University of California

Other

Bob B Buchanan

Boihon C Yee

Rosa M Lozano

Joshua H Wong

Karoly Kobrehel

Gregorio Del Val

Peggy G Lemaux

Myeong-Je Cho

Jin-an Jiao

Oscar L Frick

Sungho Shin

Frank L Buchanan, Jr

Maxime Caillau

Richard W Ermel

Corina Marx

Boihan C Yee

Susumu Morigasaki

The present invention is directed to a transgenic plant wherein at least a part of said plant includes a recombinant nucleic acid with a promoter active in the part operably linked to a nucleic acid encoding a thioredoxin polypeptide wherein the promoter is a seed or grain maturation-specific promoter and the thioredoxin polypeptide includes the amino acid sequence WCGPC. The present invention is further directed to transgenic plants that overexpress thioredoxin in seed wherein the overexpression of thioredoxin h effects a significant increase in the reduction of proteins (&#x2014;SH as compared to S&#x2014;S) of the albumin fraction of the seed.

Transgenic plants with elevated thioredoxin levels

Transgenic monocot plants and seeds comprising monocot seed-specific promoters from seed-storage protein genes and monocot seed-specific targets sequences of expression of heterologous proteins in subcellular compartments to protect the protein are disclosed. The transgenic monocot plants include maize, wheat, rice and barley. Also disclosed are methods of producing transgenic monocot seed from the transgenic plants.

Production of proteins in plant seeds

Thioredoxin, a small dithiol protein, is a specific reductant for allergenic proteins and particularly allergenic proteins present in pollen and animal and plant sources. All targeted proteins contain disulfide (S&#x2014;S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active and less digestible in the oxidized (S&#x2014;S) state. When reduced (SH state), they lose their allergenicity and/or become more digestible. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by lipoic acid chemical reductant. Skin tests carried out with sensitized dogs showed that treatment of the pollens with reduced thioredoxin prior to injection eliminated or decreased the allergenicity of the pollen. Studies showed increased digestion of the pollen proteins by pepsin following reduction by thioredoxin. Pollen proteins that have been reduced by thioredoxin are effective and safe immunotherapeutic agents for decreasing or eliminating an animal's allergic reaction that would otherwise occur upon exposure to the non-reduced pollen protein.

Alleviation of the allergenic potential of airborne and contact allergens by thioredoxin

Thioredoxin, a small dithiol protein, is a specific reductant for major food proteins, allergenic proteins and particularly allergenic proteins present in widely used foods from animal and plant sources. All targeted proteins contain disulfide (S&#x2014;S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active and less digestible in the oxidized (S&#x2014;S) state. When reduced (SH state), they lose their allergenicity and/or become more digestible. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by dithiothreitol, a chemical reductant. Skin tests and feeding experiments carried out with sensitized dogs showed that treatment of the food with reduced thioredoxin prior to ingestion eliminated or decreased the allergenicity of the food. Studies showed increased digestion of food and food proteins by pepsin and trypsin following reduction by thioredoxin.

Increasing the digestibility of food proteins by thioredoxin reduction

The present invention provides barley thioredoxin h nucleic acids and NADP-thioredoxin reductase nucleic acids, the respective encoded proteins and methods of use.

Barley gene for thioredoxin and NADP-thioredoxin reductase

Compositions and methods of use are provided herein to make and use transgenic plants with value-added traits.

Value-added traits in grain and seed transformed with thioredoxin

Disulfide proteins showed mitigated allergenicity and increased digestibility by pepsin following reduction by thioredoxin. The sulfhydryl groups newly formed on reduction by thioredoxin (at 4&deg; C.) or dithiothreitol (DTT) (at 55&deg; C.) were blocked with a physiological disulfide, such as cystamine or oxidized glutathione (GSSG) to obtain stable forms of the disarmed allergen. When derivatized with cystamine, BLG was separated from its oxidized and reduced forms on non-reducing SDS-PAGE and appeared to lack sulfhydryl groups. Although less effective GSSG, gave similar results. Allergenicity of the two derivatives was compared with that of the oxidized, reduced and reoxidized forms of BLG by skin testing dogs from a colony sensitized to cow's milk. Both the cystamine and GSSG derivatized BLG showed decreased allergenicity and increased sensitivity to pepsin as compared to controls. The reoxidized form resembled the derivatives in having lower allergenicity. The thioredoxin- and DTT-reduced forms showed hypoallergenic, hyperdigestible properties, most effectively when the reduced proteins were heated at 55&deg; C. Whole milk subjected to these procedures showed results similar to those obtained with pure BLG. Other proteins are similarly stabilized. Stable forms of such disarmed, hypoallergenic and hyperdigestible disulfide protein allergens or just hypoallergenic or just hyperdigestible protein allergens are useful in foods as well as clinical preparations.

Stabilization of hypoallergenic, hyperdigestible previously reduced proteins

Methods for producing proteins in plant seeds are disclosed. Expression of the protein is driven by a seed-specific promoter and the protein is preferably expressed as a fusion polypeptide that includes a signal peptide that causes the protein to accumulate in a subcellular compartment to protect the protein.

Methods of reducing cystine containing animal and plant proteins, and improving dough and baked goods' characteristics is provided which includes the steps of mixing dough ingredients with a thiol redox protein to form a dough and baking the dough to form a baked good. The method of the present invention preferably uses reduced thioredoxin with wheat flour which imparts a stronger dough and higher loaf volumes. Methods for reducing snake, bee and scorpion toxin proteins with a thiol redox (SH) agent and thereby inactivating the protein or detoxifying the protein in an individual are also provided. Protease inhibitors, including the, Kunitz and Bowman-Birk trypsin inhibitors of soybean, were also reduced by the NADP/thioredoxin system (NADPH, thioredoxin, and NADP-thioredoxin reductase) from either or wheat germ. When reduced by thioredoxin, the Kunitz and Bowman-Birk soybean trypsin inhibitors lose their ability to inhibit trypsin. Moreover, the reduced form of the inhibitors showed increased susceptibility to heat and proteolysis by either subtilisin or a protease preparation from germinating wheat seeds. The 2S albumin of castor seed endosperm was reduced by thioredoxin from either wheat germ or . Thioredoxin was reduced by either NADPH and NADP-thioredoxin reductase or dithiothreitol. Analyses showed that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfides that connect the large to the small subunit. A novel cystine containing protein that inhibits pullulanase was isolated. The protein was reduced by thioredoxin and upon reduction its inhibitory activity was destroyed or greatly reduced.

Use of thiol redox proteins for reducing protein intramolecular disulfide bonds, for improving the quality of cereal products, dough and baked goods and for inactivating snake, bee and scorpion toxins

Methods of reducing cystine containing animal and plant proteins, and improving dough and baked goods' characteristics is provided which includes the steps of mixing dough ingredients with a thiol redox protein to form a dough and baking the dough to form a baked good. The method of the present invention preferably uses reduced thioredoxin with wheat flour which imparts a stronger dough and higher loaf volumes. Methods for reducing snake, bee and scorpion toxin proteins with a thiol redox (SH) agent and thereby inactivating the protein or detoxifying the protein in an individual are also provided. Protease inhibitors, including the Kunitz and Bowman-Birk trypsin inhibitors of soybean, were also reduced by the NADP/thioredoxin system (NADPH, thioredoxin, and NADP-thioredoxin reductase) from either or wheat germ. When reduced by thioredoxin, the Kunitz and Bowman-Birk soybean trypsin inhibitors lose their ability to inhibit trypsin. Moreover, the reduced form of the inhibitors showed increased susceptibility to heat and proteolysis by either subtilisin or a protease preparation from germinating wheat seeds. The 2S albumin of castor seed endosperm was reduced by thioredoxin from either wheat germ or . Thioredoxin was reduced by either NADPH and NADP-thioredoxin reductase or dithiothreitol. Analyses showed that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfides that connect the large to the small subunit. A novel cystine containing protein that inhibits pullulanase was isolated. The protein was reduced by thioredoxin and upon reduction its inhibitory activity was destroyed or greatly reduced.

Thioredoxin, a small dithiol protein, is a specific reductant for allergenic proteins and particularly allergenic proteins present in pollen and animal and plant sources. All targeted proteins contain disulfide (S&mdash;S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active and less digestible in the oxidized (S&mdash;S) state. When reduced (SH state), they lose their allergenicity and/or become more digestible. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by lipoic acid chemical reductant. Skin tests carried out with sensitized dogs showed that treatment of the pollens with reduced thioredoxin prior to injection eliminated or decreased the allergenicity of the pollen. Studies showed increased digestion of the pollen proteins by pepsin following reduction by thioredoxin. Pollen proteins that have been reduced by thioredoxin are effective and safe immunotherapeutic agents for decreasing or eliminating an animal's allergic reaction that would otherwise occur upon exposure to the non-reduced pollen protein.

The present invention provides barley thioredoxin h nucleic acids and proteins. The barley thioredoxin h nucleic acid may be isolated or it may be an expression vector. The expression vector may be operably linked to a transcriptional regulatory sequence. The invention also provides for transgenic plants comprising recombinant barley thioredoxin h. The invention also provides methods of expressing and purifying barley thioredoxin h.

Barley gene for Thioredoxin and NADP-thioredoxin reductase

Thioredoxin, a small dithiol protein, is a specific reductant for major allergenic proteins present in widely used foods from animal and plant sources. All targeted allergenic proteins contain disulfide (S&mdash;S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active in the oxidized (S&mdash;S) state. When reduced (SH state), they lose their allergenicity. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by dithiothreitol, a chemical reductant. Skin tests and feeding experiments carried out with sensitized dogs showed that treatment of the food with reduced thioredoxin prior to ingestion eliminated or decreased the allergenicity of the food.

Growing community of inventors

Berkeley, CA, United States of America

Bob B Buchanan