WO2001081368A2

WO2001081368A2 - Blood-pressure reducing polypeptides containing vpp derived from microorganisms

Info

Publication number: WO2001081368A2
Application number: PCT/US2001/012776
Authority: WO
Inventors: Joseph M. Kobzeff
Original assignee: Monsanto Technology Llc
Priority date: 2000-04-21
Filing date: 2001-04-19
Publication date: 2001-11-01
Also published as: WO2001081368A3; AU2001255501A1

Abstract

The invention relates to a method of producing the anti-hypertensive tripeptide VPP. The invention also relates to pharmaceutical, nutritional, and food compositions employing VPP as the active ingredient.

Description

BLOOD-PRESSURE REDUCING POLYPEPTIDES CONTAINING VPP DERIVED FROM MICROORGANISMS

Field of Invention

This invention generally relates to a method for producing polypeptides containing the tripeptide VPP. The method encompasses growing genetically modified microorganisms containing a recombinant Bile Salt-Stimulated Lipase (BSSL) fragment and hydrolyzing the proteinaceous matter to purify the polypeptide. This polypeptide contains significant amounts of the tripeptide VPP. The VPP may then be further purified from this polypeptide.

Background of the Invention

A significant number of people are afflicted with hypertension. Hypertension is generally known as the "silent killer" because its only readily detectable symptom is an abnormally increased blood pressure. Abnormally increased blood pressure is clinically defined as a systolic blood pressure greater than 140 mmHg or a diastolic blood pressure greater than 90 mmHg. Hypertension is the primary risk factor for coronary, cerebral, and renal vascular diseases, which collectively cause over half o.f all deaths in the United States. And no single or specific cause is known for the hypertension referred to as primary (essential) hypertension. Among a number of factors for regulating blood pressure, the renin-angiotensin system plays an important role in salt-water homeostasis and the maintenance of vascular tone. In fact, stimulation or inhibition of this system, respectively, raises or lowers blood pressure, and ' may be involved in the etiology of hypertension. Hall, J.E., and Guyton, A.C. (1990), In Hypertension : Pathophysiology Diagnosis and Management, (Raven Press, Ltd., New York), pp .1105-1129. Angiotensin converting enzyme (ACE) is an important mediator in the renin- angiotensin system. ACE acts on angiotensin I, which is formed by decomposition of angiotensin secreted by the liver, by an enzyme, renin, produced in the kidney, and converts it to angiotensin II. Angiotensin II then increases blood pressure by contracting the smooth muscles of the blood vessel walls and promoting secretion of aldosterone by action on the adrenal cortex. Additionally, ACE decomposes and inactivates a protein called bradykinin. Bradykinin dilates the blood vessels and lowers blood pressure. Therefore, a common method of decreasing blood pressure in an individual is to inhibit the activity of ACE. Several compounds have been studied for their ability to reduce blood pressure by inhibiting the function of ACE in the renin-angiotensin mechanism. Current pharmaceutical treatments for hypertension include diuretics, beta-blockers, angiotensin converting enzyme inhibitors and calcium antagonists. Many ACE inhibiting substances are known and commonly used for the purpose of decreasing blood pressure in patients. Among commonly used ACE inhibiting pharmaceuticals is the synthetic chemical product known as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) , which is an oral hypotensive agent . Currently available anti-hypertensive agents, however, possess significant side effects including elevation of blood lipids and glucose. Also, special care must be taken to monitor the safety of synthetic chemical products for anti-hypertensive use.

In addition, several dietary supplements derived either from natural sources or synthesized, have been evaluated for their ability to reduce blood pressure. A number of peptides isolated from natural sources such as milk, soy, corn, gelatin, wheat, and fish protein have been identified as having functions relating to physiological regulation and ACE inhibition. See e . g. , U.S. Patent Nos . 5,238,932 and 5,071,955. Recently, two tripeptides were reported as having strong ACE inhibiting activity, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) . Both VPP and IPP have been derived from lactic acid bacteria-fermented milk. See Nakamura et al . , J. Dairy Sci . 78:777-783 (1995). Furthermore, these tripeptides have been reported to exhibit a strong antihypertensive effect in spontaneously hypertensive rats (SHR) . See Nakamura, et al . , J. Dairy Sci. , 78:1253-1257 (1995) . VPP, however, is only present in low concentration in fermented cow milk. Thus, it is difficult to obtain a substantial amount of this tripeptide from natural sources needed for use in treating high blood pressure.

Accordingly, a need exists to produce nutritional or dietary supplements that contain VPP without the unpredictability and cost associated with current production methods. VPP may then be used for pharmaceutical compositions, dietary supplements, food ingredients, and foods for the specified health uses of reducing and inhibiting hypertension and diseases related to hypertension at a low cost with no appreciable side effects.

Summary of the Invention

Among the several aspects of the invention, therefore, is provided a method for producing recombinant BSSL containing VPP comprising growing host cells under conditions where the host cells express the recombinant BSSL polypeptide.

Another aspect provides a pharmaceutical composition comprising VPP produced by the method of the invention and a pharmaceutically acceptable carrier, diluent or excipient . In yet a further aspect of the invention is provided a nutritional composition comprising VPP produced by the method of the invention and a nutritionally acceptable carrier, dilutent or excipient. Still another aspect of the invention is provided a food composition comprising WP produced by the method of the invention and a nutritionally acceptable carrier, dilutent or excipient .

Other features of the present invention will be in part apparent to those skilled in the art and in part pointed out in the detailed description provided below.

Abbreviations and Definitions

To facilitate understanding of the invention, a number of terms as used herein are defined below: The amino acid residues are abbreviated herein according to their single letters: A represents alanine; R represents arginine; N represents asparagine; D represents aspartic acid; C represents cysteine; Q represents glutamine; E represents glutamic acid; G represents glycine; H represents histidine; I represents isoleucine; L represents leucine; K represents lysine; M represents methionine; F represents phenylalanine; P represents proline; S represents serine; T represents threonine; represents tryptophan; Y represents tyrosine; and V represents valine. As used herein, "ACE" shall mean angiotensin converting enzyme .

As used herein, the terms "treatment" or "treating" relate to any treatment of hypertensive disease and include: (1) preventing hypertension from occurring in a subject who may be predisposed to the disease but who has not yet been diagnosed as having it; (2) inhibiting the disease, i.e., arresting its development; or (3) ameliorating or relieving the symptoms of the disease, i.e., causing regression of the hypertensive state.

As used herein, the terms "substantially pure" or "isolated", when referring to proteins and polypeptides, denotes those polypeptides that are separated from proteins or other contaminants with which they are naturally associated. A protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure protein will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.

As used herein, "recombinant BSSL" shall mean a non- native polypeptide derived by recombinant means or a native polypeptide with an altered amino acid sequence.

As used herein, "moiety" shall mean an identified sequence of polypeptide residues.

As used herein, "operable association" or "operably linked" are used interchangeably and shall mean a unit of coordinated and regulated gene activity by means of which the control and synthesis of a protein is determined. It consists of a DNA coding a structural gene together with one or more regulatory regions .

Detailed Description Applicant has discovered a method for the industry scale production of polypeptides containing VPP. The method comprises obtaining microorganisms genetically altered to produce a recombinant polypeptide that contains a significant amount of VPP. In accordance with this method, the proteinaceous matter is hydrolyzed from the microorganism and VPP is then further purified from the polypeptide.

Specifically, the method of the present invention can be beneficially used to produce the anti-hypertensive tripeptide VPP. This tripeptide has significant anti- hypertensive activities and therefore is useful in the diagnosis, treatment and prophylaxis of hypertension and related conditions such as left ventricular systolic dysfunction, myocardial infarction, diabetes mellitus and progressive renal impairment/failure. Furthermore, the peptide is not expected to have the negative side effects associated with the anti-hypertensive pharmaceutical products.

Any polypeptide that contains a significant amount of the tripeptide sequence VPP in its amino acid sequence may be employed in the present invention. In one embodiment, accordingly, the polypeptide Bile Salt-Stimulated Lipase ("BSSL") is typically utilized. BSSL functions as a nonspecific lipase because it hydrolyzes not only triacylglyceral, but also diacyl and monoacylglyceral, as well as cholesteryl esters (Hernell et al . , (1993) J. Pediatr. Gastroenterol . Nutr.16 :426-31) In addition, BSSL is a natural constituent of milk in a limited number of species, e.g., humans, gorillas, cats and dogs, and can accumulate to approximately 1% of total milk protein in these species (Hernell et al . , (1989) in Textbook of gastroenterology and nutrition, pp 209-217; and Hamosh et al., (1986) Fed. Proc . 45:1452).

The cDNA sequence of human milk BSSL has been characterized (Baba et al . , (1991) Biochem. 30:500-510)

( SEQ ID No: 1) . BSSL is a single chain glycoprotein. The deduced sequence of the mature protein contains 722 amino acids and is highly glycosylated (SEQ ID No : 2) . The carboxy-terminal region contains 16 proline-rich repeating units of 11 amino acids each. Additionally, each repeating unit contains the tripeptide sequence VPP. Thus, each BSSL polypeptide contains 16 repeats of the tripeptide sequence VPP. BSSL, accordingly, because it contains significant amounts of VPP and accumulates to 1% of total milk protein, is particularly advantageous for use in the present invention.

The present invention employs the use of recombinant technology to produce a recombinant BSSL polynucleotide . BSSL cDNA has been isolated from a number of sources including humans (SEQ ID NO: 1) . BSSL from any organism may be employed to the extent that the amino acid sequence contains a significant amount of the sequence VPP. Preferably, however, the BSSL employed is from humans. A recombinant polynucleotide is one in which polynucleotide sequences of different organisms have been joined together to form a single unit. Isolation of cDNA and construction of recombinant polynucleotides can be accomplished using a variety of procedures commonly known to those skilled in the art and detailed in, for example, Sambrook et al . , Molecular Cloning, A Laboratory Mannual , 2nd ed. , Cold Spring Harbor Laboratory Press, (1989) and Ausabel et al . , Short Protocols in Molecular Biology, 3rd. ed. , John Wiley & Sons (1995) . A cloning vector is a self-replicating DNA molecule that serves to transfer a DNA segment into a host cell . The three most common types of cloning vectors are bacterial plasmids, phages, and other viruses. An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into a protein.

Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells. In cloning vectors, this sequence is generally one that enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences. Various bacterial and viral origins of replication are well known to those skilled in the art and include, but are not limited to the pBR322 plasmid origin, the 2μ plasmid origin, and the SV40, polyoma, adenovirus, VSV and BPV viral origins. The polynucleotide sequence of the present invention may be used to produce proteins by the use of recombinant expression vectors containing the sequence. Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV 40 derivatives; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; and viral DNA such as vaccinia, adenovirus, fowl pox virus, retroviruses, and pseudorabies virus. In addition, any other vector that is replicable and viable in the host may be used. The nucleotide sequence of interest may be inserted into the vector by a variety of methods . In the most common method the sequence is inserted into an appropriate restriction endonuclease site(s) using procedures commonly known to those skilled in the art and detailed in, for example, Sambrook et al . , Molecular Cloning, A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al . , Short Protocols in Molecular Biology, 3rd ed. , John Wiley & Sons (1995) .

In an expression vector, the sequence of interest is operably linked to a suitable expression control sequence or promoter recognized by the host cell to direct mRNA synthesis . Promoters are untranslated sequences located generally 100 to 1000 base pairs (bp) upstream from the start codon of a structural gene that regulate the transcription and translation of nucleic acid sequences under their control . Promoters are generally classified as either inducible or constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in the environment, e.g., the presence or absence of a nutrient or a change in temperature. Constitutive promoters, in contrast, maintain a relatively constant level of transcription. In addition, useful promoters can also confer appropriate cellular and temporal specificity. Such promoters include those that are developmentally-regulated or organelle-, tissue- or cell-specific. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking is achieved by blunt end ligation or ligation at restriction enzyme sites. If suitable restriction sites are not available, then synthetic oligonucleotide adapters or linkers can be used as is known to those skilled in the art (Sambrook et al . , Molecular Cloning, A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al . , Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons (1995)) .

Common promoters used in expression vectors include, but are not limited to, CMV promoter, LTR or SV40 promoter, the E. coli lac or trp promoters, and the phage lambda PL promoter. Other promoters known to control the expression of genes in prokaryotic or eukaryotic cells can be used and are known to those skilled in the art. Expression vectors may also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may also contain sequences useful for the amplification of gene expression.

Expression and cloning vectors can and usually do contain a selection gene or selection marker. Typically, this gene encodes a protein necessary for the survival or growth of the host cell transformed with the vector. Examples of suitable markers include dihydrofolate reductase (DHFR) or neomycin or hygromycin B resistance for eukaryotic cells and tetracycline, ampicillin, or kanamycin resistance for E. coli .

In addition, expression vectors can also contain marker sequences operatively linked to a nucleotide sequence for a protein that encodes an additional protein used as a marker. The result is a hybrid or fusion protein comprising two linked and different proteins. The marker protein can provide, for example, an immunological or enzymatic marker for the recombinant protein produced by the expression vector. In a preferred embodiment of the present invention, alkaline phosphatase (AP) , green fluorescence protein (GFP) , myc, histidine tag (His) and hemagglutinin (HA) are used as markers .

Additionally, the end of the polynucleotide can be modified by the addition of a sequence encoding an amino acid sequence useful for purification of the protein produced by affinity chromatography. Various methods have been devised for the addition of such affinity purification moieties to proteins. Representative examples can be found in U.S. Patent Nos . 4,703,004, 4,782,137, 4,845,341,

5,935,824, and 5,594,115. Any method known in the art for the addition of nucleotide sequences encoding purification moieties can be used, for example, those contained in Innis et al . , PCR Protocols, Academic Press (1990) and Sambrook et al . , Molecular Cloning, 2nd ed. , Cold Spring Harbor Laboratory Press (1989) . More particularly, the present invention includes recombinant constructs comprising the isolated polynucleotide sequence of the present invention. The constructs can include a vector, such as a plasmid or viral vector, into which the sequence of the present invention has been inserted, either in the forward or reverse orientation. The recombinant construct further comprises regulatory sequences, including for example, a promoter operatively linked to the sequence. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available. In one preferred embodiment, the pCS2+, the pCEP4 (Invitrogen) and the pIRESneo (Clontech) vectors are used. It will be understood by those skilled in the art, however, that other plasmids or vectors may be used as long as they are replicable and viable or expressing the encoded protein in the host .

The polynucleotide sequences of the present invention can also be part of an expression cassette that at a minimum comprises, operably linked in the 5' to 3 ' direction, a promoter, a polynucleotide of the present invention, and a transcriptional termination signal sequence functional in a host cell . The promoter can be of any of the types discussed herein, for example, a tissue specific promoter, a developmentally regulated promoter, an organelle specific promoter, etc. The expression cassette can further comprise an operably linked targeting sequence, transit or secretion peptide coding region capable of directing transport of the protein produced. The expression cassette can also further comprise a nucleotide sequence encoding a selectable marker and a purification moiety.

A further embodiment of the present invention relates to transformed host cells containing the constructs comprising the recombinant BSSL. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell such as an insect cell or a yeast cell, or the host can be a prokaryotic cell such as a bacterial cell . Introduction of the construct into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, Polybrene mediated transfection, protoplast fusion, liposome mediated transfection, direct microinj ection into the nuclei, biolistic (gene gun) devices, scrape loading, and electroporation.

The present invention also relates to proteins encoded by the isolated BSSL polynucleotide. As used herein the term protein includes fragments, analogs and derivatives of the BSSL protein. The terms "fragment", "derivative" and "analog" as used herein mean a polypeptide that retains essentially the same biological function or activity as the recombinant BSSL protein. For example, an analog includes a proprotein which can be cleaved to produce an active mature protein. The protein of the present invention can be a natural protein, a recombinant protein or a synthetic protein or a polypeptide.

The term protein also includes forms of the BSSL protein to which one or more substituent groups have been added. A substituent is an atom or group of atoms that is introduced into a molecule by replacement of another atom or group of atoms. Such groups include, but are not limited to lipids, phosphate groups, sugars and carbohydrates. Thus, the term protein includes, for example, lipoproteins, glycoproteins, phosphoproteins and phospholipoproteins . The present invention also includes methods for the production of the recombinant BSSL protein from host cells . Proteins can be expressed in mammalian cells, plant cells, insect cells, yeast, bacteria, bacteriophage, or other appropriate host cells. Host cells are genetically transformed to produce the protein of interest by introduction of an expression vector containing the nucleic acid sequence of interest. The characteristics of suitable cloning vectors and the methods for their introduction into host cells have been previously discussed. Alternatively, cell-free translation systems can also be employed using RNA derived from the DNA of interest. Methods for cell free translation are known to those skilled in the art. (Davis et al . , Basic Methods in Molecular Biology, Elsevier Science Publishing (1986); Ausubel et al . , Short Protocols in

Molecular Biology, 2^nd Ed., John Wiley & Sons (1992)). In the preferred embodiment, host cells are HEK 293 cells or 293T cells (American Type Culture Collection) .

Host cells are grown under appropriate conditions to a suitable cell density. If the sequence of interest is operably linked to an inducible promoter, the appropriate environmental alteration is made to induce expression. If the protein accumulates in the host cell, the cells are harvested by, for example, centrifugation or filtration. The cells are then disrupted by physical or chemical means to release the protein into the cell extract from which the protein can be purified. If the host cells secrete the protein into the medium, the cells and medium are separated and the medium retained for purification of the protein. Polypeptides recovered can be purified by a variety of commonly used methods, including, but not limited to, ammonium sulfate precipitation, immuno precipitation, ethanol or acetone precipitation, acid extraction, ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography, electrophoresis, and ultra filtration. If required, protein refolding systems can be used to complete the configuration of the protein.

In one embodiment, hydrolysis is employed to purify the polypeptide according to the method of the invention. More particularly, the proteinaceous matter is measured, and mixed with a liquid such as water, preferably distilled water, or a buffer (for example, a Tris-HCl buffer or a phosphate buffer) . The proteinaceous matter may be purified from the microorganism according to any method generally known in the art. Before hydrolysis, the protein and liquid mixture is typically homogenized. Homogenization of the mixture improves the activity of cleaving agent used to hydrolyze the target polypeptide from the protein mixture. The concentration of target protein in the mixture is generally in the range of about 1 to about 50% (w/v) , and more preferably about 10%.

Once the mixture is thoroughly homogenized, it is adjusted to a pH that is favorable for the activity of the selected cleaving agent utilized in the hydrolysis reaction. For example, an acidic cleaving agent like pepsin, the most favorable pH range is about 0.1 to about 4, and more preferably about 2. In contrast, an alkaline cleaving agent would work most effectively in a pH range of about 8 about to 10. The pH adjusted mixture is raised to a boiling temperature for a period of approximately 5 to 60 minutes, preferably for about 10 to 15 minutes. This boiling step inactivates all endogenous enzymes present in the source protein. The pH in course of reaction can, if necessary, be adjusted with a base like aqueous sodium hydroxide solution, or an acid, like hydrochloric acid. Any suitable acid or base, however, may be employed to adjust the pH of the reaction solution.

After the mixture is boiled, it is typically allowed to cool to approximately 50° C or below, preferably about 37° C. Polypeptides contained in the protein solution used in the invention can be prepared by a process to hydrolyze or digest the selected protein with a cleaving agent . The target protein can be hydrolyzed via chemical or proteolytic methods. For example, a cleavage agent like a protease is added to the mixture to hydrolyze the protein material within the mixture. Commercially available proteases such as thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, or Actinase E may be used to digest the protein. Thermolysin is particularly preferred as the digesting enzyme.

The optimal concentration of the cleaving agent within the digestion mixture is dependent upon the cleaving agent selected. Although the additional amount of the enzyme thermolysin is varied depending on its titer, the final concentration within the reaction mixture is preferably about 100 μg/ml to about 1000 μg/ml, and more preferably the final thermolysin concentration is approximately 800 to 880 μg/ml based on the protein. It is also possible to add part of the thermolysin in the course of the reaction. To promote protein digestion, the temperature of the reaction mixture during the reaction may be maintained between about 30° C to about 50° C, and more preferably about 37° C. The reaction time varies depending on the amount of the enzyme, reaction temperature and reaction pH. But the time is typically about 1 to about 24 hours, and more preferably about 1 to about 6 hours .

The digestion reaction can be stopped according to a known method, for example, according to inactivation of the cleaving agent either by heating of the reaction mixture or by pH change with addition of an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide, or according to separation of the enzyme by filtration using an ultrafiltration membrane or the like. Once the cleaving agent is inactivated, the digestion mixture is cooled to approximately 0° C to 30° C, and most preferably about 4° C. The polypeptides can be isolated from the resulting digestion solution through solid-liquid separation, for example, centrifugation or filtration. The resulting liquid can be fractionated by ultrafiltration, gel filtration or the like to obtain liquid containing the desired target polypeptides of the invention. The fractionated liquid is further fractionated to obtain each objective polypeptide according to its size and retention time.

The cooled mixture is transferred into centrifuge tubes and separated into solid and liquid phases by centrifugation at approximately 1500 to about 3000 rpm. Centrifugation is conducted for a period of about 10 to 20 minutes, and more preferably for about 15 minutes. The supernatant is then removed and transferred to a clean centrifuge tube and centrifuged a second time at approximately 8000 rpm for a period of about 10 to 20 minutes, and more preferably for about 10 minutes, at a temperature below about 30° C.

The contents of the centrifuge tubes are then dried under nitrogen gas at a temperature of approximately 30° C to 80° C, and more preferably about 37° C. A mobile phase solvent is added to the centrifuge tubes and the dried contents are resuspended to form a mobile phase mixture. The mobile phase mixture is then centrifuged at about 1000 to about 10,000 rpm, and more preferably approximately 8000 rpm at a temperature below about 30° C for a period of about 10 to 20 minutes.

The resulting supernatant, comprising VPP, may then be further purified by chromatographic methods such as liquid chromatography, HPLC, FPLC, or the like. The VPP, or other desired peptides, are then isolated according to their respective retention times. In a preferred embodiment, VPP is substantially purified by any means generally known in the art . The VPP tripeptide, as stated above, has anti- hypertensive activity that effectively depress elevated blood pressure, but that, unlike current anti-hypertensive pharmaceutical compounds, do not have adverse side effects such as altering normal blood pressure, over depressing blood pressure or other side effects. Accordingly, the VPP produced by the method of the invention may be added to a number compositions and administered to treat hypertensive disease. In particular the VPP may be added to nutritional compositions (e.g. dietary supplements) , food compositions, vaccine compositions, drug and pharmaceutical compositions. Those of ordinary skill in the art of preparing pharmaceutical formulations can readily formulate pharmaceutical compositions having VPP produced by the method of the invention using known excipients, carriers or diluents (e.g. saline, glucose, starch etc.). Similarly, those of ordinary skill in the art of preparing nutritional formulations can readily formulate nutritional compositions having VPP produced by the method of the invention using nutritionally acceptable excipients. And those of ordinary skill in the art of preparing food or food ingredient compositions can readily formulate food compositions having VPP produced by the method of the invention employing nutritionally acceptable excipients.

In view of the detailed description provided above, the several objects of the invention have been accomplished. The detailed description is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variation in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

In addition, it is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents.

While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations . It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description.

Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and scope of the following claims.

All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference .

Claims

ClaimsWHAT IS CLAIMED IS:

1. A method for producing polypeptides containing the tripeptide VPP, the method comprising: (a) obtaining a recombinant expression vector comprising a nucleic acid encoding recombinant BSSL polypeptide;

(b) inserting such recombinant expression vector into a host cell; (c) growing such host cell under conditions where said host cell expresses a protein encoded by said recombinant vector; and

(d) purification of the recombinant protein from the host cell.

2. The method of claim 1 wherein the recombinant vector further comprises at least one additional sequence chosen from the group consisting of:

(a) regulatory sequences operatively coupled to the polynucleotide;

(b) selection markers operatively coupled to the polynucleotide;

(c) marker sequences operatively coupled to the polynucleotide; (d) a purification moiety operatively coupled to the polynucleotide;

(e) a secretion sequence operatively coupled to the polynucleotide; and

(f) a targeting sequence operatively coupled to the polynucleotide.

3. The method of claim 2 wherein the vector further comprises a developmentally-regulated, an organelle- specific, a tissue-specific, or cell-specific promoter.

4. The method of claim 1 wherein the host cell is selected from the group consisting of mammalian cells, plant cells, insect cells, yeast, bacteria, and bacteriophage.

5. The method of claim 4 wherein the host cell is a bacterial cell.

6. The method of claim 5 wherein the host cell is a yeast cell.

7. The method of claim 6 wherein the recombinant protein is BSSL.

8. The method of claim 1 wherein the purification is accomplished by hydrolysis.

9. The method of claim 8 wherein the hydrolysis is accomplished by chemical methods or enzymatic methods.

10. The method of claim 7 further comprising substantially purifying the VPP from the recombinant BSSL polypeptide.

11. The method of 10 wherein the VPP is purified by chromatography.

12. The method of claim 11 wherein the chromatographic method utilized is HPLC, FPLC, or affinity chromatography.

13. A nutritional composition comprising VPP and a nutritionally acceptable carrier, diluent or excipient, wherein the VPP is produced by the method of claim 1.

14. A food composition comprising VPP and a nutritionally acceptable carrier, diluent, or excipient, wherein the VPP is produced by the method of claim 1.

15. A pharmaceutical composition comprising VPP and a pharmaceutically acceptable carrier, diluent or excipient, wherein the VPP is produced by the method of claim 1.