WO2001034808A9 - Method of large-scale production and method of testing of the biological activity of a substance from soybean - Google Patents

Abstract

The lunasin peptide has been shown to exhibit inhibitory effects against malignant transformation of cells induced by chemical carcinogens and viral oncogenes. The present invention relates to a method for large-scale production of lunasin using recombinant DNA technology. The invention also includes a method for a rapid, in vitro transformation assay to detect and quantify the biological activity of this cancer preventive peptide.

Description

METHOD OF LARGE-SCALE PRODUCTION AND METHOD OF TESTING OF THE BIOLOGICAL ACTIVITY OF A SUBSTANCE FROM SOYBEAN

BACKGROUND OF THE INVENTION Related Applications

This application is a continuation-in-part of U.S. Ser. No. 60/165,334, filed November 12, 1999 which is incorporated herein by reference in its entirety. Field of Invention

This invention concerns the large scale production of a substance, e.g. lunasin, from soybean. It also describes a method to assay the biological activity of the lunasin. Description of Related Art

Discovery of the antimitotic property of the lunasin gene - The lunasin peptide has a unique poly-apartic acid carboxyl end. It was proposed to have an important biological function when it was isolated and sequenced, but not cloned from soybean seeds, by a Japanese group 13 years ago (Odani et al., 1987 J. Biol Chem, Vol.262: 10502). However, only upon the isolation and cloning of the Gm2S-l cDNA could a putative biological role for lunasin be inferred.

The Gm2S-l cDNA encodes lunasin as a small subunit component of a post- translationally processed 2S albumin (Galvez et al, 1997 Plant Physiol, Vol. 114:1567). The temporal expression of Gm2S-l coincides with the initiation of mitotic arrest and DNA endoreduplication in developing soybean cotyledon (Galvez et al., 1997). DNA endoreduplication is a unique cell cycle of Gl and S phases without cell division that occurs only in terminally differentiated storage parenchyma cells (Goldberg et al,1994 Science, Vol. 266:605). In situ hybridization and immunolocalization experiments using a lunasin antisense RNA probe and a lunasin-specific antibody, respectively, showed maximal mRNA and lunasin levels in storage parenchyma cells undergoing DNA endoreduplication and cell expansion but not in actively dividing cells of the cotyledon (Galvez and de Lumen, unpublished results).

The temporal and spatial expression of lunasin in developing seeds suggest a biological role of lunasin as an effector molecule that inhibits cytokinesis to allow DNA endoreduplication and cell expansion to occur in storage parenchyma cells during seed development. This information, together with the observation that lunasin gene expression also caused aberrant division in bacterial cells, led to the discovery by A.F. Galvez that lunasin should also disrupt eukaryotic cell division. By tagging with GFP to monitor transfected cells, it was demonstrated and reduced to practice the invention describing the antimitotic property of the lunasin gene when expressed in mammalian cells (Galvez and de Lumen, 1999 Nature Biotech 17:495). Also see U.S. patent 6,107,287, issued August 22, 2000 (filed as U.S. Ser. No. 08/938,675).

Cancer Preventive Property of Lunasin- The carboxyl end of lunasin is the region critical for its antimitotic and chromatin binding properties (Galvez and de Lumen, 1999). Lunasin contains the following structural features at its carboxyl end: a) a chromatin targeting helical domain, b) an Arg-Gly-Asp (RGD) cell adhesion motif, and c) a poly- aspartyl end (Galvez et al, 1997). The presence of the RGD motif could exogenously applied lunasin to bind to the cell membrane and to be internalized in mammalian cells. The affinity of lunasin for hypoacetylated chromatin (Galvez and de Lumen, 1999) also suggests that lunasin may be involved in chromatin modification. Regulation of the post- translational modification of chromatin has been implicated in cell-cycle control and in how tumor suppressors act as critical downstream effectors during carcinogenesis ( De Pinho, 1998). The presence of the RGD motif and its chromatin-binding property point to a potential anti-carcinogenic role for lunasin.

Using a polyclonal antibody specific to the lunasin carboxyl end epitope, immunoblot analysis showed that lunasin was a major constituent of the soybean Bowman Birk protease inhibitor (BBIC) preparation (See Figures 1 and 1A). BBIC has been shown to be cancer preventive in several in vitro and animal model studies (Examples: Yavelow et al., 1985; Troll et al., 1980, St. Clair et. al., 1990a and b; Reviews: Kennedy, 1993; Kennedy, 1995). The evidence for the anti-carcinogenic effect of BBIC was compelling enough that NCI is now conducting human clinical trials (currently in Phase II) to prove its effectivity (Kennedy et al., 1993). However, despite the accumulated in vitro and in vivo data pointing to the anticarcinogenic property of BBIC, the underlying mechanism of action has not been elucidated. More importantly, several scientific evidence have shown that BBIC or protease inhibitors (PI), in general, are unlikely to be the active anticarcinogenic component found in soybean. For instance, cooked soy products, which are devoid of any protease inhibitor activity, are equally as effective at reducing cancer development as raw soy products (Clawson, Cancer Invest. Vol. 14,608 1996). The effect of protease inhibitors appears to be indirect because dietary protease inhibitors are, in general, poorly absorbed from the gastro-intestinal (GI) tract, and never reach target organs in any measurable quantity (Clawson, 1996).

The open question is whether or not lunasin is responsible for the cancer preventive activity attributed to BBIC, considering that lunasin is a significant contaminant in the BBIC preparation? Cell transformation assays conducted at UC Berkeley showed that lunasin was on average twice more effective than equimolar amounts (125 nM) of BBIC in reducing foci formation in C3H 10 T 112 cells treated with potent chemical carcinogens, , 7, 12-dimethylbenz[a]anthracene (DMBA) (Fig.2) and 3-methylcholanthrene (MCA). More importantly, BBIC with immunodepleted lunasin, prepared by applying commercially available BBIC (Sigma T9777) through cationic exchange and inmmuno- affinity columns and then collecting flow through fractions, showed significant loss of its anti-transformation property (Fig.2). The duplicated sets of experiments showed that BBIC with immunodepleted lunasin did not inhibit foci formation upon carcinogen treatment, similar to the effect of the untreated positive control. Lunasin treatment as low as 10 nM at 24 hr. exposure was effective in reducing foci formation when compared to the untreated control (See Figure 3). These results indicate that lunasin is the major cancer preventive ingredient in the BBIC preparation.

What then is the role of BBIC in the cancer preventive property attributed to the BBIC soybean preparation? As pointed out by Clawson (1996), the effect of BBIC appears to be indirect. Digestion experiments have shown that lunasin by itself gets broken down by pancreatic digestive enzymes but resists digestion when a chymotrypsin inhibitor like BBIC is mixed with lunasin at equimolar ratios (Pascual and de Lumen, personal communication). It is most likely that BBIC's role is to prevent the digestion of lunasin in the gut to allow intact lunasin to be absorbed through the gastro-intestinal tract. Once in the circulatory system, lunasin is distributed to the various tissues and can get inside somatic cells by attaching to specific integrin receptors found in cell membranes through its RGD cell adhesion motif. Inside the cell, lunasin then preferentially binds to regions of the chromosomes enriched with hypoacetylated chromatin upon nuclear membrane breakdown at prometaphase.

A patent application for the cancer preventive properties of the lunasin peptide has been filed by University of California (U.S. Serial No. 09/303,814) and FilGen BioSciences, Inc. has obtained exclusive rights to this technology and patent.

Lunasin has a functional RGD motif that allows cell binding and internalization ~ The amino acid sequence of the lunasin peptide revealed the presence of the cell adhesion motif, Arg-Gly-Asp (RGD), at its carboxyl end which when functional could allow exogenously applied lunasin to bind and become internalized in mammalian cells. Synthetic and recombinant peptides containing the RGD motif derived from sequences of extracellular matrix proteins like fibronectin, have been shown to bind to specific membrane integrins in mammalian cells (E. Ruoslahti, M.D. Piersbacher. Cell Vol. 44, p. 517, 1986). To determine whether lunasin has a functional RGD motif, a cell adhesion assay using synthetic lunasin peptides and mice embryo fibroblast cells (C3H 10T 1/2) was conducted (assay is described in L.M. De Luca, etal, Methods ofEnzymol. Vol. 190: 81-91, 1990). The lunasin peptide adhered to C3H cells in a dose-dependent manner and that the deletion of the RGD tripeptide from lunasin (Lunasin-GRG) prevented cell adhesion (Fig. 5). When applied exogenously to the growth media, lunasin was not only adhering to the cell membrane but became internalized as well, preferentially binding to the telomeres of chromosomes during metaphase (Fig. 6). However, unlike the constitutive expression of lunasin gene in transfected cells that disrupts kinetochore formation, internalized lunasin did not affect kinetochore assembly. Immunostaining experiments showed the normal kinetochore location of the cell cycle checkpoint protein, MAD (Y. Li; R. Benezra, Science 2 , 246, 1996,; R.H. Chen, et al., Science Vol. 274, 242, 1996), in the centromere of metaphase chromosomes (Fig. 6). As a result, the exogenous application of lunasin did not affect cell division and proliferation of murine embryo fibroblast cells. Immunostaining using the lunasin polyclonal antibody also showed that internalized lunasin was initially found in the cytoplasm and then eventually bound to hypoacetylated regions of the chromosome, such as those in the telomeres, upon nuclear membrane breakdown at prometaphase (Fig.6). However, at this stage of mitosis, kinetochore assembly and spindle fiber attachment to centromeres had already transpired. This explains the non-disruptive effect of exogenously applied lunasin on cell division as compared to the antimitotic effect observed when lunasin is constitutively expressed in lunasin-transfected mammalian cells (Galvez and de Lumen, 1999).

The lunasin peptide preferentially binds to deacetylated histones and inhibits histone acetylation -- The antimitotic effect of the lunasin gene in transfected mammalian cells has been attributed to the competitive binding of lunasin to centromeres as visualized by GFP fluorescence and immunostaining (Galvez and de Lumen, 1999). On the other hand, immunostaining of exogenously applied lunasin revealed the preferential binding of lunasin mainly to the telomeres of metaphase chromosomes (Fig. 6). Telomeres are genomic regions that are also rich inhypoacetylated chromatin, comprising mainly of deacetylated histones (Braunstein et al, Genes Dev. Vol. 7, 592, 1993). The increased affinity of lunasin to these regions may be due to the greater electrostatic attraction of the negatively charged carboxyl end of lunasin to the positively charged N-terminal tails of deactelated histones. To test whether lunasin binds preferentially to deacetylated histones, an in vitro immuno-binding assay was conducted using acetylated and deacetylated forms of the H4 N-terminal tail. The full lunasin peptide (Lunasin) and lunasin with deleted RGD motif (Lunasin-GRG) were found to bind with high affinity to deacetylated H4 N-terminus but not to the tetraacetylated H4 (See Figures 7A, 7B, 7C, 7D and 7E). This result suggests that lunasin binds with high specificity to deacetylated H4 and that the RGD-motif is not important to its binding affinity. However, there was a significant reduction in deacetylated H4 binding for truncated lunasin (trLunasin) that contains only the reactive carboxyl end of the peptide. This result indicates that the N-terminus of lunasin is also important for binding to deacetylated histones most likely by stabilizing the lunasin structure to allow electrostatic interactions between the carboxyl end of lunasin and deactylated H4 to occur at higher efficiency. The lesser binding affinity of trLunasin to deacetylated H4 compared to the full lunasin peptide (Figures 7A to 7E) correlates with the observed reduced effectivity in preventing foci transformation (Fig. 5). This result provides further evidence linking the binding affinity of lunasin to deacetylated histones and its anti-transformation property.

The in vitro binding of lunasin to deacetylated histone H4 confirms the observed affinity of lunasin to regions of hypoacetylated chromatin such as the centromeres and telomeres in immunostaining experiments (Galvez and de Lumen, 1999 and Fig. 6). Deacetylated histones are substrates for histone acetylation and for chromatin remodelling which has been associated with eukaryotic transcriptional regulatory mechanisms (K. Struhl, Genes Dev. Vol. 12, 599, 1998; M. Grunstein, Nature Vol. 389,349,1997). To determine whether the preferential binding of lunasin to deacetylated histones has any biochemical effect on histone acetylation in vivo, C3H cells and the human breast cancer cell lin, MCF-7, were treated with the histone deactylase inhibitor, Na-butyrate (E.P. Candido, R. Reeves, J.R. Davie, Cell, Vol. 14, 105, 1978), in the presence or absence of lunasin. Immunoblots of acid-extracted proteins show the significant reduction of acetylated H4 and H3 in Na-butyrate treated C3H and MCF-7 cells when pretreated with 1 μM of lunasin peptide (Fig. 8). The absence of lunasin when cells were treated with Na- butyrate increased histone H4 acetylation by 200 fold in both C3H and MCF-7 cells. H3 acetylation induced by Na-butyrate treatment increased 100 fold in C3H cells and around 400 fold in MCF-7 cells. Upon addition of lunasin, there was no observed increase in H4 and H3 acetylation of C3H cells treated with Na-butyrate. In MCF-4 cells, H4 acetylation was reduced 10 fold and H3 acetylation 4 fold when lunasin was added prior to Na- butyrate treatment. These results suggest that the exogenous application of the lunasin peptide inhibit histone acetylation of mammalian cells in vivo.

Lunasin Induces Apoptosis in Cells with Inactivated Rb Tumor Suppressor -- Histone acetylation is associated with transcriptional activity in eukaryotic cells, having been observed mainly in transcriptionally active chromatin (K. Struhl, Genes Dev. 12, 599, 1998; M. Grunstein, Nature 389, 349, 1997). The inhibition of histone acetylation by lunasin provides a mechanistic model to explain the anti-carcinogenesis property of this soybean peptide. The Rb tumor suppressor, a critical downstream effector during carcinogenesis (R.A. Weinber, Cell SI, 323, 1995; M.C. Paggi, et al., J Cell.Biochem. 62 ,418, 1996), was hypothesized to repress E2F-regulated genes by binding to E2F and by recruiting a histone deacetylase (HDAC 1) to maintain a hypoacetylated state of condensed chromatin around the transcription start site (A. Brehm et al., Nature 391, 597, 1998; L. Managhi-Jaulin etal. Nature 391, 601, 1998; R.X. Luo, A.A. Posιigo,D.C. Dean, Cell 92, 463, 1998). This dual repression mechanism is thought to be abrogated upon Rb inactivation during carcinogenesis, resulting in the release of Rb binding to the E2F promoter, acetylation of the repressed chromatin structure and the induction of expression of the E2F-regulated genese involved in cell proliferation (R.A. DePinho. Nature 391, 533, 1998). By binding to deacetylated histones found in repressed chromatin, it has been hypothesized that lunasin can prevent cell proliferation and transformation even in the absence of a functional Rb by inhibiting histone acetylation and activation of E2F- regulated genes. To test this molecular model of lunasin action, C3H cells were first treated with lunasin and then transfected with E 1 A viral oncogene that specifically induces cell proliferation by binding and inactivating Rb (J.R. Nevins, Science 258, 424, 1992). As a negative control, E1A with deleted conserved region 1 (E1ADCR1) that abolishes the RB binding domain (D. Trouche, T. Kouzidares, Proc. Natl. Acad. Sci. , USA 93, 1439, 1996) was likewise used in the transfection experiments. C3H cells transfected with El ADCRl, as expected, showed normally dividing cells at 20 hr after transfection, both in the presence and absence of lunasin (Fig. 9). Transfection with the El Awt in the absence of lunasin also showed normal cell proliferation (Fig. 9). However, C3H cells initially treated with lunasin for 24 hr and then transfected with El Awt resulted in the preponderance of non-adherent cells in solution at 20 hr after transfection. Phase contrast image of the non-adherent cells showed characteristic morphology of apoptotic cells which was confirmed by the positive fluorescent staining for Annexin V-FITC (Fig. 9). Preliminary flow cytometry data also shoed increase in non-adherent and apoptotic cells by 30-40% in El A wt-transfected C3H cells treated with lunasin (data not shown) which correlates with transfection efficiency as determined by co-transfection with a plasmid vector expressing green flourescent protein (GFP).

A method for preparing biologically active BBIC from soybean has been issued (U.S. Patent 5,217,717) to Central Soya Company, Inc. and The Trustees of the University of Pennsylvania. This method involves the time consuming and expensive isolation and purification of BBIC directly from soybean seeds. In addition, the biological assay used involves measuring trypsin inhibitor activity, which has not been shown to correlate consistently with the anti-transformation property of BBIC. The scientific problem is that the mechanism of BBIC action on preventing carcinogenesis has not been elucidated and no model for BBIC mechanism has ever been accepted or proven. Lunasin inhibits in vivo acetylation of H3 and H4 histones The in vitro binding of lunasin to deacetylated histone H4 confirms the observed affinity of lunasin to regions of hypoacetylated chromatin such as the centromeres and telomeres in immunostaining experiments (Galvez and de Lumen, 1999 and submitted). Deacetylated histones are substrates for histone acetylation and for chromatin remodelling which has been associated with eukaryotic transcriptional regulatory mechanisms (K. Struhl, Genes Dev. Vol. 12, 599, 1998; Grunstein, Nature Vol. 389, 349, 1997). To determine whether the preferential binding of lunasin to deacetylated histones has any biochemical effect on histone acetylation in vivo, C3H cells and the human breast cancer cell line, MCF-7, were treated with the histone deacetylase inhibitor, Na-butyrate (Candido, et al., Cell 14, 105, 1978) in the presence or absence of lunasin. Immunoblots of acid-extracted proteins show the significant reduction of acetylated H4 and H3 in Na- butyrate treated C3H and MCF-7 cells when pretreated with 1 mM of lunasin peptide (Fig. 5). The absence of lunasin when cells were treated with Na-butyrate increased histone H4 acetylation by 200 fold in both C3H and MCF-7 cells. H3 acetylation induced by Na- butyrate treatment increased 100 fold in C3H cells and around 400 fold in MCF-7 cells. Upon addition of lunasin, there was no observed increase in H4 and H3 acetylation of C3H cells treated with Na-butyrate. In MCF-7 cells, H4 acetylation was reduced 10 fold and H3 acetylation 4 fold when lunasin was added prior to Na-butyrate treatment (Fig. 5). These results suggest that the exogenous application of the lunasin peptide inhibit histone acetylation of mammalian cells in vivo. Lunasin Induces Apoptosis in Cells with Inactivated Rb Tumor Suppressor Histone acetylation is associated with transcriptional activity in eukaryotic cells, having been observed mainly in transcriptionally active chromatin (K. Struhl, Genes Dev. 12, 599 ,1998; M. Grunstein, Nature 389, 349,1997). The inhibition of histone acetylation by lunasin provides a mechanistic model to explain the anti-carcinogenesis property of this soybean peptide. The Rb tumor suppressor, a critical downstream effector during carcinogenesis (R.A. Weinberg, Cell 81, 323, 1995; M.C. Paggi, βt al., J Cell. Biochem. 62, 418, 1996), was hypothesized to repress E2F- regulated genes by binding to E2F and by recruiting a histone deacetylase (HDAC1) to maintain a hypoacetylated state of condensed chromatin around the transcription start site (A. Brehm et al., Nature 391, 597,1998; L. Managhi-Jaulin et al. Nature 391, 601, 1998; R.X. Luo, A.A. Postigo, D.C. Dean, Cell 92, 463,1998). This dual repression mechanism is thought to be abrogated upon Rb inactivation during carcinogenesis, resulting in the release of Rb binding to the E2F promoter, acetylation of the repressed chromatin structure and the induction of expression of the E2F-regulated genes involved in cell proliferation (R.A. DePinho. Nature 391, 533, 1998 ). By binding to deacetylated histones found in repressed chromatin, it has been hypothesized that lunasin can prevent cell proliferation and transformation even in the absence of a functional Rb by inhibiting histone acetylation and activation of E2F- regulated genes. To test this molecular model of lunasin action, C3H cells were first treated with lunasin and then transfected with E 1 A viral oncogene that specifically induces cell proliferation by binding and inactivating Rb (J.R. Nevins, Science 258, 424, 1992). As a negative control, E1A with deleted conserved region 1 (E1ADCR1) that abolishes the RB binding domain (D. Trouche, T. Kouzidares, Proc. Natl. Acad. Sci., USA Vol.93, 1439, 1996) was likewise used in the transfection experiments. C3H cells transfected with E1A-DCR1, as expected, showed normally dividing cells at 20 h after transfection, both in the presence and absence of lunasin (Fig. 6). Transfection with the El Awt in the absence of lunasin also showed normal cell proliferation (Fig. 6). However, C3H cells initially treated with lunasin for 24h and then transfected with El Awt resulted in the preponderance of non-adherent cells in solution at 20 h after transfection. Phase contrast image of the non-adherent cells showed characteristic morphology of apoptotic cells which was confirmed by the positive fluorescent staining for Annexin V-FITC (Fig. 6).

The present invention provides an improved method for the large-scale production of lunasin and also provides a method to detect and quantify the biological activity of lunasin.

SUMMARY OF THE INVENTION The lunasin peptide has been shown to exhibit inhibitory effects against malignant transformation of cells induced by chemical carcinogens and viral oncogenes. The present invention relates to a method for large-scale production of lunasin using recombinant DNA technology. The invention also includes a method for a rapid, in vitro transformation assay to detect and quantify the biological activity of this cancer preventive peptide.

Lunasin has been identified to be a major if not the main active chemopreventive ingredient in BBIC. The invention described herein provides a method of producing biologically active lunasin peptide in commercial scale using recombinant

DNA technology. This method of production will provide a cheap and relatively easy way of producing commercial quantities of biologically active lunasin.

The present invention concerns an improved method to produce lunasin by recombinant DNA technology in large quantities, which method comprises:

(a) preparing lunasin gene constructs using protein expression vectors;

(b) optimizing lunasin expression parameters for large-scale production; and

(c) performing a series of isolation and purification steps to obtain large quantities of biologically active recombinant lunasin peptide.

It also concerns a method to determine the biological activity of a composition comprising lunasin which method comprises:

(a) preparing gene constructs that contain a mammalian transcription unit encoding a reporter gene, a selectable bacterial marker, and a mammalian transcription unit encoding an oncogene;

(b) pretreating normal mammalian cells exhibiting contact inhibition with compositions containing lunasin peptides;

(c) transfecting pretreated cells with gene constructs;

(d) detecting and quantifying of the level of apoptotic cells via reporter protein measurements;

(e) detecting and quantifying the number of transformed cells; and (f) determining the biological activity of compositions containing lunasin by comparison of the reporter protein measurements with standard values determined using predetermined equimolar amounts of pure, synthetic lunasin.

BRIEF DESCRIPTION OF THE FIGURES Figures 1 A and IB are Western blots showing lunasin is a major constituent of the Bowman Birk protease inhibitor (BBIC) preparation with other plant samples.

Figure 2 is a graph comparing transformation of tumerous foci of PBS, lunasin, BBI and BBI (-lunasin).

Figure 3 is a graphic presentation of lunasin treatment which is effective in reducing foci formation.

Figure 4 is a graphic representation of the reduction of mean number of foci with cells treated with 1 μM lunasin for 24 hr.

Figure 4A is a graphic representation of the helical motif in lunasin with the helical portion of conserved chromodomain regions found in other protein. Figure 5 is a graphic comparison of the mean number of transformed foci with various peptide compositions. Figure 5A is a schematic representation of lunasin peptide and its various subunits.

Figure 6 is a graphic comparison of the relative cell adhesion versus μM of lunasin and lunasin (-GRG). Figures 7A, 7B, 7C, 7D, and 7E are a series of photographic representations of staining for DAPI, lunasin, MAD showing binding of internalized lunasin to telemores.

Figure 8 is a graphic representation of the binding efficiency of various lunasin moieties.

Figure 9 is a schematic representation of immunoblot analysis of acid - extracted proteins, probed with acetylated H₄ and H₃ antibodies.

Figures 10 A, 10B, 10C, 10D, 10E and 10F are a group of photographic representations, of the effect of lunasin on El A transfected cells.

Figures 10G and 10H are graphic representatives of side scatter (SS) and forward scatter in flow cytometry. Figure 11 is a schematic representation of a large scale production of recombinant lunasin peptide of the present invention.

Figure 12 is a model describing the anticarcinogenesis property of lunasin peptide.

Figure 13 is a graphic representation of the lunasin responses in nM versus the number of scorable foci observed.

Figure 14 is a graphic representation of colony formation showing the concentration of dose response of lunasin in nM versus the number of colonies.

Figure 15 is a graphic representation of the effect of addition of IPTG and in the presence and absence of lunasin. Figure 16 is a schematic representation of the pPIC9K gene construct as used in the present invention. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS DEFINITIONS: As used herein: "Lunasin" refers to compounds comprising the natural and recombinantly produced soybean lunasin polypeptide (coincidentally purified and sequenced by Odani et al., 1987 (Ser-Lys-Trp-Gln-His-Gln-Gln-Asp-Ser-Cys-Arg-Lys-Gln-Leu-Gln-Gly-Val-Asn-Leu- Thr-Pro-Cys-Glu-Lys-His-Ile-Met-Glu-Lys-Ile-Gln-Gly-Arg-Gly-Asp-Asp-Asp-Asp- Asp- Asp-Asp-Asp-Asp (SEQ ID 1). "Lunasin" refers to the biological active lunasin peptide having 1-43 amino acids.

"Lunasin or an active variant thereof refers to the biologically active lunasin peptide having 43 amino acids, or to portions of the 1-43 amino acid chain which are also biologically active (shown herein as 22-43 amino acids meaning amino acid 22 to amino acid 43 of lunasin). See sequence data. A number of expression vectors are described and available from:

American Type Culture Collection 10801 University Boulevard Manassas, Virginia 20110-2209 USA These include for example but are not limited to: c3h having ATCC Number:CCL-226 Name:C4H/10Tl/2, Clone 8 Tissu; nih3t3 having ATCC Number: HB- 11601 Organism: mouse (B cell); mouse; ras having ATCC Number: 81560 Designations: HFBCX08 Organism: Ho; ela having ATCC Number: 107510 Designations: HTEAE54 Organism: Ho; ras having ATCC Number: 63127 Designations: Ftbeta Organism: Rat; ras having ATCC Number: 100093 Designations: HAECC01 Organism: Ho; e6 having ATCC Number: 61262 Designations: E6 Organism: Human e7 having ATCC Number: 61264 Designations: E7 Organism: Homo sap; c-fos having ATCC Number: 41041 Designations: pc-fos (mouse)-3 Organism: Mouse; V-myb having ATCC Number: 41012 Designation: pVM2; Organism-Avion;

V-myc having ATCC Number: 41029 Designation: pSV cmyc 1 [pmetD] Organism: Mouse; c-jun having ATCC Number: 87568 Designation: pSOC4-JUN; Organism: Human;

Large-Scale Lunasin Production using Recombinant DNA Technology The availability of the lunasin gene provides an avenue for producing commercial quantities of this cancer preventive peptide from soybean via recombinant DNA technology. The Pichia expression system was chosen to produce high levels of functionally active recombinant lunasin peptide. It offers high level expression, easy scale up and inexpensive growth with the advantages of expression in a eukaryotic system. Many proteins have been expressed using the Pichia system to levels as high as grams per liter. Pichia pastoris is covered by one or more of the following US patents and corresponding foreign patents owned and licensed by Research Corporation Technologies (RCT), Inc.: 4,683,293; 4,855,231; 4,895,800; 5,122,465; 4,808,537; 4,857,467; 4,929,555; 5,132,868; 4,812,405; 4,879,231; 5,002,876; 5,166,329; 4,818,700; 4,882,279; 5,004,688; 4,837,148; 4,885,242; and 5,032,516. The commercial license to use Pichia in the present invention be obtained from RCT, Inc. All articles, references, standards, patents, patent applications and the like cited in this application are incorporated herein by reference in their entirety.

The lunasin gene is subcloned into the Pichia vector, pPIC9K that was purchased from Invitrogen, who has exclusive license to sell Pichia expression kits from RTC, Inc. The lunasin-pPIC9K construct is inserted into the genome of the Pichia pastoris strain SMD 1168 by transformation using electroporation. Multi-copy integration of the lunasin expression cassette is determined by choosing transformants that grow in increasing levels of the antibiotic G418. DNA amplification using polymerase chain system and lunasin gene-specific primers are used to verify integration. About 5 multi-copy transformants are selected for small-scale expression of lunasin. The vector used allows for the secretion of lunasin to the growth media such that lysates from each transformant can be sampled at several time points to determine level of induction of lunasin. The two colonies with the highest levels of lunasin expression are used for optimization experiments in a large- scale fermentor. The optimization parameter for large-scale lunasin production using propretary lunasin-ρPIC9KP cb/ transformants is presently a trade secret and proprietary intellectual property of FilGen BioSciences, Inc. of Albany, California, and will not be made available to the public. The method of using recombinant DNA to produce cancer preventive products (lunasin and lunasin-modified and lunasin-derived peptide products) is claimed as an embodiment of the invention and a proprietary intellectual property specific to the process of producing cancer preventive lunasin peptides. The lunasin- pPIC9K construct and the resulting transformants derived after electroporation are claimed as proprietary materials, or compositions of matter related to the invention and are claimed as such.

Isolation and Purification of Lunasin - The downstream processing steps to isolate and purify recombinant lunasin from supernatant fraction of Pichia transformants include: a) applying supernatant through size exclusion columns and collecting the flowthrough comprising of low molecular weight proteins; b) applying supernatant through cation exchange columns and collecting flowthrough, to remove positively charge proteins and molecules; c) applying supernatant through anion exchange columns and then collecting elutants that are enriched for negatively charge molecules (lunasin is highly acidic with pH of 4.2); and d) applying lunasin-containing supernatants through immuno-affmity columns primed with highly antigenic lunasin antibody and the collecting elutants that comprise purified lunasin. The lunasin antibody is raised against a highly antigenic epitope that encompass the bioactive carboxyl end of lunasin. Isolation and purification of biologically active lunasin are conducted by using these methods, singly or in combination, depending on the required purity of lunasin.

The optimized method of isolating and purifying lunasin depending on the required purity of the substance for the different uses and indications of the cancer preventive lunasin products are presently considered the intellectual property of FilGen Biosciences, Inc. Development of Novel Plasmid Constructs to Detect Lunasin Biological Activity ~

The induction of apoptosis by lunasin in El A-transfected C3H cells provides evidence to a molecular model explaining lunasin' s suppression of carcinogen-mediated transformation (Fig. 10). The Rb tumor suppressor inhibits the expression of E2F- regulated genes in part by tethering a histone deacetylase (HDACl) to maintain a condensed hypoacetylated chromatin around the transcription start site. The inactivation of Rb by carcinogen treatment and oncogene expression reults in the loosening up of the repressed chromatin structure by localized histone acetylation (R.H. Giles, D.J. Peters, M.H. Breuning, Trends Genet. 14, 178, 1998). This consequently results in the activation of genes involved in cell proliferation and eventually leads to carcinogenesis. When lunasin is present in normal cells before Rb is inactivated, the deacetylated N-terminal tails of histone H3 and H4 found in repressed chromatin presumably bind to the acidic carboxyl end of lunasin. This make these deacetylated histones unavailable as substrates for histone acetylation, thus maintaining the repressed chromatin structure around the E2F promoter even when carcinogens and the viral oncogene, El A, inactivate Rb. The inhibition of expression of E2F-regulated genes triggers apoptosis instead of cell proliferation, which should be the normal occurrence when these genes are activated during carcinogenesis. The induction of apoptosis in cells with inactivated Rb by the presence of lunasin can explain the reduced number of transformed foci in normal murine fibroblast cells that have been treated with potent chemical carcinogens.

The elucidation of the molecular model of lunasin 's mechanism of action led to the conceptual synthesis of an in vitro cell culture assay to determine the biological activity of lunasin. Other viral oncoproteins like E6 and E7 from human papilloma virus also inactivate the Rb tumor suppressor in the process of transforming normal cells to proliferating, cancerous cells (J.R. Nevins, Science 258, 424, 1992). The addition of lunasin in mammalian cells transfected with these viral oncogenes is expected to result in apoptosis instead of cell proliferation. An important consideration for this to happen is the presence of a funtional p53 (to allow apoptosis to occur) in the mammalian cell line used (i.e. C3H 10T1/2 and NIH3T3 cells).

The ability of lunasin to cause apoptosis in mammalian cells trasfected with viral oncogenes that inactivate Rb can be used as a measure of its anti-carcinogenic property. This biological assay requires the development of a series of novel plasmid constructs that contain a bacterial selectable marker (ampicillin or kanamycin resistance), a transcription unit that constitutively express viral oncoproteins (El A, E6, E7 and h-ras), and a transcription unit that constitutively express reporter proteins such as GFP (green flourescent protein) and luciferase. These plasmid constructs will be trasfected into C3H and NIH3T3 cells that are pretreated with batch solutions containing equimolar amounts of recombinant lunasin peptide. After 24 hr, the cells are observed for presence of non- adherent, apoptotic cells, which are physically separated from the adherent, normal cells and quantitated by analyzing reporter gene expression through fluorescence and/or spectrophotometer readings. The relative measure of the quantity of apoptotic cells corrected for transfection efficiency will provide a measure of the biological activity in each batch of recombinant lunasin peptide produced.

Determination of Quantity and Biological Activity of Recombinant Lunasin — The method of determining the presence of the recombinant lunasin in Pichia lysates and quantifying the amounts involve the use of a FilGen proprietary antibody that has been designed and developed to detect lunasin with high accuracy, specificity and efficiency. The antibody is used to detect and quantify lunasin in each batch of supernatant containing recombinant lunasin as well as purified forms of the lunasin peptide by conducting enzyme-linked immunosorbent assay (ELISA) and immuno-blot analysis (Western analysis).

The rapid method of determining the biological activity of the isolated recombinant lunasin in Pichia lysates and or supernatants involves the use of proprietary viral oncogene plasmid constructs (as described above). The biological assay described in this invention disclosure was created upon the elucidation of the anti-carcinogenic mechanism of action of lunasin.

The biological assay for determining lunasin activity uses normal mice embryo fibroblast cells (C3H 10T1/2 andNIH3T3 cells) that will be transfected with the oncogene constructs in the prescence or abscence of a measured amount of recombinant lunasin. The number of cells that undergo apoptosis or cell death as a result of the effect of lunasin in combination with the viral oncogene will be quantified using a fluorometer or a spectrophotometer and standardized based on transfection efficiency. The values are plotted on a standard curve generated by graphing lunasin biological effect (relative number of apoptotic cells) in relation to increasing concentration of pure synthetic lunasin. Lunasin biological activity (LBA) units of each batch of recombinant lunasin are determined by taking the ratio of this plotted value t the value generated when using equivalent amounts of purified synthetic lunasin. A 100% biological activity will mean that the recombinant lunasin isolated has the same activity as pure synthetic lunasin in equivalent amounts (measured in either weight or moles). To determine the accuracy and effectivity of this rapid biological assay of lunasin activity, the solutions of recombinant lunasin peptides and appropriate controls are used in standard transformation assay using chemical carcinogens as described above in the prior art background.

A) Anion Exchange Chromatography

The pH of supernatant containing lunasin was first adjusted to pH 7.0 with 25 mM sodium acetate (Buffer A), a low ionic strength buffer that should be filtered and degassed before use. Mild non-ionic detergents and/or denaturing agents such as Tween80 at 0.1% v/v was added to the buffer to prevent aggregation and polymerization. Examples of anion exhange resins that can be used to purify lunasin includes: DEAE-sephadex, QAE-sephadex, DEAE-sepharose, QAE-sepharose, DEAE- sephacel, DEAE-cellulose, QAE-cellulose, Anion exchangers on polystyrene

(Amberlite, Dowex). Instructions for column use and care were supplied with each column. Columns were first washed with distilled water at 4°C before use. The supernatant was then injected to the column using flow rates of 2 mL per min. and the chromatograph pattern determined. The entire peak that corresponds to the eluted lunasin (5kDa peak) was collected and dialyzed against distilled water using dialysis membane with MWCO of 8 kDa to remove salts and impurities.

B) Molecular Size Exclusion Chromatography.

Examples of molecular size exclusion resin that can be used to purify lunasin includes: Sephadex G-25 (separates peptides 1-5 kDa, and Sephadex G-50 (separates proteins 1.5 - 30 kDa). Supernatant was adjusted to pH7.0 with Buffer A and 0.1% Tween or sodium dodecyl sulfate (SDS). The mixture is applied to the size exclusion columns at 4°C by pump at flow rates of 0.1 - 1.0 mL per min. Analysis of eluate fraction to determine presence of lunasin can be done by SDS-PAGE, Western, ELISA and chromatograph analysis.

After the fermentation harvest, the supernatant fraction was concentrated to half its original volume using SartoconO Slice unit. The Sartocon cross-flow filtration unit was outfitted with a 10 kDa Slice cassette. The supernate was circulated using a Watson Marlow 640A peristaltic pump. 4 volumes of filtrate were circulated per minute. The filtrate was set to have a pressure of 30 psi and no retentate pressure was observed. With these parameters, 35 ml/min of permeate passed through the filter. After the volume of the filtrate lowered to a third of its original volume, 430 ml of IX PBS (phosphate buffer solution) was added to the filtrate as apreliminary wash step. The filter was run until an additional 200 ml of permeate were acquired. The permeate and retentate fractions were tested for the presence of lunasin using Western analysis.

C) Immuno-affinity Chromatography.

A polyclonal antibody that specifically recognizes the carboxyl end epitope of Lunasin is available for immunoaffmity purification. A monoclonal antibody is currently being developed, as well. This protocol was used to successfully isolate and purify lunasin from a soybean protein mixture. It can also be used to isolate and purify lunasin from supernatant fraction or to further purify fractions from size exclusion chromatography and ion-exchange column chromatography.

DETAILED DESCRIPTION OF THE FIGURES Figures 1 A and IB show a Western blot analysis of samples containing lunasin. Approximately 5- 10 mg of total protein from different samples were run on 12% SDS-PAGE, electroblotted onto Hybond-ECL membrane (Amersham) and detected with polyclonal anti-lunasin and horseradish peroxidase labelled anti-mouse IgG secondary antibody. The left panel shows protein staining with Coomassie Blue and the right panel shows the immunoblot. M = MW markers; 1 = lunasin-pFMac, uninduced; 2 = lunasin- pFMac, induced with IPTG; 3 = soybean trypsin inhibitor (Sigma T9128); 4 = BBI (Sigma T9777); 5 = kidney bean; 6 = soybean; 7 = maize. Lunasin-pFMac is constructed by ligation of lunasin coding region into the pFMac vector (Sigma) and transfection into E. coli.

Figure 2 shows the suppression of carcinogen-induced transformation of C3H 10T 1/2 cells by the lunasin peptide. Chemical carcinogen, 7, 12- dimethylbenz[a]anthracene (DMBA) was used to induce transformation of C3H cells in 24-well plates, treated with equimolar amounts (125 nM) of lunasin peptide, the Bowman- Birk trypsin inhibitor (BBI), which was previously shown to inhibit foci formation using this assay and a BBI preparation that was immunodepleted of lunasin (BBI-lunasin). Negative controls were not treated with DMBA while the positive controls were treated with DMBA but did not receive any peptide treatment. Two sets of experiments at difference cell passages with 2 replications of each treatment were conducted. The number of foci that formed in each treatment plate was counted and a combined analysis of variance was done on the two sets of transformation experiments. There were no significant differences of means between passages. Standard error was computed from means of the four replicates. Figure 3 shows the effect of increasing doses of lunasin on foci formation induced by the chemical carcinogen 3-methylcholanthrene (MCA). Around 500 cells were plated on each well of 6-well plates and treated with increasing amounts of lunasin (0-10 μM) for 4 hr before exposure to MCA for 24 hr. Each lunasin dose was replicated twice and the number of foci that formed in each treatment plate was counted after 6 weeks. Negative controls did not receive any carcinogen or peptide treatment. Means and standard deviation of each lunasin dose are shown.

Figure 4 shows the effect of duration of lunasin exposure on foci formation induced by MCA. Around 500 cells were plated on 6-well plates and treated with 1 μM lunasin before exposure to MCA. Lunasin was added to the culture media up to the indicated time point (from 24 hr to 3 weeks). After 6 weeks, foci formation was counted in each treatment plate. Means and standard deviations from three replicates are shown.

Fig. 4A shows the structural homology of a helical motif in lunasin with a helical portion of conserved chromodomain regions found in other chromatin-binding proteins ( Aasland and Stewart, 1995). Boxed area corresponds to the helical domain, dark shaded boxes indicate highly conserved amino acid residues that includes a negatively charged residue (-) and a hydrophobic amino acid (#) flanking the helical domain, lighter shaded boxes indicate moderately conserved hydrophobic amino acid residues (%), and asterisk (*) indicates the isoleucine (I) mutated to phenylalanine (F) that results in the loss of chromatin targeting of Drosophila heterochromatin protein, DmHp 1 A (Messmer et al., 1992)

Figure 5 shows the effect of lunasin structural modifications on foci formation induced by MCA. Synthetic peptides with modifications on the reactive carboxyl end of lunasin were used in transformation assay (12). Around 500 cells were plated on each well of 6-well plates and treated with equimolar amounts (1 μM) of lunasin, modified lunasin peptides and BBIC. Negative controls were not treated with MCA while the positive controls were treated with MCA but did not receive any peptide treatment. Treatments (corresponding to one plate) were replicated four times and an analysis of variance was conducted on the number of foci that formed in each treatment plate. Treatment means were compared using Duncan's Multiple Range Test (DMRT) and treatment means with similar letters are not significantly different from each other. Figure 5 A shows the subunits of lunasin. Figure 6 shows the cell adhesion of the lunasin peptide to C3H 10T 1/2 cells using cell adhesion assay described in L.M. De Luca, et al, Methods ofEnzymol. 190: 81-91 (1990). The measure of cell adhesion is based on the intensity of blue staining arising from Giemsa staining of adherent cells as measured by absorbancy at 630 nm. The relative cell adhesion of increasing amounts of the Lunasin and Lunasin (-GRG) peptides (from 0 to 20 μM) to C3H 10T 1/2 (C3H) cells is computed by taking the ratio of the absorbancy reading of the treatment to the absorbancy of the untreated control.

Figures 7A, 7B, 7C, 7D and 7E show the internalization of the lunasin peptide in C3H 10T 1/2 cells upon exogenous application to growth media. DAPI staining of chromosomes and immunostaining of a C3H 10T 1/2 cell at the metaphase stage of mitosis. C3H cells were treated with 1 μM lunasin for 4h, trypsinized and allowed to grow for 24 hr before DAPI and immunostaining was conducted, using primary antibodies for the cell cycle checkpoint protein, MAD, and the lunasin carboxyl end epitope. The composite panel shows the metaphase location of the chromosomes (Fig.7A) (DAPI, blue fluorescence), the centromere (MAD, red fluorescence) (Fig. 7C) and the telomeres (Lunasin, green fluorescence) (Fig. 7D) with the inset (Fig. 7E) showing a magnified portion of the telomeric region with bound lunasin (green fluorescence). Bar = 8 microns. Figure 8 shows the binding affinity of lunasin to deacetylated and tetra- acetylated N-tereminal tails of histone H4. Immuno-binding assay (15) was conducted on lunasin and modified lunasin peptides to determine their binding affinity to deacytelated H4 (H4) and tetra-acetylated H4 (H4-Ac). Percentage of H4/H4-Ac bound to each peptide was determined (15) in triplicate and the means and standard deviations shown. There was consistently no binding observed between the tetra-acetylated H4 and all the peptides tested.

Figure 9 shows the immunoblot analysis of acid-extracted proteins isolated from C3H and MCF-7 cell lines treated with 2 μM lunasin and 5 mM of the histone deactylase inhibitor, Na-butyrate. Acid extracted proteins enriched for histone proteins from the different treatment combinations of lunasin and Na-butyrate were blotted onto nitrocelulose membranes and probed with anti-acetylated histone H4 and H3. Proteins were visualized using a HRP-conjugated anti-rabbit secondary antibody. Numbers underneath immunoblots correspond to densitometer readings standardized relative to the densitometer readings of the non-Na-butyrate treated controls in each immunoblot and cell line. Silver-stained gel of the acid extracted proteins shows equal loading of proteins in lanes for each cell line.

Figures 10A, 10B, IOC, 10D, 10E and 10F show the effect of lunasin on

ElA-transfected C3H cells. C3H cells were released from confluency and were either treated with 2 μM lunasin for 24 hr or not before transfected with gene constructs containing El Awt and El A ΔCRI. Phase contrast images of the cells were taken 20 hr after transfection. Arrows indicate non-adherent and apoptotic cells in lunasin-treated and

El A wt-transfected C3H cells which were stained with Annexin V-FITC and visualized under fluorescent microscope. Bars = 10 microns.

Figures 10G and 10H are a graphic representative of scatter. Figure 10G is side scatter (SS) and Figure 10H is forward scatter (FS) parameter in flow cytometry indicating percentage of apoptotic cells at gate F in a El A - transfected C3H cells in the presence and absence of lunasin.

Figure 11 is a schematic representation of the process flow diagram for the large-scale production of recombinant lunasin peptide. Figure 12 is a schematic representation of the model for the anti- carcinogenesis properties of the lunasin peptide.

Figure 13 is a graphic representation of the lunasin response shown in nM versus the number of scorable foci which are usually observerd. It shows the effect of increasing the level of lunasin concentration. Figure 14 is a graphic representation of colony formation showing the concentration of dose response of lunasin in nM versus the number of colonies of abnormal cells visually observed.

Figure 15 is a graphic representation of the effect of the addition of IPTG in the presence and absence of lunasin. As is seen, lunasin reduces colony formation. Figure 16 is a schematic representation of the pPIC9K gene. Experimental - General

The starting materials described herein are available from commercial supply houses, from recognized contracting organizations or can be prepared from published literature sources. Unless otherwise noted the material solvents, reagents, etc. are used as received without modification.

These examples are presented for the purpose of explanation and description only. They are not to be construed to be limiting in any way.

PREPARATION A Preparation of Immunoaffinity Column

Buffer Exchange of Antibody

1. Dilute Affi-Gel Hz (from Biorad, Hercules, CA ) 1 Ox coupling buffer to 1 x. Check pH with pH meter. Adjust to pH 5.5 with 1.0 M acetic acid or 1.0 M NaOH if necessary. 2. Pour out buffer from desalting column above top frit.

3. Add 20 mL lx coupling buffer. Allow buffer to drain until buffer surface level reaches top frit.

4. Dilute 1-5 mg/mL antibody sample to 3 mL with l coupling buffer. Add the 3 mL antibody sample to desalting column. Allow sample to drain to the top frit. 5. Add 4 mL lx coupling buffer to the column. Collect 1 mL fractions.

6. Do a Bradford Spot Test on your fractions to detect the presence of antibody (protein):

A) Place a piece of plastic wrap on top of a piece of white paper. Remove as many wrinkles from the wrap as possible. B) Spot 8 mL of sample (remember to use a blank composed of lx coupling buffer) on the plastic wrap. Add 2 mL Bio Rad protein assay reagent, and pipet up and down to mix.

C) Compare color change (bluish tint) after a couple of minutes. The test is sensitive to 10 mL/mL. Color change will be slight. 7. Wash column with 20 mL lx coupling buffer. To store desalting column, cap end with a column tip and add 5 mL lx coupling buffer containing 0.02% w/v NaN3.

Oxidation of IgG Sugar Residue and Consequent Removal of Sodium Periodate Note: Sodium periodate is a strong oxidizing agent and sensitive to light. The following oxidation steps are done in a chemical hood.

1. Add 1.2 mL ddH2θ to brown sodium periodate vial (25 mg), and vortex. Solution keeps for 1 week at 4°C. 2. Put buffer-exchanged antibody sample in Al foil- wrapped screw-cap tube.

3. To the tube add a volume of sodium periodate solution equal to 10% of the antibody volume in the screw-cap tube (for example 400 mL NaIO4 for 4 mL IgG sample).

4. Put tube on shaker horizontally, tape it down and let shake for 1 h at rt. 5. After the shaking, immediately run the solution through a desalting column, limiting column runs to 3 mL each,and wash with 20 mL lx coupling buffer between runs. Use the Bradford Spot Test for each run to detect antibody (protein).

6. Collect fractions containing antibody together in a 15 mL (or appropriate size) tube. Coupling of IgG to Affi-Gel Hz Gel

Note: If using less than 5 mL gel for coupling, wash only what is needed. Keep the rest in isopropanol at 4°C. Also, 1-5 mg antibody per mL of gel is recommended.

1. Pipet gel slurry to 15 mL tube and allow slurry to settle.

2. Pipet out supernatant and add 10 mL lx coupling buffer. Vortex. Allow to settle. Repeat.

3. Remove supernatant and add 5 mL lx coupling buffer.

4. Transfer slurry to coupling reaction tube.

5. Add oxidized, desalted IgG sample to gel in reaction tube. Tape down tube on shaker and shake overnight. 6. Pour gel/antibody slurry into chromatography column. Collect column eluant and measure volume.

7. Wash column with 10 mL PBS pH 7.0 containing 0.5 M NaCl.

8. Add PBS pH 7.0 with 0.5 M NaCl and 0.02% NaN3 until the buffer is above the gel bed. Cap both ends of column and store at 4^°C.

Application of Lunasin Sample to Immunoaffinity Column

1. Remove column from 4°C and allow to warm to room temperature. Add 2-4 bed volumes of buffer chosen for antigen elution to affinity column. The following acid elution buffers can be used: 0.2 M glycine-HCl, pH 2.5, 0.1 M acetic acid, 0.15 M sodium citrate, pH 3.0, and 0.5 M formic acid

2. Wash column with 5 bed volumes of PBS pH 7.0. 3. Apply sample to affinity column.

4. Wash column with 2 bed volumes of PBS pH 7.0 (0.5 M NaCl) to remove unbound protein.

5. Wash column with 1-2 bed volumes of PBS pH 7.0.

6. Add 2 bed volumes of eluant to column to remove antigen from column. Collect the fractions. Allow the elution buffer to reach the gel bed. Collect eluant for further analysis. Analysis of eluate fraction to determine presence of lunasin is done by SDS-PAGE, Western, ELISA and chromatograph analysis.

EXAMPLE 1 LARGE-SCALE DNA SYNTHESIS OF LUNASIN (a) The lunasin gene is subcloned into a Pichia expression vector, pPIC9K. The lunasin-pPIC9K gene construct is used to transform different strains of Pichia pastoris and Transformants that express lunasin at high concentrations in the supernatant are selected to optimize fermentation process. The ideal incubation conditions (temperature, pH and level of aeration), amount of inducing compound (methanol) and the optimal time to harvest cells are determined experimentally. Using optimal fermentation conditions, a selected transformant is grown in large-scale fermentation tanks before inducing lunasin expression by adding optimal amounts of methanol. The supernatant containing recombinant lunasin is separated by microfiltration and undergoes isolation and purification steps to obtain biologically active recombinant lunasin peptide. Purification steps include size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and immuno-affinity chromatography. Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophilization and further tested for biological activity.

(b) Example 1 (a) is repeated except that the lunasin gene is subcloned into a bacterial expression vector. The optimum fermentation conditions specific to bacterial expression systems are obtained experimentally and utilized in large-scale fermentation tanks. Lunasin containing supernatant and/or bacterial cell lysates are separated using microfiltration procedures specific to protein extraction from bacterial cells. Purification steps include size exclusion chromatography, ion exchange chromatography. Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophilization and further tested for biological activity. The amount of product obtained is comparable to that of Example 1 (a).

(c) Example 1 (a) is repeated except that the lunasin genes is subcloned into a mammalian expression vector. The optimum fermentation conditions specific to mammalian expression systems are obtained experimentally and utilized in large-scale fermentation tanks. Lunasin containing supernatant and/or mammalian cell lysates are separated using microfiltration procedures specific to protein extraction from mammalian cells. Purification steps include size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and immuno-affinity chromatography. Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophilization and further tested for biological activity. The amount of product obtained is comparable to that of Example 1 (a).

(d) Example 1(a) is repeated except that the lunasin genes is subcloned into an insect expression vector. The optimum fermentation conditions specific to mammalian expression systems are obtained experimentally and utilized in large-scale fermentation tanks. Lunasin containing insect cell lysates are separated using micofiltration procedures specific to protein extraction from insect cells. Purification steps include size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and immuno-affinity chromatography. Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophliization and further tested for biological activity. The amount of product obtained is comparable to that of Example 1 (a).

(e) Example 1 (a) is repeated except that the lunasin gene is subcloned into another yeast expression vector. The optimum fermentation conditions specific to yeast expression systems are obrained experimentally and utilized in large-scale fermentation tanks. Lunasin containing supernatant and/or yeast cell lysates are separated using microfiltration procedures specific to protein extraction from yeast cells. Purification steps include size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and immuno-affinity chromatography. Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophilization and further tested for biological activity. The amount of product obtained is comparable to that of Example 1 (a).

(f) Example 1 (a) is repeated except that the lunasin gene is subcloned into a baculovirus expression vector. The optimum fermentation conditions specific to baculovirus expression systems are obtained experimentally and utilized in large-scale fermentation tanks. Lunasin containing supernatant and/or baculovirus cell lysates are separated using microfiltration procedures specific to protein extraction from baculovirus cells. Purification steps include size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, and immuno-affinity chromatography.

Purified lunasin fractions are quantified and analyzed by Western analysis and ELISA, concentrated by lyophilization and further tested for biological activity. The amount of product obtained is comparable to that of Example 1 (a).

EXAMPLE 2 ASSAY TO DETERMINE BIOLOGICAL ACTIVITY

(a) Bacterial plasmid constructs are made that contain the following DNA fragments: a reporter gene comprising of a transcription unit that could express green fluorescent protein (GFP) in mammalian cells; a bacterial selectable marker comprising of an ampicillin and/or kanamycin resistance gene; and a transcription unit that could express the oncogene, E1A, in mammalian cells. C3H 10T1/2 murine cells are first pretreated with a known molar amount of recombinant lunasin peptide upon release of the cells from confiuency. After 18-20 hrs, the cells are transfected with the GFP-E1 A gene construct and incubated for another 20-24 hr. Non-adherent, apoptotic cells are collected from the growth media and concentrated by centrifugation. Samples are transferred intoa microtiter plate or cuvettes and the amount of fluoresence specific to GFP is measured in fluorometer or a spectrophotometer. The amount of GFP fluorescence (corrected by transfection efficiency) provides a direct measure of the number of apoptotic cells induced by the presence of biologically active recombinant lunasin peptide in the growth media. The values are compared with data generated from using equimolar amounts of pure synthetic lunasin. Lunasin biological activity (LBA) units of each batch of recombinant lunasin is determined by taking the ratio of the GFP fluorescence induced by recombinant lunasin from that of purified synthetic lunasin. (b) Example 2(a) is repeated except that green flourescent protein (GFP) is replaced with luciferase. The amount of product analyzed is comparable to that of Example 2(a).

(c) Example 2(a) is repeated except that green flourescent protein (GFP) is replaced with glucorunidase (GUS protein). The amount of product analyzed is comparable to that of Example 2(a).

(d) Example 2(a) is repeated except that C3H 10T1/2 murine cells are replaced with NIH 3T3 cells. The amount of product analyzed is comparable to that of Example 2(a). (e) Example 2(a) is repeated except that C3H 10T1/2 murine cells are replaced with other normal, non-tumorigenic mammalian cells that exhibit contact inhibition. The amount of product analyzed is comparable to that of Example 2(a).

(f) Example 2(a) is repeated except that the oncogene E1A is replaced with E6. The amount of product analyzed is comparable to that of Example 2(a). (g) Example 2(a) is repeated except that the oncogene El A is replaced with E7.

The amount of product analyzed is comparable to that of Example 1 (a).

(h) Example 2(a) is repeated except that the oncogene El A is replaced with the gene encoding the large T-antigen of the simian virus 40 (SV40). The amount of product analyzed is comparable to that of Example 2(a). (i) Example2 (a) is repeated except that the oncogene El A is replaced with h-ras.

The amount of product analyzed is comparable to that of Example 2 (a).

(j) Example 2(a) is repeated except that the oncogene El A is replaced with c-myc. The amount of product analyzed is comparable to that of Example 2(a).

(k) Example 2(a) is repeated except that the oncogene El A is replaced with c-myb. The amount of product analyzed is comparable to that of Example 2(a).

(1) Example 2(a) is repeated except that the oncogene El A is replaced with c-fos. The amount of product analyzed is comparable to that of Example 2(a).

(m) Example 2(a) is repeated except taht the onco gene El A is replaced with c-jun. The amount of product analyzed is comparable to that of Example 2(a). (n) Example 2(a) is repeated except that the oncogene E 1 A is replaced with other oncogenes that induce carcinogenesis in mammalian cells. The amount of product analyzed is comparable to that of Example 2(a). EXAMPLE 3 DETECTION OF LUNASIN BIOLOGICAL ACTIVITY

The induction of apoptosis by lunasin in ElA-transfected C3H cells provides evidence to a molecular model explaining lunasin's suppression of carcinogen-mediated transformation (Galvez and de Lumen, submitted). The Rb tumor suppressor inhibits the expression of E2F-regulated genes in part by tethering a histone deacetylase (HDACl) to maintain a condensed hypoacetylated chromatin around the transcription start site. The inactivation of Rb by carcinogen treatment and oncogene expression results in the loosening up of the repressed chromatin structure by localized histone acetylation (R.H. Giles, D.J. Peters, M.H. Breuning, Trends Genet. 14, 178, 1998). This consequently results in the activation of genes involved in cell proliferation and eventually leads to carcinogenesis. When lunasin is present in normal cells before Rb is inactivated, the deacetylated N-terminal tails of histone H3 and H4 found in repressed chromatin presumably bind to the acidic carboxyl end of lunasin. This makes these deacetylated histones unavailable as substrates for histone acetylation, thus maintaining the repressed chromatin structure around the E2F promoter even when carcinogens and the viral oncogene, El A, inactivate Rb. The inhibition of expression of E2F-regulated genes triggers apoptosis instead of cell proliferation, which should be the normal occurrence when these genes are activated during carcinogenesis. The induction of apoptosis in cells with inactivated Rb by the presence of lunasin can explain the reduced number of transformed foci in normal murine fibroblast cells that have been treated with potent chemical carcinogens.

The elucidation of the molecular model of lunasin's mechanism of action led to the conceptual synthesis of an in vitro cell culture assay to determine the biological activity of lunasin. Other viral oncoproteins like E6 and E7 from human papilloma virus, as well SV40 T-antigen also inactivate the Rb tumor suppressor in the process of transforming normal cells to proliferating, cancerous cells (J.R. Nevins, Science 258, 424, 1992). The addition of lunasin in mammalian cells transfected with these viral oncogenes is expected to result in apoptosis instead of cell proliferation. An important consideration for this to happen is the presence of a functional p53 (to allow apoptosis to occur) in the mammalian cell line used (i.e. C3H 10T1/2 and NIH 3T3 cells). EXAMPLE 4 APOPTOSIS ASSAY

(a) The ability of lunasin to cause apoptosis in mammalian cells transfected with viral oncogenes that inactivate Rb is used as a measure of its anti-carcinogenic property. The viral oncogenes are transfected into C3H and NIH 3T3 cells that are pretreated with batch solutions containing equimolar amounts of recombinant lunasin peptide. After 24h, the cells are observed for presence of apoptotic cells, which are physically separated from the normal cells and quantitated by analyzing cells through flow cytometry and/or fluorescence and spectrophotometer readings. The relative measure of the quantity of apoptotic cells corrected for transfection efficiency provides a measure of the biological activity in each batch of recombinant lunasin peptide produced.

To determine whether this result is achieved, lunasin was added to actively dividing C3H10T1/2 and NIH 3T3 cells. The cells were then transfected with EIA oncogene, 20 hours after lunasin treatment and release from confluency. As a control, the cells were also transfected with inactivated EIA which does not bind to Rb tumor suppressor. Twenty-four hours after transfection, the cells were analyzed by flow cytometry to determine presence of apoptotic cells. Results of flow cytometry analysis show that it is possible to differentiate apoptotic and viable cells by measuring cell granularity. EIA transfection of lunasin-treated C3H cells resulted in more cells undergoing apoptosis than those transfected with the mutated EIA. The flow cytometry data also showed increase in apoptotic cells by 30-40% in ElA-transfected cells treated with lunasin (see Figures 10G and 10H) which correlates with transfection efficiency as determined by co- transfection with a plasmid vector expressing green fluorescent protein (GFP).

An extension of this experiment is to use stably transfected cells containing viral oncogenes controlled by inducible promoters. After pretreatment of lunasin, oncogene expression is induced and then level of apoptosis is measured to determine biological activity of lunasin.

(b) Similarly when 4 (a) is repeated except that lunasin 1-43 (SEQ.ID.l) is replaced with an equivalently active amount of: protein'having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6), protein having 22 to 43 amino acids (SEQ.ID.7), protein having 22 to 42 amino acids (SEQ.ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and protein having 22 to 38 amino acids (SEQ.ID.12), in each example a comparable result is obtained. (c) Similarly when 5 (a) is repeated except that EIA oncogene is replaced with an equivalently active amount of c3h, NIH 3T3, hras, ela, ras, ras, e7, e6, e-fos, V-myb, V- myc, or c-jun, in each case a comparable result is obtained.

EXAMPLE 5 COLONY FORMATION ASSAY

(a) Loss of cell adherence can also be used as a biological assay to determine Lunasin cancer preventive property. Cell transformation and carcinogenesis leads to the loss of contact inhibition and cell adherence. Normal cells usually form a monolayer of cells at the bottom of plates because of contact inhibition. Upon transformation, cells lose their contact inhibition property and become non-adherent, starting to grow on top of each other and forming distinct colonies in the soft agar plate.

To test the utility of colony formation assay to determine lunasin biological activity, NIH 2-12 cell lines were used. This cell line is characterized by the presence of stably- transfected gene construct in its genome, comprising of a lac inducible promoter and an h-ras oncogene (Liu et al., Cancer Res. 52:983, 1992). Upon induction of IPTG, the cells lose contact inhibition and start to form scorable foci in soft agar within tw weeks (Liu et al., Brit. J. Cancer 77:1777, 1998). The protocol for this assay is outlined below.

Colony Formation (Soft Agar) Assay

1. Prepare 40 ml of 5% agar in PBS with a magnetic stir bar in the bottle.

2. 0.6% base layer agar:

Use 6-well tray, add 1ml of base agar which contains: Agar (5%) 120 ul

Medium (MEM with serum) 880 ul

3. 0.33% upper soft agar:

Prepare cells (1 x 10⁴/100 ul) a. G418 treatment

G418 (8 mg/ml) 40 ul

Agar (0.6%) 550 ul

Medium (MEM with serum) 310 ul Note: Lunasin is added to the medium Total 900 ul b. G418 + IPTG treatment

G418 40 ul

IPTG (IM) 20 ul

Agar (0.6%) 550 ul Medium (MEM with serum) 290 ul

Note: Lunasin is added to the medium

Total 900 ul

4. To make soft agar a. Melt the 5% agar using magnetic stir bar in water bath on the heat plate. b. Warm the medium to 42(C. c. Prepare 0.6% agar. d. Make 0.33% agar by adding the above components. e. Split the 0.33% agar into 3 ml tubes (900 ul /each), and keep them in a heat block at 40(C. f. Add cells (1 x 104/100 ul) to each tube, and transfer to the 6-well tray immediately, g. Add some water outside the wells to prevent drying out. h. Maintain the cells at 37°C for at least 2 weeks.

Using the soft agar assay and NIH2-12 cell lines, the biological activity of 1 uM synthetic Lunasin was measured by counting colony formation two weeks after IPTG induction of h-ras in the presence or absence of Lunasin. Results show that the treatment of Lunasin reduced significantly the colony formation upon induction of h-ras by the addition of IPTG (Fig. 11). This indicates that cell adhesion assay and the use of viral oncogenes, i.e. h-ras, can be used to determine biological activity of lunasin.

An extension of this assay is to transiently transfect normal cells (i.e. NIH 3T3, C3H cells) with viral oncogenes (i.e., h-ras) and then measure foci formation in soft agar in the presence or absence of lunasin.

(b) Similarly when 5(a) is performed in the same manner except that the lunasin 1-43 (SEQ.ID.l) is replaced with an equivalently active amount of: protein having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6), protein having 22 to 43 amino acids (SEQ.ID.7), protein having 22 to 42 amino acids (SEQ.ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and . protein having 22 to 38 amino acids (SEQ.ID.12), in each example a comparable product is obtained .

(c) Similarly when 5(a) is performed in an equivalent manner except that the lac is replaced with an equivalently active amount of tac, the comparable product is obtained.

(d) Similarly when 5(a) is repeated except that the oncogene is replaced with an equivalently active amount of c3h, NIH 3T3, hras, ela, ras, ras, e7, e6, e-fos, N-myb, V- myc, or c-jun, in each example a comparable result is obtained.

(e) Similarly when 5(a) is repeated except that the stably transfected construct is replaced with a transiently transfected construct, a comparable result is obtained.

(f) Similarly when 5(e) is repeated, except that lac is replaced with an equivalently active amount of tac, a comparable result is obtained. (g) Similarly when 5(e) is repeated except that the h-ras is replaced with an equivalently active amount of c3h, ΝIH 3T3, ela, ras, ras, e7, e6, e-fos, N-myb, N-myc, or c-jun, in each example a comparable result is obtained. EXAMPLE 6 STANDARD TRANSFORMATION ASSAY

(a) The standard transformation assay using normal cells (C3H and NIH 3T3) was previously described ( CA. Reznikoff, et al, Cancer Res.33, 3239 (1973) and can also be used to determine Lunasin biological activity. Polycyclic hydrocarbons like 7, 12- dimethylbenz[a]anthracene (DMBA) and 3-methylcholanthrene (MCA) and transfection with viral oncogenes can both be used in the transformation assay . C3H cell line used for the transformation experiments were between the 9^th and 15^th passage and were grown in DMEM + 10% FBS.

(b) Similarly when 6 (a) is repeated except that the lunasin 1-43 (SEQ.ID.l) is replaced with an equivalently active amount of: protein having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6), protein having 22 to 43 amino acids (SEQ.ID.7), protein having 22 to 42 amino acids (SEQ. ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and protein having 22 to 38 amino acids (SEQ.ID.12), in each example a comparable product is obtained. EXAMPLE 7

CHEMICAL CARCINOGENESIS

Confluent plates of C3H cells were trypsinized and resuspended in fresh media to 250-300 cells /mL dilution. For the 24 well plate experiments, 1 mL of cell suspension (containing approximately 300 cells) is added to each well. For the 6 well plates, 2 mL was added to each well (approximately 500 cells). Cells were allowed to adhere overnight in 37°C incubator and after 20h equimolar amounts of the different peptide treatments, including BBI (Sigma T9777) as a positive control, were administered. After 4h, the chemical carcinogen (1.5 mg/mL of DMBA or 5 mg/mL of MCA) was added to the media. Cells were exposed to the carcinogen for 20h after which the cells were washed with 1XPBS and fresh media was added. For negative control cells, acetone was added to the media. Medium was changed every 5 days until the cells reach confluency, then once weekly. After 6 weeks, cells were washed with 0.9% NaCl, fixed by incubating in methanol for 20 min at -20°C, stained with Giemsa and scored for transformed foci.

Plating efficiency was determined by taking the cell count from each carcinogen treatment as a percentage of the cell count from the untreated control, 5 days after the treatment.

Treatments were replicated at least twice and experiments were conducted in duplicate at different cell passages. EXAMPLE 8

VIRAL ONCOGENESIS

Confluent plates of NIH 3T3 cells were trypsinized and around 2.5 x 10⁵ cells were resuspended in fresh DMEM + 10% FBS media using 60 mm cell culture plates. For lunasin treatment, 0.1- 10 mM lunasin was added to the media. After 20 h, C3H cells were transfected with viral oncogene constructs (i.e. ras oncogene) using standard protocols for liposome transfection. Fresh medium was added every 5 days until the cells reach confluency, then once weekly. After 6 weeks, cells were washed with 0.9% NaCl, fixed by incubating in methanol for 20 min at -20°C, stained with Giemsa and scored for transformed foci. Results show that lunasin can suppress ras-induced transformation of NIH 3T3 cells in vitro by 40%, with effective concentration range of lunasin at 0.1 - 10 mM (Fig. 12).

Determination of Quantity and Biological Activity of Recombinant Lunasin The method of determining the presence of the recombinant lunasin in Pichia lysates and quantifying the amounts involve the use of a FilGen proprietary antibody that has been designed and developed to detect lunasin with high accuracy, specificity and efficiency.

The antibody will be used to detect and quantify lunasin in each batch of supernatant containing recombinant lunasin as well as purified forms of the lunasin peptide by conducting enzyme-linked immunosorbent assay (ELISA) and immuno-blot analysis

(Western analysis).

The rapid method of determining the biological activity of the isolated recombinant lunasin in Pichia lysates and/or supernatants involves the use of viral oncogene plasmid constructs. The biological assay for determining lunasin activity uses normal mice embryo fibroblast cells (C3H 10Tl/2 andNIH3T3 cells). The cells are transiently transfected with constitutively expressed oncogenes in the presence or absence of a measured amount of recombinant lunasin or in cases of stable transfectants, induce oncogene expression. The number of cells that undergo apoptosis, loss of cell adherence or foci foraiation are measured and quantified. The values arel plotted on a standard curve generated by graphing lunasin biological effect (i.e. number of apoptotic cells, colony formation, foci formation) in relation to increasing concentrations of pure synthetic lunasin. Lunasin biological activity (LBA) units of each batch of recombinant lunasin will be determined by taking the ratio of this plotted value to the value generated when using equivalent amounts of purified synthetic lunasin. A 100% biological activity will mean that the recombinant lunasin isolated has the same activity as pure synthetic lunasin in equivalent amounts (measured in either weight or moles).

To determine utility of this methodology to measure lunasin biological activity, the colony formation assay was used. A standard curve was generated that shows highly significant negative correlation between the number of colonies in h-ras induced NIH2- 12 cells and increasing dose of synthetic lunasin (Fig. 16). This shows that it should be possible to test and measure biological activity of Lunasin derived from recombinant sources by comparing them to standard curve generated using colony formation assay. While only a few general embodiments of the invention have been shown and described herein, it will become apparent to those skilled in the art that various modifications and changes can be made in the large-scale production of lunasin and in an assay to determine presence and biological activity of lunasin without departing from the spirit and scope of the present invention. All such modifications and changes coming within the scope of the appended claims are intended to be carried out thereby.

Claims

I CLAIM

1. An improved method to produce lunasin or an active variant thereof by recombinant DNA technology in large quantities, which method comprises:

(a) preparing lunasin gene constructs or variants of lunasin gene constructs using protein expression vectors;

(b) optimizing lunasin expression parameters for large-scale production; and

(c) performing a series of isolation and purification steps to obtain large quantities of biologically active recombinant lunasin peptide.

2. The improved method of Claim 1 wherein in step (a) the construct is selected from the group consisting of mammalian, bacteria, insect, plant and bactulovirus expression vectors.

3. The improved method of Claim 2 wherein the protein expression vector is a mammalian expression vector.

4. The improved method of Claim 2 wherein the protein expression vecor is a bacteria expression vector.

5. The improved method of Claim 2 wherein the protein expression vector is a plant expression vector.

6. The improved method of Claim 2 wherein the protein expression vector is baculovirus. 7. The improved method of Claim 1 wherein: in step (a) the lunasin gene expression vectors will produce the lunasin protein selected from the group selected from protein having 1 to 43 amino acids (SEQ.ID.l), protein having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6). protein having 22 to 43 amino acids (SEQ.ID.

7), protein having 22 to 42 amino acids (SEQ.ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and protein having 22 to 38 amino acids (SEQ.ID.12).

8. The improved method of Claim 1 wherein the isolation and purification steps are selected from the group consisting of size exclusion chromatography, ion exchange chromatography reverse phase chromatography, immuno-affinity chromatography and combinations thereof.

9. The method of Claim 1 wherein the protein expression vector is Pichia pastoris.

10. The improved method of Claim 7 wherein in step (a) the gene construct is for lunasin (1-43) (SEQ.ID.l), in step (b) the cells are grown at a pH of about 3 to 5, the induction of lunasin is at low cell weight of about 150 to 200 g/1, the harvest of lunasin ocurs about 40 to 60 hr after induction, minimal or no anti-foaming agent is present, and the lunasin protein produced has 43 amino acids,

11. A method to determine the biological activity of a composition comprising lunasin or variants thereof which method comprises:

(a) obtaining a gene construct of a mammalian transcription unit encoding an oncogene;

(b) preparing gene constructs that contain a mammalian transcription unit encoding a reporter gene;

(c) pretreating normal mammalian cells exhibiting cell adhesion and contact inhibition with compositions containing lunasin peptides or variants thereof;

(d) transfecting pretreated cells with gene constructs of steps (a) and (b);

(e) detecting and quantifying lunasin biological activity by measuring the level of apoptotic cells via flow cytometry, spectrophotometry, fluorometry and microscopy measurements; and

(f) determining the biological activity of compositions containing lunasin by comparison of the flow cytometry, spectrophotometry, fluorometry, and microscopy with standard values determined using predetermined equimolar amounts of pure, synthetic lunasin.

12. The improved method of Claim 11 wherein: in step (b) the gene construct comprise: (A) a reporter gene itself comprising

(B) a transcription unit which expresses an oncogene.

13. The improved method of Claim 11 wherein: the lunasin protein or variants thereof are selected from the group consisting of: protein having 1 to 43 amino acids (SEQ.ID.l), protein having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6). protein having 22 to 43 amino acids (SEQ.ID.7), protein having 22 to 42 amino acids (SEQ.ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and protein having 22 to 38 amino acids (SEQ.ID.12).

14. The improved method of Claim 11 wherein: in step (b) the reporter gene is selected from the group consisting of green fluorescent protein (GFP), luciferase, and glucorunidase (GUS protein).

15. The improved method of Claim 11 wherein: in step (c) wherein the mammalian cell is selected from C3H 10T1/2 or NIH 3T3 murine cells.

16. The improved method of Claim 11 wherein: in step (a) the oncogene is selected from the group consisting of:

EIA, E6, E7, the gene encoding the large T-antigen of simian virus 40 (SV40), h-ras, c-myc, c-myb, c-fos, c-jun, mdm2 and combinations thereof.

17. The improved method of Claim 11 wherein the oncogene is selected for EIA, h-ras, the large T-antigen of simion virus 40(SV40), or E7.

18. The improved method of Claim 11 wherein the protein is lunasin 1-43; the reporter gene is green fluorescent protein; and the oncogene is EIA.

19. The improved method of Claim 11 wherein the protein is lunasin 22 to 43, the reporter gene is green fluorescent protein and the oncogene is the large T-antigen of simion virus 40(SV40).

20. The improved method of Claim 11 wherein the gene construct is stably transfected in NIH 3T3 or C 3H 1OT1/2 cells.???

21. A method to determine the biological activity of a composition comprising lunasin or active variant thereof, which method comprises:

(a) obtaining a cell line having the property of cell adherence and contact inhibition having a lad or laclq stably integrated into the genome;

(b) stably transfect a gene construct comprising lac or tac inductable promotor and h-ras oncogene; (c) inducing expression of the oncogene by addition of IPTG; and

(d) determining by visual observation the number of scorable foci in soft agar wherein the prescence of lunasin or active variant thereof results in a lower number of scorable foci and is dependent on the dose level.

22. The method of Claim 21 wherein the lunasin variant is selected from the group of: protein having 1 to 43 amino acids (SEQ.ID.l), protein having 22 to 43 amino acids (SEQ.ID.2), protein having 22 to 42 amino acids (SEQ.ID.3), protein having 22 to 41 amino acids (SEQ.ID.4), protein having 22 to 40 amino acids (SEQ.ID.5), protein having 22 to 39 amino acids (SEQ.ID.6), protein having 22 to 38 amino acids (SEQ.ID.7), protein having 1 to 42 amino acids (SEQ.ID.8), protein having 1 to 41 amino acids (SEQ.ID.9), protein having 1 to 40 amino acids (SEQ.ID.10), protein having 1 to 39 amino acids (SEQ.ID.l 1), and protein having 1 to 38 amino acids (SEQ.ID.12).

23. The method of Claim 21 wherein the oncogene of step (a) is selected from the group consisting of: EIA, E6, E7, the gene encoding the large T-antigen of simian virus 40 (SV40), h-ras, c- myc, c-myb, c-fos, c-jun, mdm2 and combinations thereof.

24. A method to determine the biological activity of a composition comprising lunasin or active variant thereof, which method comprises: (a) obtaining a cell line having the property of cell adherence and contact inhibition having a lad or laclq transiently integrated into the genome;

(b) transiently transfecting a gene construct comprising lac or laclq inductable promoter and the h-ras oncogene (c) inducing expression of the oncogene by addition of IPTG; and

(d) determining by visual observation the number of scorable foci in soft agar wherein the prescence of lunasm or active variant thereof results in a lower number of scorable foci and is dependent on the dose level.

25. The method of Claim 24 wherein the lunasin variant is selected from the group of: protein having 1 to 43 amino acids (SEQ.ID.l), protein having 1 to 42 amino acids (SEQ.ID.2), protein having 1 to 41 amino acids (SEQ.ID.3), protein having 1 to 40 amino acids (SEQ.ID.4), protein having 1 to 39 amino acids (SEQ.ID.5), protein having 1 to 38 amino acids (SEQ.ID.6). protein having 22 to 43 amino acids (SEQ.ID.7), protein having 22 to 42 amino acids (SEQ.ID.8), protein having 22 to 41 amino acids (SEQ.ID.9), protein having 22 to 40 amino acids (SEQ.ID.10), protein having 22 to 39 amino acids (SEQ.ID.l 1), and protein having 22 to 38 amino acids (SEQ.ID.12).

26. The method of Claim 23 wherein the oncogene of step (a) is selected from the group consisting of: EIA, E6, E7, the gene encoding the large T-antigen of simian virus 40 (SV40), h-ras, c- myc, c-myb, c-fos, c-jun, mdm2 and combinations thereof.