Starch encapsulation

This application is a continuation of U.S. patent application Ser. No. 09/625,406, filed Jul. 25, 2000, now abandoned which is a continuation of U.S. patent application Ser. No. 08/941,445, filed Sep. 30, 1997 and now U.S. Pat. No. 6,107,060, which claims priority to provisional patent application Ser. No. 60/026,855 filed Sep. 30, 1996. Said provisional application is incorporated herein by reference to the extent not inconsistent herewith.

Polysaccharide Enzymes

Both prokaryotic and eukaryotic cells use polysaccharide enzymes as a storage reserve. In the prokaryotic cell the primary reserve polysaccharide is glycogen. Although glycogen is similar to the starch found in most vascular plants it exhibits different chain lengths and degrees of polymerization. In many plants, starch is used as the primary reserve polysaccharide. Starch is stored in the various tissues of the starch bearing plant. Starch is made of two components in most instances; one is amylose and one is amylopectin. Amylose is formed as linear glucans and amylopectin is formed as branched chains of glucans. Typical starch has a ratio of 25% amylose to 75% amylopectin. Variations in the amylose to amylopectin ratio in a plant can effect the properties of the starch. Additionally starches from different plants often have different properties. Maize starch and potato starch appear to differ due to the presence or absence of phosphate groups. Certain plants' starch properties differ because of mutations that have been introduced into the plant genome. Mutant starches are well known in maize, rice and peas and the like.

The changes in starch branching or in the ratios of the starch components result in different starch characteristic. One characteristic of starch is the formation of starch granules which are formed particularly in leaves, roots, tubers and seeds. These granules are formed during the starch synthesis process. Certain synthases of starch, particularly granule-bound starch synthase, soluble starch synthases and branching enzymes are proteins that are “encapsulated” within the starch granule when it is formed.

The use of cDNA clones of animal and bacterial glycogen synthases are described in International patent application publication number GB92/101881. The nucleotide and amino acid sequences of glycogen synthase are known from the literature. For example, the nucleotide sequence for the E. coli glgA gene encoding glycogen synthase can be retrieved from the GenBank/EMBL (SWISSPROT) database, accession number J02616 (Kumar et al., 1986, J. Biol. Chem., 261:16256–16259). E. coli glycogen biosynthetic enzyme structural genes were also cloned by Okita et al. (1981, J. Biol. Chem., 256(13):6944–6952). The glycogen synthase glgA structural gene was cloned from Salmonella typhimurium LT2 by Leung et al. (1987, J. Bacteriol., 169(9):4349–4354). The sequences of glycogen synthase from rabbit skeletal muscle (Zhang et al., 1989, FASEB J., 3:2532–2536) and human muscle (Browner et al., 1989, Proc. Natl. Acad. Sci., 86:1443–1447) are also known.

The use of cDNA clones of plant soluble starch synthases has been reported. The amino acid sequences of pea soluble starch synthase isoforms I and II were published by Dry et al. (1991, Plant Journal, 2:193202). The amino acid sequence of rice soluble starch synthase (SSTS) was described by Baba et al. (1993, Plant Physiology,). This last sequence (rice SSTA) incorrectly cites the N-terminal sequence and hence is misleading. Presumably this is because of some extraction error involving a protease degradation or other inherent instability in the extracted enzyme. The correct N-terminal sequence (starting with AELSR SEQ. ID NO:38) is present in what they refer to as the transit peptide sequence of the rice SSTS.

The sequence of maize branching enzyme I was investigated by Baba et al., 1991, BBRC, 181:8794. Starch branching enzyme II from maize endosperm was investigated by Fisher and Shrable (1993, Plant Physiol., 102:10451046). The use of cDNA clones of plant, bacterial and animal branching enzymes have been reported. The nucleotide and amino acid sequences for bacterial branching enzymes (BE) are known from the literature. For example, Kiel et al. cloned the branching enzyme gene glgB from Cyanobacterium synechococcussp PCC7942 (1989, Gene (Amst), 78(1):918) and from Bacillus stearothermophilus (Kiel et al., 1991, Mol. Gen. Genet., 230(12):136–144). The genes glc3 and gha1 of S. cerevisiae are allelic and encode the glycogen branching enzyme (Rowen et al., 1992, Mol. Cell Biol., 12(1):22–29). Matsumomoto et al. investigated glycogen branching enzyme from Neurospora crassa (1990, J. Biochem., 107:118–122). The GenBank/EMBL database also contains sequences for the E. coli glgB gene encoding branching enzyme.

Starch synthase (EC 2.4.1.11) elongates starch molecules and is thought to act on both amylose and amylopectin. Starch synthase (STS) activity can be found associated both with the granule and in the stroma of the plastid. The capacity for starch association of the bound starch synthase enzyme is well known. Various enzymes involved in starch biosynthesis are now known to have differing propensities for binding as described by Mu-Forster et al. (1996, Plant Phys. 111: 821–829). Granule-bound starch synthase (GBSTS) activity is strongly correlated with the product of the waxy gene (Shure et al., 1983, Cell 35: 225–233). The synthesis of amylose in a number of species such as maize, rice and potato has been shown to depend on the expression of this gene (Tsai, 1974, Biochem Gen 11: 83–96; Hovenkamp-Hermelink et al., 1987, Theor. Appl. Gen. 75: 217–221). Visser et al. described the molecular cloning and partial characterization of the gene for granule-bound starch synthase from potato (1989, Plant Sci. 64(2):185192). Visser et al. have also described the inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs (1991, Mol. Gen. Genet. 225(2):289296).

The other STS enzymes have become known as soluble starch synthases, following the pioneering work of Frydman and Cardini (Frydman and Cardini, 1964, Biochem. Biophys. Res. Communications 17: 407–411). Recently, the appropriateness of the term “soluble” has become questionable in light of discoveries that these enzymes are associated with the granule as well as being present in the soluble phase (Denyer et al., 1993, Plant J. 4: 191–198; Denyer et al., 1995, Planta 97: 57–62; Mu-Forster et al., 1996, Plant Physiol. 111: 821–829). It is generally believed that the biosynthesis of amylopectin involves the interaction of soluble starch synthases and starch branching enzymes. Different isoforms of soluble starch synthase have been identified and cloned in pea (Denyer and Smith, 1992, Planta 186: 609–617; Dry et al., 1992, Plant Journal, 2: 193–202), potato (Edwards et al., 1995, Plant Physiol. 112: 89–97; Marshall et al., 1996, Plant Cell 8: 1121–1135) and in rice (Baba et al., 1993, Plant Physiol. 103: 565–573), while barley appears to contain multiple isoforms, some of which are associated with starch branching enzyme (Tyynela and Schulman, 1994, Physiol. Plantarum 89: 835–841). A common characteristic of STS clones is the presence of a KXGGLGDV (SEQ. ID NO:39) consensus sequence which is believed to be the ADP-Glc binding site of the enzyme (Furukawa et al., 1990, J. Biol. Chem. 265: 2086–2090; Furukawa et al., 1993, J. Biol. Chem. 268:23837–23842).

In maize, two soluble forms of STS, known as isoforms I and II, have been identified (Macdonald and Preiss, 1983, Plant Physiol. 73: 175–178; Boyer and Preiss, 1978, Carb. Res. 61: 321–334; Pollock and Preiss, 1980, Arch Biochem. Biophys. 204: 578–588; Macdonald and Preiss, 1985 Plant Physiol. 78: 849–852; Dang and Boyer, 1988, Phytochemistry 27: 1255–1259; Mu et al., 1994, Plant J. 6: 151–159), but neither of these has been cloned. STSI activity of maize endosperm was recently correlated with a 76-kDa polypeptide found in both soluble and granule-associated fractions (Mu et al., 1994, Plant J. 6: 151–159). The polypeptide identity of STSII remains unknown. STSI and II exhibit different enzymological characteristics. STSI exhibits primer-independent activity whereas STSII requires glycogen primer to catalyze glucosyl transfer. Soluble starch synthases have been reported to have a high flux control coefficient for starch deposition (Jenner et al., 1993, Aust. J. Plant Physiol. 22: 703–709; Keeling et al., 1993, Planta 191: 342–348) and to have unusual kinetic properties at elevated temperatures (Keeling et al., 1995, Aust. J. Plant Physiol. 21807–827). The respective isoforms in maize exhibit significant differences in both temperature optima and stability.

Plant starch synthase (and E. coli glycogen synthase) sequences include the sequence KTGGL (SEQ ID NO:40) which is known to be the adenosine diphosphate glucose (ADPG) binding domain. The genes for any such starch synthase protein may be used in constructs according to this invention.

Branching enzyme [α1,4Dglucan: α1,4Dglucan 6D(α1,4Dglucano) transferase (E.C. 2.4.1.18)], sometimes called Q-enzyme, converts amylose to amylopectin. A segment of a α1,4Dglucan chain is transferred to a primary hydroxyl group in a similar glucan chain.

Bacterial branching enzyme genes and plant sequences have been reported (rice endosperm: Nakamura et. al., 1992, Physiologia Plantarum, 84:329–335 and Nakamura and Yamanouchi, 1992, Plant Physiol., 99:1265–1266; pea: Smith, 1988, Planta, 175:270–279 and Bhattacharyya et al., 1989, J. Cell Biochem., Suppl. 13D:331; maize endosperm: Singh and Preiss, 1985, Plant Physiology, 79:34–40; VosScherperkeuter et al., 1989, Plant Physiology, 90:75–84; potato: Kossmann et al., 1991, Mol. Gen. Genet., 230(12):39–44; cassava: Salehuzzaman and Visser, 1992, Plant Mol Biol, 20:809–819).

In the area of polysaccharide enzymes there are reports of vectors for engineering modification in the starch pathway of plants by use of a number of starch synthesis genes in various plant species. That some of these polysaccharide enzymes bind to cellulose or starch or glycogen is well known. One specific patent example of the use of a polysaccharide enzyme shows the use of glycogen biosynthesis enzymes to modify plant starch. In U.S. Pat. No. 5,349,123 to Shewmaker a vector containing DNA to form glycogen biosynthetic enzymes within plant cells is taught. Specifically, this patent refers to the changes in potato starch due to the introduction of these enzymes. Other starch synthesis genes and their use have also been reported.

Hybrid (Fusion) Peptides

Hybrid proteins (also called “fusion proteins”) are polypeptide chains that consist of two or more proteins fused together into a single polypeptide. Often one of the proteins is a ligand which binds to a specific receptor cell. Vectors encoding fusion peptides are primarily used to produce foreign proteins through fermentation of microbes. The fusion proteins produced can then be purified by affinity chromatography. The binding portion of one of the polypeptides is used to attach the hybrid polypeptide to an affinity matrix. For example, fusion proteins can be formed with beta galactosidase which can be bound to a column. This method has been used to form viral antigens.

Another use is to recover one of the polypeptides of the hybrid polypeptide. Chemical and biological methods are known for cleaving the fused peptide. Low pH can be used to cleave the peptides if an acid-labile aspartate-proline linkage is employed between the peptides and the peptides are not affected by the acid. Hormones have been cleaved with cyanobromide. Additionally, cleavage by site-specific proteolysis has been reported. Other methods of protein purification such as ion chromatography have been enhanced with the use of polyarginine tails which increase the overall basicity of the protein thus enhancing binding to ion exchange columns.

A number of patents have outlined improvements in methods of making hybrid peptides or specific hybrid peptides targeted for specific uses. U.S. Pat. No. 5,635,599 to Pastan et al. outlines an improvement of hybrid proteins. This patent reports a circularly permuted ligand as part of the hybrid peptide. This ligand possesses specificity and good binding affinity. Another improvement in hybrid proteins is reported in U.S. Pat. No. 5,648,244 to Kuliopulos. This patent describes a method for producing a hybrid peptide with a carrier peptide. This nucleic acid region, when recognized by a restriction endonuclease, creates a nonpalindromic 3-base overhang. This allows the vector to be cleaved.

An example of a specifically targeted hybrid protein is reported in U.S. Pat. No. 5,643,756. This patent reports a vector for expression of glycosylated proteins in cells. This hybrid protein is adapted for use in proper immunoreactivity of HIV gp120. The isolation of gp120 domains which are highly glycosylated is enhanced by this reported vector.

U.S. Pat. Nos. 5,202,247 and 5,137,819 discuss hybrid proteins having polysaccharide binding domains and methods and compositions for preparation of hybrid proteins which are capable of binding to a polysaccharide matrix. U.S. Pat. No. 5,202,247 specifically teaches a hybrid protein linking a cellulase binding region to a peptide of interest. The patent specifies that the hybrid protein can be purified after expression in a bacterial host by affinity chromatography on cellulose.

The development of genetic engineering techniques has made it possible to transfer genes from various organisms and plants into other organisms or plants. Although starch has been altered by transformation and mutagenesis in the past there is still a need for further starch modification. To this end vectors that provide for encapsulation of desired amino acids or peptides within the starch and specifically within the starch granule are desirable. The resultant starch is modified and the tissue from the plant carrying the vector is modified.

This invention provides a hybrid polypeptide comprising a starch-encapsulating region (SER) from a starch-binding enzyme fused to a payload polypeptide which is not endogenous to said starch-encapsulating region, i.e. does not naturally occur linked to the starch-encapsulating region. The hybrid polypeptide is useful to make modified starches comprising the payload polypeptide. Such modified starches may be used to provide grain feeds enriched in certain amino acids. Such modified starches are also useful for providing polypeptides such as hormones and other medicaments, e.g. insulin, in a starch-encapsulated form to resist degradation bv stomach acids. The hybrid polypeptides are also useful for producing the payload polypeptides in easily-purified form. For example, such hybrid polypeptides produced by bacterial fermentation, or in grains or animals, may be isolated and purified from the modified starches with which they are associated by artknown techniques.

The term “polypeptide” as used herein means a plurality of identical or different amino acids, and also encompasses proteins.

The term “hybrid polypeptide” means a polypeptide composed of peptides or polypeptides from at least two different sources, e.g. a starch-encapsulating region of a starch-binding enzyme, fused to another polypeptide such as a hormone, wherein at least two component parts of the hybrid polypeptide do not occur fused together in nature.

The term “payload polypeptide” means a polypeptide not endogenous to the starch-encapsulating region whose expression is desired in association with this region to express a modified starch containing the payload polypeptide.

When the payload polypeptide is to be used to enhance the amino acid content of particular amino acids in the modified starch, it preferably consists of not more than three different types of amino acids selected from the group consisting of: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

When the payload polypeptide is to be used to supply a biologically active polypeptide to either the host organism or another organism, the payload polypeptide may be a biologically active polypeptide such as a hormone, e.g., insulin, a growth factor, e.g. somatotropin, an antibody, enzyme, immunoglobulin, or dye, or may be a biologically active fragment thereof as is known to the art. So long as the polypeptide has biological activity, it does not need to be a naturally-occurring polypeptide, but may be mutated, truncated, or otherwise modified. Such biologically active polypeptides may be modified polypeptides, containing only biologically-active portions of biologically-active polypeptides. They may also be amino acid sequences homologous to naturally-occurring biologically-active amino acid sequences (preferably at least about 75% homologous) which retain biological activity.

The starch-encapsulating region of the hybrid polypeptide may be a starch-encapsulating region of any starch-binding enzyme known to the art, e.g. an enzyme selected from the group consisting of soluble starch synthase I, soluble starch synthase II, soluble starch synthase III, granule-bound starch synthase, branching enzyme I, branching enzyme IIa, branching enzyme IIBb and glucoamylase polypeptides.

When the hybrid polypeptide is to be used to produce payload polypeptide in pure or partially purified form, the hybrid polypeptide preferably comprises a cleavage site between the starch-encapsulating region and the payload polypeptide. The method of isolating the purified payload polypeptide then includes the step of contacting the hybrid polypeptide with a cleaving agent specific for that cleavage site.

This invention also provides recombinant nucleic acid (RNA or DNA) molecules encoding the hybrid polypeptides. Such recombinant nucleic acid molecules preferably comprise control sequences adapted for expression of the hybrid polypeptide in the selected host. The term “control sequences” includes promoters, introns, preferred codon sequences for the particular host organism, and other sequences known to the art to affect expression of DNA or RNA in particular hosts. The nucleic acid sequences encoding the starch-encapsulating region and the payload polypeptide may be naturally-occurring nucleic acid sequences, or biologically-active fragments thereof, or may be biologically-active sequences homologous to such sequences, preferably at least about 75% homologous to such sequences.

Host organisms include bacteria, plants, and animals. Preferred hosts are plants. Both monocotyledonous plants (monocots) and dicotyledonous plants (dicots) are useful hosts for expressing the hybrid polypeptides of this invention.

This invention also provides expression vectors comprising the nucleic acids encoding the hybrid proteins of this invention. These expression vectors are used for transforming the nucleic acids into host organisms and may also comprise sequences aiding in the expression of the nucleic acids in the host organism. The expression vectors may be plasmids, modified viruses, or DNA or RNA molecules, or other vectors useful in transformation systems known to the art.

By the methods of this invention, transformed cells are produced comprising the recombinant nucleic acid molecules capable of expressing the hybrid polypeptides of this invention. These may be prokaryotic or eukaryotic cells from unicellular organisms, plants or animals. They may be bacterial cells from which the hybrid polypeptide may be harvested. Or, they may be plant cells which may be regenerated into plants from which the hybrid polypeptide may be harvested, or, such plant cells may be regenerated into fertile plants with seeds containing the nucleic acids encoding the hybrid polypeptide. In a preferred embodiment, such seeds contain modified starch comprising the payload polypeptide.

The term “modified starch” means the naturally-occurring starch has been modified to comprise the payload polypeptide.

A method of targeting digestion of a payload polypeptide to a particular phase of the digestive process, e.g., preventing degradation of a payload polypeptide in the stomach of an animal, is also provided comprising feeding the animal a modified starch of this invention comprising the payload polypeptide, whereby the polypeptide is protected by the starch from degradation in the stomach of the animal. Alternatively, the starch may be one known to be digested in the stomach to release the payload polypeptide there.

Preferred recombinant nucleic acid molecules of this invention comprise DNA encoding starch-encapsulating regions selected from the starch synthesizing gene sequences set forth in the tables hereof.

Preferred plasmids of this invention are adapted for use with specific hosts. Plasmids comprising a promoter, a plastid-targeting sequence, a nucleic acid sequence encoding a starch-encapsulating region, and a terminator sequence, are provided herein. Such plasmids are suitable for insertion of DNA sequences encoding payload polypeptides and starch-encapsulating regions for expression in selected hosts.

Plasmids of this invention can optionally include a spacer or a linker unit proximate the fusion site between nucleic acids encoding the SER and the nucleic acids encoding the payload polypeptide. This invention includes plasmids comprising promoters adapted for a prokaryotic or eukaryotic hosts. Such promoters may also be specifically adapted for expression in monocots or in dicots.

A method of forming peptide-modified starch of this invention includes the steps of: supplying a plasmid having a promoter associated with a nucleic acid sequence encoding a starch-encapsulating region, the nucleic acid sequence encoding the starch-encapsulating region being connected to a nucleic acid region encoding a payload polypeptide, and transforming a host with the plasmid whereby the host expresses peptide-modified starch.

This invention furthermore comprises starch-bearing grains comprising: an embryo, nutritive tissues; and, modified starch granules having encapsulated therein a protein that is not endogenous to starch granules of said grain which are not modified. Such starch-bearing grains may be grains wherein the embryo is a maize embryo, a rice embryo, or a wheat embryo.

All publications referred to herein are incorporated by reference to the extent not inconsistent herewith.

FIG. 1A shows the plasmid pEXS114 which contains the synthetic GFP (Green Fluorescent Protein) subcloned into pBSK from Stratagene.

FIG. 1B shows the plasmid pEXS115.

FIG. 2A shows the waxy gene with restriction sites subcloned into a commercially available plasmid.

FIG. 2B shows the p ET-21A plasmid commercially available from Novagen having the GFP fragment from pEXS115 subcloned therein.

FIG. 3A shows pEXS114 subcloned into pEXSWX, and the GFP-FLWX map.

FIG. 3B shows the GFP-Bam HIWX plasmid.

FIG. 4 shows the SGFP fragment of pEXS115 subcloned into pEXSWX, and the GFP-NcoWX map.

FIG. 5 shows a linear depiction of a plasmid that is adapted for use in monocots.

FIG. 6 shows the plasmid pEXS52.

FIG. 7A–7F shows the six introductory plasmids used to form pEXS51 and pEX560; FIG. 7A shows pEXS adh1. FIG. 7B shows pEXS adh1-nos3′. FIG. 7C shows pEXS33. FIG. 7D shows pEXS10zp. FIG. 73 shows pEXS10zp-adh1. FIG. 7F shows pEXS10zp-adh1-nos3′.

FIGS. 8 a and 8 b show the plasmids pEXS50 and pEXS51, respectively, containing the MS-SIII gene which is a starch-soluble synthase gene.

FIG. 9 a shows the plasmid pEXS60 which excludes the intron shown in pEXS50, and FIG. 9 b shows the plasmid pEXS61 which excludes the intron shown in pEXS60.

The present invention provides, broadly, a hybrid polypeptide, a method for making a hybrid polypeptide, and nucleic acids encoding the hybrid polypeptide. A hybrid polypeptide consists of two or more subparts fused together into a single peptide chain. The subparts can be amino acids or peptides or polypeptides. One of the subparts is a starch-encapsulating region. Hybrid polypeptides may thus be targeted into starch granules produced by organisms expressing the hybrid polypeptides.

A method of making the hybrid polypeptides within cells involves the preparation of a DNA construct comprising at least a fragment of DNA encoding a sequence which functions to bind the expression product of attached DNA into a granule of starch, ligated to a DNA sequence encoding the polypeptide of interest (the payload polypeptide). This construct is expressed within a eukaryotic or prokaryotic cell. The hybrid polypeptide can be used to produce purified protein or to immobilize a protein of interest within the protection of a starch granule, or to produce grain that contains foreign amino acids or peptides.

The gene for X can be placed in the 5′ or 3′ position within the DNA construct described below.

CS is a central site which may be a leaving site, a cleavage site, or a spacer, as is known to the art. A cleavage site is recognized by a cleaving enzyme. A cleaving enzyme is an enzyme that cleaves peptides at a particular site. Examples of chemicals and enzymes that have been employed to cleave polypeptides include thrombin, trypsin, cyanobromide, formic acid, hydroxyl amine, collagenase, and alasubtilisin. A spacer is a peptide that joins the peptides comprising the hybrid polypeptide. Usually it does not have any specific activity other than to join the peptides or to preserve some minimum distance or to influence the folding, charge or water acceptance of the protein. Spacers may be any peptide sequences not interfering with the biological activity of the hybrid polypeptide.

The starch-encapsulating region (SER) is the region of the subject polypeptide that has a binding affinity for starch. Usually the SER is selected from the group consisting of peptides comprising starch-binding regions of starch synthases and branching enzymes of plants, but can include starch binding domains from other sources such as glucoamylase and the like. In the preferred embodiments of the invention, the SER includes peptide products of genes that naturally occur in the starch synthesis pathway. This subset of preferred SERs is defined as starch-forming encapsulating regions (SFER). A further subset of SERs preferred herein is the specific starch-encapsulating regions (SSER) from the specific enzymes starch synthase (STS), granule-bound starch synthase (GBSTS) and branching enzymes (BE) of starch-bearing plants. The most preferred gene product from this set is the GBSTS. Additionally, starch synthase I and branching enzyme II are useful gene products. Preferably, the SER (and all the subsets discussed above) are truncated versions of the full length starch synthesizing enzyme gene such that the truncated portion includes the starch-encapsulating region.

The DNA construct for expressing the hybrid polypeptide within the host, broadly is as follows:

As is known to the art, a promoter is a region of DNA controlling transcription. Different types of promoters are selected for different hosts. Lac and T7 promoters work well in prokaryotes, the 35S CaMV promoter works well in dicots, and the polyubiquitin promoter works well in many monocots. Any number of different promoters are known to the art and can be used within the scope of this invention.

Also as is known to the art, an intron is a nucleotide sequence in a gene that does not code for the gene product. One example of an intron that often increases expression in monocots is the Adh1 intron. This component of the construct is optional.

The transit peptide coding region is a nucleotide sequence that encodes for the translocation of the protein into organelles such as plastids. It is preferred to choose a transit peptide that is recognized and compatible with the host in which the transit peptide is employed. In this invention the plastid of choice is the amyloplast.

It is preferred that the hybrid polypeptide be located within the amyloplast in cells such as plant cells which synthesize and store starch in amyloplasts. If the host is a bacterial or other cell that does not contain an amyloplast, there need not be a transit peptide coding region.

A terminator is a DNA sequence that terminates the transcription.

X is the coding region for the payload polypeptide, which may be any polypeptide of interest, or chains of amino acids. It may have up to an entire sequence of a known polypeptide or comprise a useful fragment thereof. The payload polypeptide may be a polypeptide, a fragment thereof, or biologically active protein which is an enzyme, hormone, growth factor, immunoglobulin, dye, etc. Examples of some of the payload polypeptides that can be employed in this invention include, but are not limited to, prolactin (PRL), serum albumin, growth factors and growth hormones, i.e., somatotropin. Serum albumins include bovine, ovine, equine, avian and human serum albumin. Growth factors include epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), fibroblast growth factor (FGF), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and recombinant human insulin-like growth factors I (rHuIGF-I) and II (rHuIGF-II). Somatotropins which can be employed to practice this invention include, but are not limited to, bovine, porcine, ovine, equine, avian and human somatotropin. Porcine somatotropin includes delta-7 recombinant porcine somatotropin, as described and claimed in European Patent Application Publication No. 104,920 (Biogen). Preferred payload polypeptides are somatotropin, insulin A and B chains, calcitonin, beta endorphin, urogastrone, beta globin, myoglobin, human growth hormone, angiotensin, proline, proteases, beta-galactosidase, and cellulases.

The hybrid polypeptide, the SER region and the payload polypeptides may also include post-translational modifications known to the art such as glycosylation, acylation, and other modifications not interfering with the desired activity of the polypeptide.

Developing a Hybrid Polypeptide

The SER region is present in genes involved in starch synthesis. Methods for isolating such genes include screening from genomic DNA libraries and from cDNA libraries. Genes can be cut and changed by ligation, mutation agents, digestion, restriction and other such procedures, e.g., as outlined in Maniatis et al., Molecular Cloning, Cold Spring Harbor Labs, Cold Spring Harbor, N.Y. Examples of excellent starting materials for accessing the SER region include, but are not limited to, the following: starch synthases I, II, III, IV, Branching Enzvmes I, IIA and B and granule-bound starch synthase (GBSTS). These genes are present in starch-bearing plants such as rice, maize, peas, potatoes, wheat, and the like. Use of a probe of SER made from genomic DNA or cDNA or mRNA or antibodies raised against the SER allows for the isolation and identification of useful genes for cloning. The starch enzyme-encoding sequences may be modified as long as the modifications do not interfere with the ability of the SER region to encapsulate associated polypeptides.

When genes encoding proteins that are encapsulated into the starch granule are located, then several approaches to isolation of the SER can be employed, as is known to the art. One method is to cut the gene with restriction enzymes at various sites, deleting sections from the N-terminal end and allowing the resultant protein to express. The expressed truncated protein is then run on a starch gel to evaluate the association and dissociation constant of the remaining protein. Marker genes known to the art, e.g., green fluorescent protein gene, may be attached to the truncated protein and used to determine the presence of the marker gene in the starch granule.

Once the SER gene sequence region is isolated it can be used in making the gene fragment sequence that will express the payload polypeptide encapsulated in starch. The SER gene sequence and the gene sequence encoding the payload polypeptide can be ligated together. The resulting fused DNA can then be placed in a number of vector constructs for expression in a number of hosts. The preferred hosts form starch granules in plastids, but the testing of the SER can be readily performed in bacterial hosts such as E. coli.

The nucleic acid sequence coding for the payload polypeptide may be derived from DNA, RNA, genomic DNA, cDNA, mRNA or may be synthesized in whole or in part. The sequence of the payload polypeptide can be manipulated to contain mutations such that the protein produced is a novel, mutant protein, so long as biological function is maintained.

When the payload polypeptide-encoding nucleic acid sequence is ligated onto the SER-encoding sequence, the gene sequence for the payload polypeptide is preferably attached at the end of the SER sequence coding for the N-terminus. Although the N-terminus end is preferred, it does not appear critical to the invention whether the payload polypeptide is ligated onto the N-terminus end or the C-terminus end of the SER. Clearly, the method of forming the recombinant nucleic acid molecules of this invention, whether synthetically, or by cloning and ligation, is not critical to the present invention.

The central region of the hybrid polypeptide is optional. For some applications of the present invention it can be very useful to introduce DNA coding for a convenient protease cleavage site in this region into the recombinant nucleic acid molecule used to express the hybrid polypeptide. Alternatively, it can be useful to introduce DNA coding for an amino acid sequence that is pH-sensitive to form the central region. If the use of the present invention is to develop a pure protein that can be extracted and released from the starch granule by a protease or the like, then a protease cleavage site is useful. Additionally, if the protein is to be digested in an animal then a protease cleavage site may be useful to assist the enzymes in the digestive tract of the animal to release the protein from the starch. In other applications and in many digestive uses the cleavage site would be superfluous.

The central region site may comprise a spacer. A spacer refers to a peptide that joins the proteins comprising a hybrid polypeptide. Usually it does not have any specific activity other than to join the proteins, to preserve some minimum distance, to influence the folding, charge or hydrophobic or hydrophilic nature of the hybrid polypeptide.

Construct Development

Once the ligated DNA which encodes the hybrid polypeptide is formed, then cloning vectors or plasmids are prepared which are capable of transferring the DNA to a host for expressing the hybrid polypeptides. The recombinant nucleic acid sequence of this invention is inserted into a convenient cloning vector or plasmid. For the present invention the preferred host is a starch granule-producing host. However, bacterial hosts can also be employed. Especially useful are bacterial hosts that have been transformed to contain some or all of the starch-synthesizing genes of a plant. The ordinarily skilled person in the art understands that the plasmid is tailored to the host. For example, in a bacterial host transcriptional regulatory promoters include lac, TAC (a functional hybrid derived from the TRP and lac promoters), trp and the like. Addtionally, DNA coding for a transit peptide most likely would not be used and a secretory leader that is upstream from the structural gene may be used to get the polypeptide into the medium. Alternatively, the product is retained in the host and the host is lysed and the product isolated and purified by starch extraction methods or by binding the material to a starch matrix (or a starch-like matrix such as amylose or amylopectin, glycogen or the like) to extract the product.

The preferred host is a plant and thus the preferred plasmid is adapted to be useful in a plant. The plasmid should contain a promoter, preferably a promoter adapted to target the expression of the protein in the starch-containing tissue of the plant. The promoter may be specific for various tissues such as seeds, roots, tubers and the like; or, it can be a constitutive promoter for gene expression throughout the tissues of the plant. Well-known promoters include the 10 kD zein (maize) promoter, the CAB (chlorophyll a/b binding protein) promoter, patatin, 35S and 19S cauliflower mosaic virus promoters (very useful in dicots), the polyubiquitin promoter (useful in monocots) and enhancements and modifications thereof known to the art.

The cloning vector may contain coding sequences for a transit peptide to direct the plasmid into the correct location. Examples of transit peptide-coding sequences are shown in the sequence tables. Coding sequences for other transit peptides can be used. Transit peptides naturally occurring in the host to be used are preferred. Preferred transit peptide coding regions for maize are shown in the tables and figures hereof. The purpose of the transit peptide is to target the vector to the correct intracellular area.

Attached to the transit peptide-encoding sequence is the DNA sequence encoding the N-terminal end of the payload polypeptide. The direction of the sequence encoding the payload polypeptide is varied depending on whether sense or antisense transcription is desired. DNA constructs of this invention specifically described herein have the sequence encoding the payload polypeptide at the N-terminus end but the SER coding region can also be at the N-terminus end and the payload polypeptide sequence following. At the end of the DNA construct is the terminator sequence. Such sequences are well known in the art.

The cloning vector is transformed into a host. Introduction of the cloning vector, preferably a plasmid, into the host can be done by a number of transformation techniques known to the art. These techniques may vary by host but they include microparticle bombardment, micro injection, Agrobacterium transformation, “whiskers” technology (U.S. Pat. Nos. 5,302,523 and 5,464,765), electroporation and the like. If the host is a plant, the cells can be regenerated to form plants. Methods of regenerating plants are known in the art. Once the host is transformed and the proteins expressed therein, the presence of the DNA encoding the payload polypeptide in the host is confirmable. The presence of expressed proteins may be confirmed by Western Blot or ELISA or as a result of a change in the plant or the cell.

Uses of Encapsulated Protein

There are a number of applications of this invention. The hybrid polypeptide can be cleaved in a pure state from the starch (cleavage sites can be included) and pure protein can be recovered. Alternatively, the encapsulated payload polypeptide within the starch can be used in raw form to deliver protein to various parts of the digestive tract of the consuming animal (“animal” shall include mammals, birds and fish). For example if the starch in which the material is encapsulated is resistant to digestion then the protein will be released slowly into the intestine of the animal, therefore avoiding degradation of the valuable protein in the stomach. Amino acids such as methionine and lysine may be encapsulated to be incorporated directly into the grain that the animal is fed thus eliminating the need for supplementing the diet with these amino acids in other forms.

The present invention allows hormones, enzymes, proteins, proteinaceous nutrients and proteinaceous medicines to be targeted to specific digestive areas in the digestive tracts of animals. Proteins that normally are digested in the upper digestive tract encapsulated in starch are able to pass through the stomach in a nondigested manner and be absorbed intact or in part by the intestine. If capable of passing through the intestinal wall, the payload polypeptides can be used for medicating an animal, or providing hormones such as growth factors, e.g., somatotropin, for vaccination of an animal or for enhancing the nutrients available to an animal.

If the starch used is not resistant to digestion in the stomach (for example the sugary 2 starch is highly digestible), then the added protein can be targeted to be absorbed in the upper digestive tract of the animal. This would require that the host used to produce the modified starch be mutated or transformed to make sugary 2 type starch. The present invention encompasses the use of mutant organisms that form modified starch as hosts. Some examples of these mutant hosts include rice and maize and the like having sugary 1, sugary 2, brittle, shrunken, waxy, amylose extender, dull, opaque, and floury mutations, and the like. These mutant starches and starches from different plant sources have different levels of digestibility. Thus by selection of the host for expression of the DNA and of the animal to which the modified starch is fed, the hybrid polypeptide can be digested where it is targeted. Different proteins are absorbed most efficiently by different parts of the body. By encapsulating the protein in starch that has the selected digestibility, the protein can be supplied anywhere throughout the digestive tract and at specific times during the digestive process.

Another of the advantages of the present invention is the ability to inhibit or express differing levels of glycosylation of the desired polypeptide. The encapsulating procedure may allow the protein to be expressed within the granule in a different glycosylation state than if expressed by other DNA molecules. The glycosylation will depend on the amount of encapsulation, the host employed and the sequence of the polypeptide.

Improved crops having the above-described characteristics may be produced by genetic manipulation of plants known to possess other favorable characteristics. By manipulating the nucleotide sequence of a starch-synthesizing enzyme gene, it is possible to alter the amount of key amino acids, proteins or peptides produced in a plant. One or more genetically engineered gene constructs, which may be of plant, fungal, bacterial or animal origin, may be incorporated into the plant genome by sexual crossing or by transformation. Engineered genes may comprise additional copies of wildtype genes or may encode modified or allelic or alternative enzymes with new properties. Incorporation of such gene construct(s) may have varying effects depending on the amount and type of gene(s) introduced (in a sense or antisense orientation). It may increase the plant's capacity to produce a specific protein, peptide or provide an improved amino acid balance.

Cloning Enzymes Involved in Starch Biosynthesis

Known cloning techniques may be used to provide the DNA constructs of this invention. The source of the special forms of the SSTS, GBSTS, BE, glycogen synthase (GS), amylopectin, or other genes used herein may be any organism that can make starch or glycogen. Potential donor organisms are screened and identified. Thereafter there can be two approaches: (a) using enzyme purification and antibody/sequence generation following the protocols described herein; (b) using SSTS, GBSTS, BE, GS, amylopectin or other cDNAs as heterologous probes to identify the genomic DNAs for SSTS, GBSTS, BE, GS, amylopectin or other starch-encapsulating enzymes in libraries from the organism concerned. Gene transformation, plant regeneration and testing protocols are known to the art. In this instance it is necessary to make gene constructs for transformation which contain regulatory sequences that ensure expression during starch formation. These regulatory sequences are present in many small grains and in tubers and roots. For example these regulatory sequences are readily available in the maize endosperm in DNA encoding Granule Bound Starch Synthesis (GBSTS), Soluble Starch Synthases (SSTS) or Branching Enzymes (BE) or other maize endosperm starch synthesis pathway enzymes. These regulatory sequences from the endosperm ensure protein expression at the correct developmental time (e.g., ADPG pyrophosphorylase).

In this method we measure starch-binding constants of starch-binding proteins using native protein electrophoresis in the presence of suitable concentrations of carbohydrates such as glycogen or amylopectin. Starch-encapsulating regions can be elucidated using site-directed mutagenesis and other genetic engineering methods known to those skilled in the art. Novel genetically-engineered proteins carrying novel peptides or amino acid combinations can be evaluated using the methods described herein.

Method for Identification of Starch-encapsulatinq Proteins

Starch-Granule Protein Isolation:

Homogenize 12.5 g grain in 25 ml Extraction buffer (50 mM Tris acetate, pH 7.5, 1 mM EDTA, 1 mM DTT for 3×20 seconds in Waring blender with 1 min intervals between blending). Keep samples on ice. Filter through mira cloth and centrifuge at 6,000 rpm for 30 min. Discard supernatant and'scrape off discolored solids which overlay white starch pellet. Resuspend pellet in 25 ml buffer and recentrifuge. Repeat washes twice more. Resuspend washed pellet in −20° C. acetone, allow pellet to settle at −20° C. Repeat. Dry starch under stream of air. Store at −20° C.

Protein Extraction:

Mix 50 mg starch with 1 ml 2% SDS in eppendorf. Vortex, spin at 18,000 rpm, 5 min, 4° C. Pour off supernatant. Repeat twice. Add 1 ml sample buffer (4 ml distilled water, 1 ml 0.5 M Tris-HCl, pH 6.8, 0.8 ml glycerol, 1.6 ml 10% SDS, 0.4 ml B-mercaptoethanol, 0.2 ml 0.5% bromphenol blue). Boil eppendorf for 10 min with hole in lid. Cool, centrifuge 10,000 rpm for 10 min. Decant supernatant into new eppendorf. Boil for 4 minutes with standards. Cool.

SDS-Page Gels: (non-denaturing) 10% 4% Resolve Stack Acryl/Bis 40% stock 2.5 ml 1.0 ml 1.5 M Tris pH 8.8 2.5 ml — 0.5 M Tris pH 8.8 — 2.5 ml 10% SDS 100 μl 100 μl Water 4.845 ml 6.34 ml Degas 15 min add fresh 10% Ammonium Persulfate 50 μl 50 μl TEMED 5 μl 10 μl

Mini-Protean II Dual Slab Cell; 3.5 ml of Resolve buffer per gel. 4% Stack is poured on top. The gel is run at 200V constant voltage. 10×Running buffer (250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3).

Method of Measurement of Starch-Encapsulating Regions: Solutions: Extraction Buffer: 50 mM Tris-acetate pH 7.5, 10 mM EDTA, 10% sucrose, 2.5 mM DTT-fresh. Stacking Buffer: 0.5 M Tris-HCl, pH 6.8 Resolve Buffer: 1.5 M Tris-HCl, pH 8.8 10 × Lower 30.3 g Tris + 144 g Glycine qs to 1 L. (pH is ~8.3, Electrode Buffer: no adjustment). Dilute for use. Upper Electrode Same as Lower Buffer: Sucrose Solution: 18.66 g sucrose + 100 ml dH 2 O 30% Acryl/ 146 g acrylamide + 4 g bis + 350 ml dH 2 O. Bring up Bis Stock to 500 ml. Filter and store at 4 C in the dark for up (2.67% C): to 1 month. 15% Acryl/ 6 g acrylamide + 1.5 g bis + 25 ml dH 2 O. Bring Bis Stock up to 50 ml. Filter and store at 4 C in the dark for (20% C): up to 1 month. Riboflavin 1.4 g riboflavin + 100 ml dH 2 O. Store in dark for up Solution: to 1 month. SS Assay mix: 25 mM Sodium Citrate, 25 mM Bicine-NaO (pH 8.0), 2 mM EDTA, 1 mM DTT-fresh, 1 mM Adenosine 5′ Diphosphoglucose-fresh, 10 mg/ml rabbit liver glycogen Type III-fresh. Iodine Solution: 2 g iodine + 20 g KI, 0.1 N HCl up to 1 L.

Extract:

4 ml extraction buffer+12 g endosperm. Homogenize. filter through mira cloth or 4 layers cheesecloth, spin 20,000 g (14,500 rpm, SM-24 rotor), 20 min., 4° C. remove supernatant using a glass pipette. 0.85 ml extract+0.1 ml glycerol+0.05 ml 0.5% bromophenol blue. vortex and spin 5 min. full speed microfuge. Use directly or freeze in liquid nitrogen and store at −80° C. for up to 2 weeks.

Cast Gels:

Attach Gel Bond PAG film (FIfC Industries, Rockland, Me.) to (inside of) outer glass plate using two-sided scotch tape, hydrophilic side up. The tape and the film is lined up as closely and evenly as possible with the bottom of the plate. The film is slightly smaller than the plate. Squirt water between the film and the plate to adhere the film. Use a tissue to push out excess water. Set up plates as usual, then seal the bottom of the plates with tacky adhesive. The cassette will fit into the casting stand if the gray rubber is removed from the casting stand. The gel polymerizes with the film, and stays attached during all subsequent manipulations.

Cast 4.5% T resolve mini-gel (0.75 mm): 2.25 ml dH 2 O

+3.75 ml sucrose solution

+2.5 ml resolve buffer

+1.5 ml 30% Acryl/Bis stock

+various amounts of glycogen for each gel (i.e., 0–1.0%)

DEGAS 15 NIN.

+50 μl 10% APS

+5 μl TEMED

POLYMERIZE FOR 30 NIN. OR OVERNIGHT

Cast 3.125% T stack: 1.59 ml dH 2 O

+3.75 ml sucrose solution

+2.5 ml stack buffer

+2.083 ml 15% Acryl/Bis stock

15 μl 10% APS

+35 μl riboflavin solution

+30 μl TEMED

POLYMERIZE FOR 2.5 HOURS CLOSE TO A LIGHT BULB cool in 4° C. before pulling out combs. Can also not use combs, and just cast a centimeter of stacker.

The Foregoing Procedure:

Can run at different temperatures; preincubate gels and solutions.

Pre-run for 15 min. at 200 V

Load gel: 7 μl per well, or 115 μl if no comb.

Run at 140 V until dye front is close to bottom. Various running temperatures are achieved by placing the whole gel rig into a water bath. Can occasionally stop the run to insert a temperature probe into the gel.

Enzyme assay: Cut gels off at dye front. Incubate in SS. Assay mix overnight at room temperature with gentle shaking. Rinse gels with water. Flood with I2/KI solution.

Take pictures of the gels on a light box, and measure the pictures. Rm=mm from top of gel to the active band/mm from top of gel to the bottom of the gel where it was cut (where the dye front was). Plot % glycogen vs. 1/Rm. The point where the line intersects the x axis is −K (where y=0).

Testing and Evaluation Protocol for SER Region Length:

Following the procedure above for selection of the SER region requires four basic steps. First DNA encoding a protein having a starch-encapsulation region must be selected. This can be selected from known starch-synthesizing genes or starch-binding genes such as genes for amylases, for example. The protein must be extracted. A number of protein extraction techniques are well known in the art. The protein may be treated with proteases to form protein fragments of different lengths. The preferred fragments have deletions primarily from the N-terminus region of the protein. The SER region is located nearer to the C-terminus end than the N-terminus end. The protein is run on the gels described above and affinity for the gel matrix is evaluated. Higher affinity shows more preference of that region of the protein for the matrix. This method enables comparison of different proteins to identify the starch-encapsulating regions in natural or synthetic proteins.

SER Fusion Vector:

The following fusion vectors are adapted for use in E. coli . The fusion gene that was attached to the probable SER in these vectors encoded for the green fluorescent protein (GFP). Any number of different genes encoding for proteins and polypeptides could be ligated into the vectors. A fusion vector was constructed having the SER of waxy maize fused to a second gene or gene fragment, in this case GFP.

pEXS114 (see FIG. 1 a ): Synthetic GFP (SGFP) was PCR-amplified from the plasmid HBT-SGFP (from Jen Sheen; Dept. of Molecular Biology; Wellman 11, MGH; Boston, Mass. 02114) using the primers EXS73 (5′-GACTAGTCATATG GTG AGC AAG GGC GAG GAG-3′) [SEQ ID NO:1] and EXS74 (5′-CTAGATCTTCATATG CTT GTA CAG CTC GTC CAT GCC-3′) [SEQ ID NO:2]. The ends of the PCR product were polished off with T DNA polymerase to generate blunt ends; then the PCR product was digested with Spe I. This SGFP fragment was subcloned into the EcoRV-Spe I sites of pBSK (Stratagene at 11011 North Torrey Pines Rd. La Jolla, Calif.) to generate pEXS114.

pEXS115 [see FIG. 1 b ]: Synthetic GFP (SGFP) was PCR-amplified from the plasmid HBT-SGFP (from Jen Sheen) using the primers EXS73 (see above) and EXS75 (5′-CTAGATCTTGGCCATGGC CTT GTA CAG CTC GTC CAT GCC-3′) [SEQ ID NO:3]. The ends of the PCR product were polished off with T DNA polymerase to generate blunt ends; then the PCR product was digested with Spe I. This SGFP fragment was subcloned into the EcoRV-Spe I sites of pBSK (Stratagene) generating pEXSI 115.

pEXSWX (see FIG. 2 a ): Maize WX subcloned NdeI-Not I into pET-21a (see FIG. 2 b ). The genomic DNA sequence and associated amino acids from which the mRNA sequence can be generated is shown in TABLES 1a and 1b below and alternatively the DNA listed in the following tables could be employed.

GFP constructs:

1. GFP only in pET-21a:

pEXS115 is digested with Nde I and Xho I and the 740 bp fragment containing the SGFP coding sequence is subcloned into the Nde I and Xho I sites of pET-21a (Novagen 601 Science Dr. Madison Wis.). (See FIG. 2 b GFP-21a map.)

2. GFP subcloned in-frame at the 5′ end of full-length mature WX:

The 740 bp Nde I fragment containing SGFP from pEXS114 is subcloned into the Nde I site of pEXSWX. (See FIG. 3 a GFP-FLWX map.)

3. GFP subcloned in-frame at the 5′ end of N-terminally truncated WX:

WX truncated by 700 bp at N-terminus.

The 1 kb BamH I fragment encoding the C-terminus of WX from pEXSWX is subcloned into the Bgl II site of pEXS 115. Then the entire SGFP-truncated WX fragment is subcloned into pET21a as a Nde I-HindIII fragment. (See FIG. 3 b GFP-BamHIWX map.)

4. GFP subcloned in-frame at the 5′ end of truncated WX: WX truncated by 100 bp at N-terminus.

The 740 bp Nde I-Nco I fragment containing SGFP from pEXS115 is subcloned into pEXSWX at the Nde I and Nco I sites. (See FIG. 4 GFP-NcoWX map.)

Plasmid Transformation into Bacteria:

Escherichia coli competent cell preparation:

1. Inoculate 2.5 ml LB media with a single colony of desired E. coli strain: selected strain was XLIBLUE DL2IDE3 from (Stratagene); included appropriate antibiotics. Grow at 37° C., 250 rpm overnight.

2. Inoculate 100 ml of LB media with a 1:50 dilution of the overnight culture, including appropriate antibiotics. Grow at 37° C., 250 rpm until OD 600 =0.3–0.5.

3. Transfer culture to sterile centrifuge bottle and chill on ice for 15 minutes.

4. Centrifuge 5 minutes at 3,000×g (4° C. ).

5. Resuspend pellet in 8 ml ice-cold Transformation buffer. Incubate on ice for 15 minutes.

6. Centrifuge 5 minutes at 3,000×g (4° C. ).

7. Resuspend pellet in 8 ml ice-cold Transformation buffer 2. Aliquot, flash-freeze in liquid nitrogen, and stored at −70° C.

Transformation Buffer 1 Transformation Buffer 2 RbCl 1.2 g MOPS (10 mM) 0.209 g MnCl 2 4H 2 O 0.99 g RbCl 0.12 g K-Acetate 0.294 g CaCl 2 2H 2 O 1.1 g CaCl 2 2H 2 O 0.15 g Glycerol 15 g Glycerol 15 g dH 2 O 100 ml dH 2 O 100 ml pH to 6.8 with NaOH pH to 5.8 with 0.2 M Filter sterilize acetic acid Filter sterilize Escherichia coli transformation by rubidium chloride heat shock method: Hanahan, D. (1985) in DNA cloning: a practical approach (Glover, D. M. ed.), pp. 109–135, IRL Press.

1. Incubate 1–5 μl of DNA on ice with 150 μl E. coli competent cells for 30 minutes.

2. Heat shock at 42° C. for 45 seconds.

3. Immediately place on ice for 2 minutes.

4. Add 600 μl LB media and incubate at 37° C. for 1 hour.

5. Plate on LB agar including the appropriate antibiotics.

This plasmid will express the hybrid polypeptide containing the green fluorescent protein within the bacteria.

Expression of Construct in E. coli:

1. Inoculate 3 ml LB with E. coli containing plasmid of interest. Include appropriate antibiotics. 37° C., 250 rpm, overnight.

2. Inoculate 100 ml LB with 2 ml of overnight culture. Include appropriate antibiotics.

Grow at 37° C., 250 rpm.

3. At OD 600 about 0.4–0.5, place at room temperature, 200 rpm. 4. At OD 600 about 0.6–0.8, induce with 100 μl 1M 1PTG. Final 1PTG concentration is 1 mM.

5. Grow at room temperature, 200 rpm, 4–5 hours.

6. Collect cells by centrifugation.

7. Flash freeze in liquid nitrogen and store at −70° C. until use.

Cells can be resuspended in dH 2 O and viewed under UV light (λ max =395 nm) for intrinsic fluorescence. Alternatively, the cells can be sonicated and an aliquot of the cell extract can be separated by SDS-PAGE and viewed under UV light to detect GFP fluorescence. When the protein employed is a green fluorescent protein, the presence of the protein in the lysed material can be evaluated under UV at 395 nm in a light box and the signature green glow can be identified.

Plasmid Extraction from Bacteria:

The following is one of many common alkaline lysis plasmid purification protocols useful in practicing this invention.

1. Inoculate 100–200 ml LB media with a single colony of E. coli transformed with the one of the plasmids described above. Include appropriate antibiotics. Grow at 37° C., 250 rpm overnight. 2. Centrifuge 10 minutes at 5,000×g (4° C.). 3. Resuspend cells in 10 ml water, transfer to a 15 ml centrifuge tube, and repeat centrifugation. 4. Resuspend pellet in 5 ml 0.1 M NaOH, 0.5% SDS. Incubate on ice for 10 minutes. 5. Add 2.5 ml of 3 M sodium acetate (pH 5.2), invert gently, and incubate 10 minutes on ice. 6. Centrifuge 5 minutes at 15,000–20,000×g (4° C.). 7. Extract supernatant with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). 8. Centrifuge 10 minutes at 6,000–10,000×g (4° C.). 9. Transfer aqueous phase to clean tube and precipitate with I volume of isopropanol. 10. Centrifuge 15 minutes at 12,000×g (4° C.). 11. Dissolve pellet in 0.5 ml TE, add 20 μl of 10 mg/ml Rnase, and incubate 1 hour at 37° C. 12. Extract twice with phenol:chloroform:isoamyl alcohol (25:24:1). 13. Extract once with chloroform. 14. Precipitate aqueous phase with 1 volume of isopropanol and 0.1 volume of 3 M sodium acetate. 15. Wash pellet once with 70% ethanol. 16. Dry pellet in SpeedVac and resuspend pellet in TE.

This plasmid can then be inserted into other hosts.

This experiment employs a plasmid having a maize promoter, a maize transit peptide, a starch-encapsulating region from the starch synthase I gene, and a ligated gene fragment attached thereto. The plasmid shown in FIG. 6 contains the DNA sequence listed in Table 8.

Plasmid pEXS52 was constructed according to the following protocol:

Materials Used to Construct Transgenic Plasmids are as Follows:

Plasmid pBluescript SK-

plasmid pMF6 (contain nos3′ terminator)

Plasmid pHKH1 (contain maize adh1 intron)

Plasmid MstsI(6-4) (contain maize stsI transit peptide, use as a template for PCT stsI transit peptide out)

Plasmid MstsIII in pBluescript SK-

Primers EXS29 (GTGGATCCATGGCGACGCCCTCGGCCGTGG) [SEQ ID NO:22] EXS35 (CTGAATTCCATATGGGGCCCCTCCCTGCTCAGCTC) [SEQ ID NO:23] both used for PCT stsI transit peptide

Primers EXS31 (CTCTGAGCTCAAGCTTGCTACMTTCTTTCCTTAATG) [SEQ ID NO:24] EXS32 (GTCTCCGCGGTGGTGTCCTTGCTTCCTAG) [SEQ ID NO:25] both used for PCR maize 10KD zein promoter (Journal: Gene 71:359–370 [1988])

Maize A632 genomic DNA (used as a template for PCR maize 10KD zein promoter).

1. PCR 1.1Kb maize 10KD zein promoter

primers: EXS31, EXS32 template: maize A632 genomic DNA

2. Clone 1.1Kb maize, 10KD zein promoter PCR product into pBluescript SK-plasmid at SacI and SacII site (See FIG. 7 ).

NdeI is removed by fill in and blunt end ligation from maize 10KD zein promoter in pBluescriptSK.

Maize adh1 intron is released from plasmid pHKH1 at XbaI and BamHI sites. Maize adh1 intron (XbaI/BamHI fragment) is cloned into pBluescriptSK- at XbaI and BamHI sites (see FIG. 7 ).

Step 4: Clone maize 10KD zein promoter and maize adh1 intron into pBluescriptSK-(named as pEXS 10zp-adh 1).

Maize 10KD zein promoter is released from plasmid pEXS 10zp-NdeI at SacI and SacII sites. Maize 10KD zein promoter (SacI/SacII fragment) is cloned into plasmid pEXSadh1 (contain maize adh1 intron) at SacI and SacII sites (see FIG. 7 ).

Maize nos3′ terminator is released from plasmid pMF6 at EcoRI and HindIII sites. Maize nos3′ terminator (EcoRI/HindIII fragment) is cloned into plasmid pEXSadh1 at EcoRI and HindIII (see FIG. 7 ).

Maize nos3′ terminator is released from plasmid pEXSadh1-nos3′ at EcoRI and ApaI sites. Maize nos3′ terminator (EcoRI/ApaI fragment) is cloned into plasmid pEXS10zp-adh1 at EcoRI and Apal sites (see FIG. 7 ).

1. PCR 150bp maize STSI transit peptide

primer: EXS29, EXS35 template: MSTSI(6-4) plasmid

2. Clone 150bp maize STSI transit peptide PCR product into plasmid pEXS10zpadh1-nos3′ at EcoRI and BamHI sites (see FIG. 7 ).

There is a mutation (stop codon) on maize STSI transit peptide in plasmid pEXS33. Site-directed mutagenesis is carried out to change stop codon to non-stop codon. New plasmid (containing maize 10KD zein promoter, maize STSI transit peptide, maize adh1 intron, maize nos3′ terminator) is named as pEXS33(m).

NotI site is removed from pEXS33 by NotI fillin, blunt end ligation to form pEXS50 (see FIG. 8 ).

Maize adh1 intron is removed by NotI/BamHI digestion, filled in with Klenow fragment, blunt end ligation to form pEXS60 (see FIG. 9 ).

Maize STSIII is released from plasmid maize STSIII in pBluescript SK- at NdeI and EcoRI sites. Maize STSIII (NdeI-EcoRI fragment) is cloned into pEXS50, pEXS60 separately, named as pEXS51, pEXS61 (see FIGS. 8 and 9 , respectively).

Step 12: Clone the gene in Table 8 into pEXS51 at NdeI/NotI site to form pEXS52. Other similar plasmids can be made by cloning other genes (STSI, II, WX, glgA, glgB, glgC, BEI, BEII, etc.) into pEXS51, pEXS61 at NdeI/NotI site.

Plasmid EXS52 was transformed into rice. The regenerated rice plants transformed with pEXS52 were marked and placed in a magenta box.

Two siblings of each line were chosen from the magenta box and transferred into 2.5 inch pots filled with soil mix (topsoil mixed with peat-vermiculite 50/50). The pots were placed in an aquarium (fish tank) with half an inch of water. The top was covered to maintain high humidity (some holes were made to help heat escape). A thermometer monitored the temperature. The fish tank was placed under fluorescent lights. No fertilizer was used on the plants in the first week. Light period was 6 a.m.–8 p.m., minimum 14 hours light. Temperature was minimum 68° F. at night, 80°–90° F. during the day. A heating mat was used under the fish tank to help root growth when necessary. The plants stayed in the above condition for a week. (Note: the seedlings began to grow tall because of low light intensity.)

After the first week, the top of the aquarium was opened and rice transformants were transferred to growth chambers for three weeks with high humidity and high light intensity.

Alternatively, water mix in the greenhouse can be used to maintain high humidity. The plants grew for three weeks. Then the plants were transferred to 6-inch pots (minimum 5-inch pots) with soil mix (topsoil and peat-Vet, 50/50). The pots were in a tray filled with half an inch of water. 15-16-17 (N-K-P) was used to fertilize the plants (250 ppm) once a week or according to the plants' needs by their appearances. The plants remained in 14 hours light (minimum) 6 a.m.–8 p.m. high light intensity, temperature 85°–90°/70° F. day/night.

The plants formed rice grains and the rice grains were harvested. These harvested seeds can have the starch extracted and analyzed for the presence of the ligated amino acids C, V, A, E, L, S, R, E [SEQ ID NO:27] in the starch within the seed.

SER Vector for Plants:

The plasmid shown in FIG. 6 is adapted for use in monocots, i.e., maize. Plasmid pEXS52 ( FIG. 6 ) has a promoter, a transit peptide (from maize), and a ligated gene fragment (TGC GTC GCG GAG CTG AGC AGG GAG) [SEQ ID NO:26] which encodes the amino acid sequence C V A E L S R E [SEQ ID NO:27].

This gene fragment naturally occurs close to the N-terminal end of the maize soluble starch synthase (MSTSI) gene. As is shown in TABLE 8, at about amino acid 292 the SER from the starch synthase begins. This vector is preferably transformed into a maize host. The transit peptide is adapted for maize so this is the preferred host. Clearly the transit peptide and the promoter, if necessary, can be altered to be appropriate for the host plant desired. After transformation by “whiskers” technology (U.S. Pat. Nos. 5,302,523 and 5,464,765), the transformed host cells are regenerated by methods known in the art, the transformant is pollinated, and the resultant kernels can be collected and analyzed for the presence of the peptidc in the starch and the starch granule.

The following preferred genes can be employed in maize to improve feeds: phytase gene, the somototrophin gene, the following chained amino acids: AUG AUG AUG AUG AUG AUG AUG AUG [SEQ ID NO:28]; and/or, AAG AAG AAG AAG AAG AAG AAG AAG AAG AAG AAG AAG {SEQ ID NO:29]; and/or AAA AAA AAA AAA AAA AAA [ID NO:30]; or a combination of the codons encoding the lysine amino acid in a chain or a combination of the codons encoding both lysine and the methionine codon or any combination of two or three of these amino acids. The length of the chains should not be unduly long but the length of the chain does not appear to be critical. Thus the amino acids will be encapsulated within the starch granule or bound within the starch formed in the starch-bearing portion of the plant host.

This plasmid may be transformed into other cereals such as rice, wheat, barley, oats, sorghum, or millet with little to no modification of the plasmid. The promoter may be the waxy gene promoter whose sequence has been published, or other zein promoters known to the art.

Additionally these plasmids, without undue experimentation, may be transformed into dicots such as potatoes, sweet potato, taro, yam, lotus cassava, peanuts, peas, soybean, beans, or chickpeas. The promoter may be selected to target the starch-storage area of particular dicots or tubers, for example the patatin promoter may be used for potato tubers.

Various methods of transforming monocots and dicots are known in the industry and the method of transforming the genes is not critical to the present invention. The plasmid can be introduced into Agrobacterium tumefaciens by the freeze-thaw method of An et al. (1988) Binary Vectors, in Plant Molecular Biology Manual A3, S. B. Gelvin and R. A. Schilperoot, eds. (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 1–19. Preparation of Agrobacterium inoculum carrying the construct and inoculation of plant material, regeneration of shoots, and rooting of shoots are described in Edwards et al., “Biochemical and molecular characterization of a novel starch synthase from potatoes,” Plant J. 8, 283–294 (1995).

A number of encapsulating regions are present in a number of different genes. Although it is preferred that the protein be encapsulated within the starch granule (granule encapsulation), encapsulation within non-granule starch is also encompassed within the scope of the present invention in the term “encapsulation.” The following types of genes are useful for this purpose.

Use of Starch-Encapsulating Regions of Glycogen Synthase:

E. coli glycogen synthase is not a large protein: the structural gene is 1431 base pairs in length, specifying a protein of 477 amino acids with an estimated molecular weight of 49,000. It is known that problems of codon usage can occur with bacterial genes inserted into plant genomes but this is generally not so great with E. coli genes as with those from other bacteria such as those from Bacillus . Glycogen synthase from E. coli has a codon usage profile much in common with maize genes but it is preferred to alter, by known procedures, the sequence at the translation start point to be more compatible with a plant consensus sequence:

glgA G A T A A T G C A G [SEQ ID NO:31] cons A A C A A T G G C T [SEQ ID NO:32]

Use of Starch-Encapsulating Regions of Soluble Starch Synthase:

cDNA clones of plant-soluble starch synthases are described in the background section above and can be used in the present invention. The genes for any such SSTS protein may be used in constructs according to this invention.

Use of Starch-Encapsulating Regions of Branching Enzyme:

cDNA clones of plant, bacterial and animal branching enzymes are described in the background section above can be used in the present invention. Branching enzyme [1,4Dglucan: 1,4Dglucan 6D(1,4Dglucano) transferase (E.C. 2.4.1.18)] converts amylose to amylopectin, (a segment of a 1,4Dglucan chain is transferred to a primary hydroxyl group in a similar glucan chain) sometimes called Q-enzyme.

The sequence of maize branching enzyme I was investigated by Baba et al. (1991) BBRC, 181:87–94. Starch branching enzyme II from maize endosperm was investigated by Fisher et al. (1993) Plant Physiol, 102:1045–1046. The BE gene construct may require the presence of an amyloplast transit peptide to ensure its correct localization in the amyloplast. The genes for any such branching enzyme of GBSTS protein may be used in constructs according to this invention.

Use of Starch-Binding Domains of Granule-Bound Starch Synthase:

The use of cDNA clones of plant granule-bound starch synthases are described in Shure et al. (1983) Cell 35:225–233, and Visser et al. (1989) Plant Sci. 64(2):185–192. Visser et al. have also described the inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs (1991) Mol. Gen. Genetic 225(2):289–296; (1994) The Plant Cell 6:43–52.) Shimada et al. show antisense in rice (1993) Theor. Appl. Genet. 86:665–672. Van der Leij et al. show restoration of amylose synthesis in low-amylose potato following transformation with the wild-type waxy potato gene (1991) Theor. Appl. Genet. 82:289–295.

The amino acid sequences and nucleotide sequences of granule starch synthases from, for example, maize, rice, wheat, potato, cassava, peas or barley are well known. The genes for any such GBSTS protein may be used in constructs according to this invention.

Construction of Plant Transformation Vectors:

Plant transformation vectors for use in the method of the invention may be constructed using standard techniques

Use of Transit Peptide Sequences:

Some gene constructs require the presence of an amyloplast transit peptide to ensure correct localization in the amyloplast. It is believed that chloroplast transit peptides have similar sequences (Heijne et al. describe a database of chloroplast transit peptides in (1991) Plant Mol. Biol. Reporter, 9(2): 104–126). Other transit peptides useful in this invention are those of ADPG pyrophosphorylase (1991) Plant Mol. Biol. Reporter, 9:104–126), small subunit RUBISCO, acetolactate synthase, glyceraldehyde3Pdehydrogenase and nitrite reductase.

The consensus sequence of the transit peptide of small subunit RUBISCO from many genotypes has the sequence:

[SEQ ID NO:33]

The corn small subunit RUBISCO has the sequence:

MAPTVMMASSATATRTNPAQAS AVAFQGLKSTASLPVARRSSR SLGNVASNGGRIRC [SEQ ID NO:34]

The transit peptide of leaf glyceraldehyde3Pdehydrogenase from corn has the sequence:

[SEQ ID NO:35]

The transit peptide sequence of corn endosperm-bound starch synthase has the sequence:

[SEQ ID NO:36]

The transit peptide sequence of corn endosperm soluble starch synthase has the sequence:

MATPSAVGAACLLLARXAWPAAVGDRARPRRLQRV [SEQ ID NO:37] LRRR

Engineering New Amino Acids or Peptides into Starch-Encapsulating Proteins:

The starch-binding proteins used in this invention may be modified by methods known to those skilled in the art to incorporate new amino acid combinations. For example, sequences of starch-binding proteins may be modified to express higher-than-normal levels of lysine, methionine or tryptophan. Such levels can be usefully elevated above natural levels and such proteins provide nutritional enhancement in crops such as cereals.

In addition to altering amino acid balance, it is possible to engineer the starch-binding proteins so that valuable peptides can be incorporated into the starch-binding protein. Attaching the payload polypeptide to the starch-binding protein at the N-terminal end of the protein provides a known means of adding peptide fragments and still maintaining starch-binding capacity. Further improvements can be made by incorporating specific protease cleavage sites into the site of attachment of the payload polypeptide to the starch-encapsulating region. It is well known to those skilled in the art that proteases have preferred specificities for different amino-acid linkages. Such specificities can be used to provide a vehicle for delivery of valuable peptides to different regions of the digestive tract of animals and man.

In yet another embodiment of this invention, the payload polypeptide can be released following purification and processing of the starch granules. Using amylolysis and/or gelatinization procedures it is known that the proteins bound to the starch granule can be released or become available for proteolysis. Thus recovery of commercial quantities of proteins and peptides from the starch granule matrix becomes possible.

In yet another embodiment of the invention it is possible to process the starch granules in a variety of different ways in order to provide a means of altering the digestibility of the starch. Using this methodology it is possible to change the bioavailablility of the proteins, peptides or amino acids entrapped within the starch granules.

Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.