inches to about three feet.

91. The method of claim 74, further comprising before step (a): cutting plant material from the plant at a height in the range of from about six inches to about three feet.

92. The method of claim 73, further comprising before step (a): cutting plant material from the plant into pieces that have a mean chop size in the range of from about two to about sixteen square inches.

93. The method of claim 74, further comprising before step (a): cutting plant material from the plant into pieces that have a mean chop size in the range of from about two to about sixteen square inches.

94. A method of increasing the number of harvests in a growing season, comprising: a) growing a plant to a desirable height; b) harvesting biomass from the plant; c) allowing the plant to generate new biomass; d) harvesting the new biomass; and e) repeating steps c) and d).

95. The method of claim 94, wherein the plant is grown to a height of no more than 4 feet.

96. The method of claim 94, wherein the plant is grown to a height of no more than 3 feet.

97. The method of claim 94, wherein the plant is grown to a height of no more than 2 feet.

98. The method of claim 94, wherein the plant is grown to a height of no more than 1 foot.

99. The method of claim 94, wherein the plant is grown to a height of no more than 6 inches.

100. A method of increasing the yield of biomass in a growing season, comprising: a) harvesting the younger biomass from a plant; b) allowing the plant to regenerate new biomass; c) harvesting the new biomass; and d) repeating steps b) and c).

AMENDED CLAIMS

[received by the International Bureau on 15 February 2001 (15.02.01).; original claims 1-100 replaced by new claims 1-52 (7 pages)]

1. A method of minimizing the presence of ribulose 1 ,5-diphosphate carboxylase to obtain a plant product suitable for isolation of one or more bioactive species, said method comprising: cutting plant materials from a plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle.

2. A method for obtaining a virus of interest, comprising: inoculating a plant with the virus of interest; cutting plant material from the plant, 'wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light dark cycle; and isolating the virus of interest from the plant material.

3. A method according to Claim 1 or 2, further comprising: maintaining a plant in an environment substantially without light for a selected period during which a quantity of ribulose 1,5-diphosphate carboxylase is reduced.

4. The method of Claim 1 or 2, wherein the cutting period is a period of a light/dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is substantially at a minimum.

5. The method of Claim 4, wherein the cutting period occurs during a dark period of the light dark cycle.

6. The method of Claim 1, wherein the cutting period occurs after sunset and prior to sunrise.

7. The method of any one of Claims 1-3, wherein the cutting step is carried out in a greenhouse.

8. The method of any one of Claims 1-3, wherein the cutting step is carried out outside.

9. The method of any one of Claims 1-3, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about one foot to about three feet.

10. The method of Claim 9, wherein the cutting step is carried out to cut the plant material from the plant at a height of about two feet.

11. The method of Claim 1 , wherein the cutting step is carried out to cut the plant material from the plant at a height of about six inches or less.

12. The method of any one of Claims 1-3, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about eight square inches.

13. The method of Claim 12, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 4 inches x 4 inches.

14. The method of Claim 12, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size of about 2 inches x 2 inches

15. The method of Claim 1 or 3, further comprising prior to the cutting step: inoculating the plant with a virus.

16. The method of Claim 2 or 15, wherein the virus is a recombinant virus.

17. The method of Claim 16, wherein the virus is a viral vector capable of carrying a heterologous nucleic acid sequence.

18. The method of Claim 16, wherein the virus contains a gene encoding for a protein of interest.

19. The method of Claim 18, further comprising after the cutting step: isolating the protein of interest from the plant material.

20. The method of Claim 2 or 15, wherein the virus is a tobacco mosaic virus (TMV) or a TMV derivative.

21. The method of Claim 15, wherein the inoculating step is carried out after growth of primary leaves of the plant.

22. The method of Claim 15, wherein the inoculating step is carried out when the plant is approximately 12-18 inches in height.

23. The method of Claim 15, wherein the inoculating step is carried out when a growth rate of the plant is substantially at a maximum.

24. The method of any one of Claims 1-3, wherein the plant is a tobacco plant.

25. The method of Claim 24, wherein the cutting step is carried out to cut the tobacco plant material from the plant at a height in the range from about one foot to about three feet.

26. The method of any one of Claims 1-3, wherein the plant material comprises leaf tissue.

27. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of:

(a) cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light dark cycle during which a quantity of ribulose 1,5-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle; (b) homogenizing the plant material 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°C;

(e) centrifuging the green juice homogenate to produce a supernatant; and

(f) purifying the protein or peptide from the supernatant.

28. A method for obtaining a fusion protein or peptide from a plant comprising the sequential steps of:

(b) homogenizing the plant material 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°C;

(e) centrifuging the green juice homogenate to produce a pellet;

(f) resuspending the pellet in a liquid solution;

(h) centrifuging the liquid solution of step (g) containing the resuspended pellet; and (i) purifying the fusion protein or fusion peptide.

29. The method of Claim 27 or 28, wherein the pH of the green juice homogenate is adjusted to between about 4.0 and 5.2.

30. 52. The method of Claim 27 or 28, wherein the pH of the green juice homogenate is adjusted to about 5.0.

31. The method of Claim 27 or 28 wherein the green juice homogenate is heated to a temperature of between about 45° and 50°C.

32. The method of Claim 27, wherein the supernatant produced in step (e) is further subjected to ultrafiltration.

33. A method according to Claim 32, 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.

34. The method of Claim 32, further comprising the step of subjecting a permeate produced by the ultrafiltration to a second ultrafiltration.

35. A method according to Claim 34, wherein said second ultrafiltration produces in a permeate containing molecules selected from the group consisting of sugars, polysaccharides, vitamins, alkaloids, flavor compounds and peptides.

36. The method of Claim 34, further comprising the step of purifying a concentrate resulting from the second ultrafiltration.

37. The method of Claim 27 or 28 wherein said purifying is performed by chromatography, affinity-based method of purification, or salt precipitation.

38. The method of Claim 27 or 28, wherein said protein or peptide of interest is selected from the group consisting of IL- 1, IL-2, LL-3, IL-4, LL-5, LL-6, IL-7, IL-8, IL-9, LL-10, LL-11, LL-12,, EPO, G-CSF, GM-CSF, M-CSF, Factor VHL Factor IX, tPA, receptors, receptor antagonists, antibodies, single-chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone.

39. The method of Claim 27 or 28, wherein said protein or peptide of interest is an antimicrobial peptide or protein and is selected from the group consisting of protegrins, magainins, cecropins, melittins, indolicidins, defensins, β-defensins, cryptdins, clavainins, plant defensins, nicin and bactenecins.

40. The method of Claim 27, wherein the soluble protein or peptide is a recombinant protein or peptide.

41. The method of Claim 27, wherein the soluble protein or piptide is a non-native protein or peptide.

42. A method for obtaining a soluble protein or peptide from a plant comprising the sequential steps of:

(a) homogenizing the plant material 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 °C;

(d) centrifuging the green juice homogenate to produce a supernatant; and

(e) purifying the protein or peptide from the supernatant.

43. The method of Claim 42, further comprising before step (a): cutting plant material from the plant, wherein the cutting step is carried out during a cutting period, wherein the cutting period is a period of a light dark cycle during which a quantity of ribulose 1 ,6-diphosphate carboxylase in the plant is reduced from a maximum quantity in the plant during a light portion of the light/dark cycle.

44. The method of Claim 27 or 28, wherein the cutting step is carried out to cut the plant material from the plant at a height in the range of from about six inches to about three feet.

45. The method of Claim 27 or 28, wherein the cutting step is carried out to cut the plant material into pieces that have a mean chop size in the range of from about two to about sixteen square inches.

46. A method of increasing the number of harvests in a growing season, comprising: a) growing a plant to a desirable height; b) harvesting biomass from the plant; c) allowing the plant to generate new biomass; d) harvesting the new biomass; and e) repeating steps c) and d).

47. The method of Claim 46, wherein the plant is grown to a height of no more than 4 feet.

48. The method of Claim 47, wherein the plant is grown to a height of no more than 3 feet.

49. The method of Claim 48, wherein the plant is grown to a height of no more than 2 feet.

50. The method of Claim 49, wherein the plant is grown to a height of no more than 1 foot.

51. The method of Claim 50, wherein the plant is grown to a height of no more than 6 inches.

52. A method of increasing the yield of biomass in a growing season, comprising: a) harvesting the younger biomass from a plant; b) allowing the plant to regenerate new biomass; c) harvesting the new biomass; and d) repeating steps b) and c).