Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C5/00—Other raw materials for the preparation of beer
  • C12C5/02—Additives for beer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits

Definitions

• This invention relates to a novel gene sequence encoding a foam- related protein. More specifically, the invention relates to a nucleic acid sequence and the 17 kDa Foam Protein it encodes, which protein is useful to enhance the production of foaming beverages, including beer.
• foam properties are an important parameter in high quality beverages.
• Formation of a stable head of foam on pouring a beverage such as beer is an important quality parameter considered by consumers as a vital characteristic.
• Beer proteins range in molecular weight from small polypeptides to more than 150 kDa.
• the present invention describes the purification of a 17 kDa Foam Protein from beer, from barley, from first wort and from rye; the characterization of the 17 kDa Foam Protein with respect to sequence and structure; the establishment of ELISA assays for quantification of the 17 kDa Foam Protein; and the use of the 17 kDa Foam Protein to enhance foam production.
• the 17 kDa Foam Protein has been demonstrated herein to have a positive effect on foam potential and on foam stability.
• the instant invention includes a novel 17 kDa Foam Protein, nucleic acid sequences encoding the protein, methods for enhancing foam quality, and methods for producing enhanced amounts of the protein in host cells, including yeast, plant cells and plants.
• the invention further includes anti-17 kDa Foam Protein antibodies useful in immunoassays for the analysis of 17 kDa Foam Protein content of a sample, and immunoassay kits including the antibodies, and optionally 17 kDa Foam Protein standards.
• Products of the invention include foaming products, such as beverages which foam on pouring.
• Preferred products of the invention include brewed and fermented products, in particular, beer, supplemented with enhanced amounts of 17 kDa Foam Protein and having improved foam quality characteristics such as improved foam potential, foam stability, and foam half-life.
• foam quality is enhanced by supplementation of 17 kDa Foam Protein.
• a product may include an amount of 17 kDa Foam Protein naturally and under normal processing conditions
• the method and products of the invention include an additional amount of 17 kDa Foam Protein, supplementing the amount normally present, and resulting in an enhanced amount of the 17 kDa Foam Protein in the product.
• Such supplementation is achieved by adding purified and isolated 17 kDa Foam Protein to the product during its manufacturing steps, or by providing raw materials (for example, barley or wheat grain or yeast) that contain or produce enhanced amounts of 17 kDa Foam Protein.
• a foaming product is manufactured from raw materials such as barley or wheat grain or yeast, which raw materials are transformed with a supplemental nucleic acid sequence encoding 17 kDa Foam Protein.
• foam quality is enhanced by supplementation with a combination of 17 kDa Foam Protein and LTP-1 protein.
• Figure 1 is a diagrammatic representation of the structure of the 17 kDa Foam Protein polypeptide showing predicted disulfide bridges and protein domains A, B, C, and D.
• Figure 2A is a SDS-poiyacrylamide gel showing 17 kDa Foam Protein purified from beer (lane C).
• Figure 2B is a Western blot showing selective reactivity of anti-17 kDa Foam Protein antibody with the isolated and purified protein from beer (lane C).
• Figure 3 is a diagram showing the distribution of 17 kDa protein between flotate and remanent during repeated flotations in a foam tower.
• Figure 4 is a graph showing the effect of removing 17 kDa protein from lager beer.
• Figure 5 is a plasmid map of a self-replicating yeast expression plasmid carrying the coding sequence of 17 kDa Foam Protein.
• Figure 6 is a plasmid map of a yeast integration plasmid carrying the coding sequence of 17 kDa Foam Protein.
• Figures 7A-7C are graphs showing the relationship between foam half-life and the total amount of 17 kDa Foam Protein and foam-type LTP1 in 50 Danish lager beers at different carbonization levels (g CO 2 /liter): 4.8-5.0 (A); 5.1- 5.3 (B); 5.4-5.6 (C).
• Figure 8 is a Western blot of barley, rye and wheat extracts probed with anti-17 kDa Foam Protein (barley) antibody and showing antibody recognition of components in wheat and rye having approximately the same molecular mass (17 kDa).
• barley Foam Protein
• Figure 9 is a diagram showing a barley transformation cassette carrying the coding sequence of the 17 kDa Foam Protein.
• the present invention includes an isolated and purified 17 kDa Foam Protein having foam-enhancing properties, and useful in the production of foaming products, including beer.
• the invention further includes nucleic acid sequences encoding 17 kDa Foam Protein, anti-17 kDa Foam Protein antibodies, and assays for the detection and quantitation of 17 kDa Foam Protein in a sample.
• Methods of the invention include methods for the enhancement of foam quality in a foaming product, and methods for the production of a foaming product, particularly beer, having a content of 17 kDa Foam Protein that is enhanced over the amount of 17 kDa Foam Protein naturally present in the product.
• the content of 17 kDa Foam Protein, and hence the foam quality of a product is enhanced by adding purified 17 kDa Foam Protein directly to the product during processing steps, and/or by providing raw materials genetically engineered to produce increased amounts of 17 kDa Foam Protein.
• a combination of enhanced 17 kDa Foam Protein and LTP-1 is used to enhance foam quality.
• 17 kDa Foam Protein A novel protein isolated from beer foam, first wort, barley or rye having a molecular weight of approximately 17 kilodaltons (kDa) and having foam enhancing properties as described below.
• Figure 1 shows a diagrammatic representation of the predicted 2-dimensional structure of the 17 kDa Foam Protein. While the amino acid sequence set forth in Tables 1 and 2 (SEQ ID NOS: 10 and 12) define one embodiment of the 17 kDa Foam Protein as obtained from beer foam, first wort, and barley, it is anticipated that 17 kDa Foam Proteins (homologues) having similar foam enhancing properties and homologous amino acid sequences will be similarly isolated from other grains by conventional methods.
• 17 kDa Foam Protein includes foam enhancing proteins derived from cereal grain and having the following characteristics of primary structure useful for purposes of identification:
• a molecular mass of about 17 kDa that is, approximately 15 to 20 kilodaltons (kDa)
• a primary amino acid sequence which can be aligned with distinct homology to the non-repetitive C-terminal domain of the sulfur-rich prolamin storage proteins found in cereals. More specifically, a primary sequence showing significant homology (e.g., greater than 25%) to the C-terminal domain of the monomeric ⁇ -type prolamins (such as ⁇ -gliadin in wheat and ⁇ -hordein of barley).
• the 17 kDa foam proteins are distinguished from these latter ⁇ -type prolamins by the lack of an N-terminal proline- and glutamine-rich domain composed of degenerate pentapeptide repeats;
• the 17 kDa foam proteins of the invention can also be identified by its cross-reactivity with anti-17 kDa foam protein antibodies raised against the purified 17 kDa protein purified from barley.
• 17 kDa foam protein can be purified from cereal grain with the use of 17 kDa specific antibodies for identification purposes.
• the 17 kDa foam proteins are members of a newly identified class of storage polypeptides ( ⁇ -type) present in cereal grain, belonging to the prolamin storage protein family. As such, they are found in the endosperm tissue of mature cereal grain and are synthesized during grain development.
• Foam Enhancing Properties As described more fully in the Examples below, 17 kDa Foam Protein, when added to a product such as water, milkshakes, soft drinks, alcopops or beer, causes the product to have enhanced foam quality. Parameters of foam quality that are enhanced include foam potential (P), foam stability (S), and foam half-life (F), as described more fully below.
• P foam potential
• S foam stability
• F foam half-life
• Nucleic Acid Sequences Encoding 17 kDa Foam Protein Nucleic acid sequences encoding 17 kDa Foam Protein were determined by methods described more fully in the Examples below. The nucleic acid sequence of an isolated barley cDNA and its deduced amino acid sequence combined with the determined sequence of the purified protein are shown in Table 1. Nucleotides 1-57 are back-translated from the determined amino acid sequence, using the codon usage bias of the barley 1-3,1-4 ⁇ -glucanase as described by Jensen et al., 1996 PNAS USA 93:3487-3491. A genomic nucleic acid sequence encoding barley 17 kDa Foam Protein and its deduced amino acid sequence is shown in Table 2.
• the deduced amino acid sequence of the 17 kDa Foam Protein encoded by the cDNA and genomic sequences show close homology, but are not identical.
• the given 1336 nucleotide sequence comprises a 522 nucleotide sequence encoding the precursor 17 kDa Foam Protein polypeptide and 674 and 140 nucleotides of 5' and 3' flanking sequences, respectively.
• the 3' flanking sequence contains consensus sequences for three polyadenylation signals (AATAAA).
• the deduced amino acid sequence of the precursor 17 kDa Foam Protein is predicted to have a 19 amino acid signal peptide sequence.
• nucleic acid libraries including genomic DNA and cDNA libraries constructed from a cereal plant, using sequence-specific primers based on the nucleotide sequence of the barley or wheat 17 kDa Foam Protein genes, or alternatively using degenerate primers back- translated from the determined deduced amino acid sequence of 17 kDa Foam Protein from wheat or barley;
• the cDNA or genomic sequence may be identified in a library constructed from a cereal plant by screening the library with a barley cDNA encoding 17 kDa Foam Protein (for example, that shown in Table 1) as a probe, using standard hybridization conditions (5xSSC, 5x Denhardts solution, 0.5% SDS at 65°C) followed by washing at increasing stringency, with a final 30 minute wash at high stringency (65°C in 0.2% SSC, 0.5% SDS).
• homologs As discussed above, a 17 kDa Foam Protein homolog is defined to include proteins derived from cereal plants having the functional and structural characteristics of the described wheat or barley 17 kDa Foam Proteins. Homologous nucleic acid sequences hybridize to the described nucleic acid sequences encoding barley and wheat 17 kDa Foam Protein, under standard and stringent hybridization conditions, for example, those recited above. Homologous amino acid sequences contain the characteristics of primary structure listed above.
• nucleic acid sequence encoding 17 kDa Foam Protein which contains codons that are modified according to optimal codon frequencies for a particular cellular host. For example, modification for expression in yeast is preferred for production of enhanced 17 kDa Foam Protein in yeast, using known preferred codon frequencies for yeast.
• Redundancy in the genetic code permits variation in the gene sequences shown in Tables 1, 2 and 4.
• specific codon preferences are recognized for a specific host such that the disclosed sequence can be adapted as preferred for the desired host. For example, rare codons having a frequency of less than about 20% in known sequences of the desired host are preferably replaced with higher frequency codons.
• Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and other such well characterized sequences which may be deleterious to gene expression.
• the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
• the genomic sequence may additionally be modified by the removal of introns.
• the nucleic acid sequence encoding the 17 kDa Foam Protein is delivered to host cells, including yeast and plant cells, for transient transfections or for incorporation into the cells by known methods.
• the gene is used to stably transform plant cells for expression of the protein in vivo.
• the gene containing the coding sequence for the 17 kDa Foam Protein may be attached to regulatory elements needed for the expression of the gene in a particular host cell or system.
• regulatory elements include, for example, promoters, terminators, and other elements that permit desired expression of the protein in a particular plant host, in a particular tissue or organ of a host such as starchy endosperm, aleurone or embryo tissues of the barley kernel, during grain development or germination or in response to a particular signal.
• the 17 kDa Foam Protein gene may additionally be attached to a regulatory signal peptide directing its expression into the secretory pathway within the cell.
• the 17 kDa Foam Protein gene coding sequence is attached to regulatory sequences directing its expression into the secretory pathway within the starchy endosperm of the developing barley endosperm.
• the protein is also preferentially adapted for production in yeast.
• the isolated nucleic acid sequence of the invention can be incorporated into DNA constructs and used to transform or transfect a host cell.
• DNA vectors can be used, depending on the host cell and desired expression.
• suitable vectors include, but are not limited to, self-replicating or integration plasmids suitable for expression in prokaryotic or eukaryotic cells.
• a typical gene construct includes a promoter, the coding sequence of interest, and a terminator sequence coupled in operative association. Additional known regulatory elements can also be included in the construct.
• Suitable gene constructs for the stable transformation of host cells include those having constitutive promoters such as the Ubi 1 gene promoter (Christensen, et al., 1992, Plant Mol. Biol, 18: 675-689) driving expression of selectable markers such as the phosphinothricin acetyl transferase gene (bar) (De
• the plasmid can include other gene sequences such as resistance genes (required for the selection and amplification of host transformed cells), reporter genes or other elements.
• resistance genes Required for the selection and amplification of host transformed cells
• reporter genes reporter genes or other elements. Examples of suitable plant transformation selection systems for cereals or other plants are described by Yoder and Goldsbrough, 1994, Bio/Technology, 12: 263-267, and Tingay, et al., 1997, 77ze Plant Journal 11: 1369-1376 are incorporated herein by reference.
• a DNA construct of the invention includes a promoter sequence which may be a "homologous” or “heterologous” promoter.
• a heterologous defines a nucleic acid sequence not normally found in the genome associated with the 17 kDa Foam Protein-expressing gene.
• a heterologous sequence is one derived from a different cell type, different plant species, different organism, or one normally associated with a different gene.
• a heterologous promoter is one which does not drive transcription of the 17 kDa Foam Protein-expressing gene in its natural, non-transformed genome.
• a "homologous" promoter is one normally associated with the 17 kDa Foam Protein gene.
• a promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site. A promoter may be inducible, increasing the rate of transcription in response to an agent, or constitutive, whereby the rate of transcription is not regulated by an inducing agent. A promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operably linked coding region in a specific tissue type or types, for example, plant seeds, leaves, roots, or meristem.
• An heterologous promoter may initiate transcription of an operably linked gene coding sequence at an earlier time or developmental stage in a given tissue, than initiated by the native promoter of the same gene. Within a given host cell or tissue, certain promoters may drive transcription more strongly, resulting in a higher accumulation of transcript, thereby enhancing synthesis of the gene product.
• a promoter useful in the invention is operably linked to a nucleotide sequence encoding 17 kDa Foam Protein such that transcription of the 17 kDa Foam Protein sequence is driven by the promoter.
• the promoter is operably linked to a nucleotide sequence encoding a signal peptide, which in turn is operably linked to a sequence encoding the mature 17 kDa Foam Protein.
• promoters can be used to express the 17 kDa Foam Protein gene in a host cell.
• tissue-specific promoter which drives gene expression in endosperm tissue of developing grain.
• suitable endosperm specific promoter sequences include the promoters of the following genes: Bl hordein gene ( ⁇ hor2-4, GenBank Ace No: X87232), ⁇ -hordein gene ( ⁇ hor ⁇ -1, GenBank Ace No: XI 3508), C hordein gene ( ⁇ hor 1-14, GenBank Ace No: M36941 and ⁇ hor 1-17, GenBank Ace No: X60037); D hordein gene (phor 3-1, GenBank Ace No: X84369); ⁇ -amylase gene ( ⁇ -amyl, GenBank Ace No: D63574) and protein Z gene (Pazl, GenBank Ace No: X51726).
• Additional regulatory elements include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell.
• Such regulatory elements include, but are not limited to, 3' termination/polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al, 1983, Nucl.
• Suitable methods include chemical transfection methods such as the use of calcium phosphate, microorganism-mediated gene transfer such as transfection using an Agrobacterium-mediated transfection system (Horsh, et al., 1985, Science, 227:1229-31), electroporation, micro-injection, and biolistic bombardment.
• chemical transfection methods such as the use of calcium phosphate
• microorganism-mediated gene transfer such as transfection using an Agrobacterium-mediated transfection system (Horsh, et al., 1985, Science, 227:1229-31), electroporation, micro-injection, and biolistic bombardment.
• Agrobacterium-mediated Transformation The most widely used method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium.
• A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
• the Ti and Ri plasmids for A. tumefaciens and A. rhizogenes, respectively, include genes responsible for this genetic transformation. See, for example, Kado, 1991, Crit. Rev. Plant Sci. 10:1.
• a generally applicable method of plant transformation is microprojectile-mediated transformation, wherein DNA is carried on the surface of microprojectiles measuring about 1 to 4 ⁇ m in diameter.
• the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s, sufficient to penetrate the plant cell walls and membranes.
• Electroporation of protoplasts and whole cells and tissues has also been described. See, for example, D'Halluin, et al., 1992, Plant Cell 4:1495-1505; and Spencer et al., 1994, Plant Mol. Biol. 24:51-61.
• 17 kDa Foam Protein can be commercially produced by expression in yeast, either laboratory yeast (e.g.,Saccharomyces cervisiae) or in brewer's yeast (e.g., Saccharomyces carlsbergensis).
• yeast either laboratory yeast (e.g.,Saccharomyces cervisiae) or in brewer's yeast (e.g., Saccharomyces carlsbergensis).
• yeast either laboratory yeast (e.g.,Saccharomyces cervisiae) or in brewer's yeast (e.g., Saccharomyces carlsbergensis).
• yeast signal peptide e.g., Saccharomyces carlsbergensis
• yeast expression vector for example under the control of an inducible promotor, and used to transform yeast cells.
• a 17 kDa Foam Protein expression cassette including the 17 kDa Foam Protein coding sequence, yeast signal peptide coding sequence, and promoter is stably integrated into the yeast genome.
• yeast cells containing the 17 kDa Foam Protein expression cassette during fermentation 17 kDa Foam Protein can be directly secreted into the wort.
• An example of one such expression system is more fully described below in Example 17.
• 17 kDa Foam Protein secreted into the yeast cell growth medium can also be isolated and purified as described in the Example 16 and 17 below, and added to the product during one or more steps in the brewing process.
• Suitable host cells for transformation with the nucleic acid sequence of the invention or its coding sequence include cells that will benefit from the expression of enhanced amounts of 17 kDa Foam Protein, and cells that are useful as raw materials for the production of foaming products.
• Host cells may be adapted for the production, isolation and purification of large amounts of 17 kDa Foam Protein polypeptide.
• Preferred host cells include plant cells, such as barley or other cereal grains, and yeast cells, that are useful in a commercial process to produce foaming products, such as beer.
• Host cells such as bacterial, yeast, or eukaryotic cell lines are transformed with the nucleic acid sequence of the invention such that the transformed cells produce enhanced levels of active 17 kDa Foam Protein.
• the active protein is then added to a brewing mixture, for example, to malt, malt extract, wort, or clarified product to enhance final product foam quality.
• the nucleic acid sequence encoding 17 kDa Foam Protein is used to transform plants whose grain is used in fermentation processes, including barley, wheat, sorghum and other cereals.
• Other host cells including bacteria, yeast, and eukaryotic cell lines are transformed with the nucleic acid sequence of the invention, and used to produce an 17 kDa Foam Protein that can be added to the system during beer production.
• transgenic plant cells, callus, tissues, kernels, and transgenic plants are tested for the presence of the 17 kDa Foam Protein-expressing gene by DNA analysis (Southern blot or PCR), for expression of the gene by immunoassay (ELISA or Western blot), or for functional protein activity by a foam activity assay. Examples of some such conventional methods are shown in the Examples below.
• transgenic plant cells or tissue can be assayed for the presence of 17 kDa Foam Protein mRNA transcripts by hybridization to 17 kDa Foam Protein DNA probes.
• the cDNA sequence encoding the barley 17 kDa Foam Protein [Sequence ID No.9] is a useful hybridization probe for identifying the presence of 17 kDa Foam Protein mRNA in a test sample.
• Such probes are preferably about 10-35 consecutive nucleic acids, but the size may be larger.
• Transcripts from plant tissue transformed with a construct comprising the 17 kDa Foam Protein gene fused to heterologous 5 ' or 3' untranslated region (UTR) sequences can be selectively detected and quantitated by RNA-PCR using primers pairs located in the coding region and the 5' or 3' UTR.
• UTR untranslated region
• a 17 kDa Foam Protein gene construct, fused to a heterologous promoter and/or terminator, can be detected in the genome of transformed tissue by PCR using primer pairs located in the 17 kDa Foam Protein coding region and the heterologous promoter or terminator sequences.
• the PCR product can be used as a hybridization probe for Southern blot analysis of genomic DNA from transformed plants. Transformed plants are compared with untransformed plants to distinguish the introduced constructs from the endogenous 17 kDa Foam Protein gene.
• Samples, including transgenic cells, tissue, plants, and foaming products are screened for content of the 17 kDa Foam Protein by immunological assays, including an Enzyme Linked Immunoassay (ELISA).
• ELISA Enzyme Linked Immunoassay
• Polyclonal antibodies used in an ELISA are, for example, generated against the purified 17 kDa Foam Protein or as described below for Example 8.
• ELISA ELISA
• wells of a microtiter plate are coated with anti- 17 kDa Foam Protein antibodies.
• An aliquot of a sample (the antigen) is added in serial dilution to each antibody-coated well.
• Labelled anti-17 kDa Foam Protein antibodies such as biotinylated antibodies, are then added to the microtiter plate.
• the concentration of bound labelled (e.g., biotinylated) antibody is determined by the interaction of the biotin with streptavidin coupled to peroxidase. The activity of the bound peroxidase is easily determined by known methods.
• the amount of 17 kDa Foam Protein in a sample is quantitated with reference to the ELISA performed with pure antigen standards, where the detection range should lie in the range of 0.2 - 10 ng/ml. Any known method for producing antibodies and using such antibodies in an ELISA assay can be used to determine the amount of 17 kDa Foam Protein in samples, and expressed in transgenic plant cells and tissues of the invention.
• Immunoassay Kits are any known method for producing antibodies and using such antibodies in an ELISA assay can be used to determine the amount of 17 kDa Foam Protein in samples, and expressed in transgenic plant cells and tissues of the invention.
• An immunoassay kit of the invention includes an anti-17 kDa Foam Protein antibody.
• the kit also contains 17 kDa Foam Protein standards for quantitative analysis.
• the kit can include other anti-Foam Protein antibodies such as anti-LTPl antibodies and/or additional reference antibodies or standards.
• the invention provides for improvements in the production of foaming products, such as beer. Improvements in beer are particularly in the quality of the beer foam.
• a manufacturing process for the production of beer can include the following processing steps:
• beers are manufactured from grains, including barley grains, which are naturally low in fermentable sugars. Hydrolysis of starch to sugars is needed prior to fermentation with yeasts. To effect this hydrolysis, grains are wetted and allowed to germinate, during which time the germinating kernels produce hydro lytic enzymes (malting). At the end of malting, the malt is kilned and stored. The malt is then ground and suspended in water at the start of mashing, during which the major part of starch hydrolysis occurs to produce the wort. The wort is boiled and separated from insoluble materials. The wort is then fermented, for example by adding yeast to the wort. Fermentation will convert the wort to beer. To clarify and finish the product, insolubles are filtered, and other constituents are added.
• the quality of the foam depends on both the brewing process and the raw materials used.
• the quality of foam is enhanced by supplementing the brewing process with 17 kDa Foam Protein added during one or more processing steps and/or by providing raw materials such as barley grain or brewer's yeast producing enhanced amounts of 17 kDa Foam Protein.
• Danish Lager beer brewed with adjuncts contains about 10-50 mg/1 of 17 kDa Foam Protein.
• the effect of further addition of 17 kDa Foam Protein is dependent on the composition of the beer and the ratio of the beer's foam components. However, in general, addition of about 25 mg/1 or more of 17 kDa Foam Protein is expected to have a beneficial effect on the foam properties of the beer.
• the quality of foam in a foaming product is enhanced by providing enhanced amounts of 17 kDa Foam Protein in combination with enhanced amounts of LTP protein.
• a combination of enhanced amounts of 17 kDa Foam Protein and enhanced amounts of LTP 1 are provided by genetically engineering plants such as barley, rye and wheat to produce high amounts of these Foam Proteins.
• a cereal plant e.g., a barley plant carrying transgenes encoding both LTPl and 17 kDa Foam Protein.
• two independent transgenic lines e.g. barley
• the two lines are crossed to generate and breed lines which are homozygous for both transgenes, thereby producing a plant, e.g. barley plant, providing even greater enhancement of foam potential and stability than achieved by either transgene alone.
• the Head Hunter an opto-electrical foam assay system using digital video image analysis, was used to measure foam potential and foam half-life of small, degassed samples. This system creates foam on 10 ml samples by a short, vigorous, standardized shaking procedure. The decay with time of the foam column generated in this way is then automatically monitored (Haugsted, et al., 1990 Monatsschr. Brauwissenschaft 43:336-339; Haugsted and Erdal, 1991 Proc. 23rd EBC Congress (Lisbon): 449-456).
• the foam potential (P) is the amount of foam formed initially, in ml per ml sample.
• the foam half-life (F) is the time, in seconds, after which the foam column is reduced to half the initial volume.
• foam assays on the Head Hunter were conducted at least in duplicate.
• 150 g of beer is fully converted to foam, and the decay of foam into beer is followed. After an initial lag phase of about 30 seconds, this decay is a first order process (Hallgren, et al, 1991 J. Am. Soc. Brew. Chem. 49:78-86).
• the foam half-life, in seconds, is then determined.
• CO 2 in beer was determined by a titration procedure. Initially, CC ⁇ was absorbed in a carbonate-free solution of sodium hydroxide. Then, CO was liberated again via addition of sulphuric acid and forced into a solution of barium hydroxide of known concentration. After precipitation of barium carbonate, the surplus of barium hydroxide was determined by titration with hydrochloric acid using phenolphtalein as an indicator, and CO 2 in beer was finally calculated from this determination.
• the protein content was determined from amino acid analyses or by the dye binding method of Bradford (Bradford, 916 Anal. Biochem 72:248-254).
• SDS-PAGE and Western blots SDS-polyacrylamide gel electrophoresis (PAGE) was performed after boiling the samples for 5 minutes in NuPAGE sample buffer from Novex supplemented with 10 mM dithiothreitol using a Novex XCell IITM mini cell, NuPAGETM 10% Bis-Tris gels (1.0 mm x 10 wells) and NuPAGE MES-SDS Running Buffer from Novex. Blotting onto nitrocellulose was then performed in the Xcell IITM blot module using the NuPAGETM Transfer Buffer from Novex. After washing with water, the nitrocellulose was incubated 30 minutes with calf serum to block non-specific binding and then incubated overnight with antibodies.
• PAGE SDS-polyacrylamide gel electrophoresis
• the Promega Western blot AP system (Catalogue No. W3930) was then applied to detect specific antibody binding.
• a mixture of nitro blue tetrazolium and 5-bromo-4- chloro-3 -indolyl phosphate was used as color development substrate for alkaline phosphatase.
• Amino acid compositions were determined on an amino acid analyzer (LKB, model Alpha PlusTM) after hydrolysis in 6 M HCI at 110°C for 24 hours in evacuated tubes.
• N-terminal amino acid sequencing was performed on a gas-phase sequence (Applied Biosystems model 470 A), using the program provided by the company.
• the phenylthiohydantoin-labelled amino acids from the sequencer were identified on-line by reversed-phase HPLC using an Applied Biosystems model 120 A phenylthiohydantoin analyzer.
• foam was produced from 15 liters of beer by sparging with nitrogen, as described in S ⁇ rensen, et al., 1993, MBAA Techn. Quart. 30:136-145.
• the foam fraction was diluted with water and reintroduced into the foam tower. Two additional flotations were carried out to get rid of components which were not foam-active themselves but merely carried over with the foam.
• the third flotate was used in the following studies. By gel chromatography on Sephadex G75, the foam fractions were separated according to size into three fractions: a high molecular weight fraction (HMW), a low molecular weight fraction (LMW), and a very low molecular weight fraction.
• HMW high molecular weight fraction
• LMW low molecular weight fraction
• very low molecular weight fraction a very low molecular weight fraction.
• the very low molecular weight fraction consisted of small peptides and carbohydrates, free amino acids, and iso- ⁇ acids.
• Foam analyses in the Head Hunter revealed that the foam active components are present in the HWM and LMW foam fractions, while the very low-molecular weight foam fraction does not contribute to foam formation nor foam stability. When dissolved in water, the HMW foam fraction forms a low but most stable foam, while the LMW foam fraction provides a very high foam potential.
• the HMW foam fraction contains mainly carbohydrate (90%) but also some protein Z, a barley albumin. Foam measurements on protein Z isolated from beer showed that protein Z creates a low but very stable foam when dissolved in water. Further, protein Z isolated from beer has a stabilizing effect on the foam created by other proteins.
• protein Z was distributed with approximately 1/3 in the first flotate and 2/3 in the remanent. In the following flotations, most of protein Z from the beer remained in the remanent. Thus, protein Z does not concentrate in the foam upon foaming of beer.
• Protein Z has also been purified from malt and antibodies have been raised against this protein. Based on these antibodies a sandwich ELISA for the quantification of protein Z has been established. Using these antibodies, an affinity column directed towards protein Z has been synthesized and used for selective removal of protein Z from beer. Removal of protein Z does not influence the foam potential, but affects the foam stability, depending on the composition of the beer.
• the LMW foam fraction consists of 10% carbohydrate and 90% protein.
• a major component in LMW foam is the protein Lipid Transfer Protein 1 (LTPl), a barley protein with a molecular weight of 10,000.
• LTPl Lipid Transfer Protein 1
• the LMW foam fraction also contains substantial amounts of other peptides of which the major part can be separated from LTPl by ion exchange chromatography.
• LTPl from foam forms a very high foam which is not particularly stable.
• Foam-LTPl also increases foam potential when dissolved in beer, the effect, however, being not as pronounced as in water since beer already contains foam positive agents.
• LTPl has also been purified from barley (barley-LTPl), and foam measurements have shown that foam-LTPl is considerably more foam active than is barley-LTPl . It was, therefore, of interest to compare the two varieties and investigate when the transformation of LTPl into a more foam active form takes place.
• the LMW foam fraction also contains substantial amounts of other peptides of which the major part can be separated from LTPl by ion exchange chromatography. Amino acid analysis of this pool, "Pool 1", showed high concentrations of proline and glutamate/glutamine, indicating that it contains substantial amounts of hordein peptides. When dissolved in water, Pool 1 contributes to foam potential as well as foam stability. Pool 1 is also able to increase foam potential when dissolved in beer.
• 17 kDa Foam Protein was purified from 5 liters of beer by addition of ammonium sulfate to 55% saturation. After 16 hours at 4° C, the suspension was centrifuged and the precipitate was dissolved in water and dialyzed in a SpectraporTM membrane against water. The dialysate was passed through a 270 ml SP-SepharoseTM column equilibrated with 20 mM sodium acetate, pH 4.5. The run-through fraction from this column was adjusted to pH 3.9 by addition of HCI and the ionic strength was reduced to 0.03 mS by addition of water.
• the resulting fraction was subjected to ion exchange chromatography on a 270 ml SP- SepharoseTM column equilibrated with 5mM sodium formate, pH 3.9.
• ELISA revealed the 17 kDa Foam Protein to be eluted by a linear gradient from 0 to 0.25 M NaCl.
• Fractions containing the 17 kDa Foam Protein were concentrated by vacuum evaporation and applied to a 350 ml SephadexTM G50 column equilibrated with 20mM sodium acetate, 100 mM sodium chloride, pH 4.9.
• the resultant fractions were analyzed by ELISA and those containing the 17 kDa Foam Protein were pooled, dialyzed against water and lyophilized.
• 17 kDa Foam Protein was isolated from 2 kg barley flour (Alexis) by extraction with 10 L water at 45°C for 1 hour. The mixture was centrifuged and ammonium sulfate (50% saturation) was added to the supernatant to precipitate protein. After 16 hours at 4°C, the suspension was centrifuged and the precipitate was dissolved in water and dialyzed in a Spectrapor membrane against water. 17 kDa Foam Protein was purified from barley essentially as described above for purification from beer in Example 2.
• 17 kDa Foam Protein was isolated from first wort from a production scale mashing (Carlsberg Pilsner) using the purification procedure described above for Example 2. On SDS-PAGE, the preparation showed a distinct double band of 17-18 kDa.
• 17 kDa Foam Protein isolated from barley and beer as described above was subjected to N-terminal sequencing.
• 17 kDa Foam Protein from barley was sequenced 40 cycles while 17 kDa Foam Protein from beer was found to be N- terminally blocked.
• 17 kDa Foam Protein contains 7 methionyl residues corresponding to 8 cyanogen bromide fragments. After chemical cleavage with cyanogen bromide, five of these fragments were isolated and sequenced and the sequence showed homology with that of the 17 kDa Foam Protein encoded by the cDNA and genomic clones shown in Tables 1 and 2.
• the amino acid sequence of each of these fragments is indicated in the sequence Table 1 by underlining, and is listed below.
• the nucleotide sequence of the barley gene coding for the 17 kDa Foam Protein was determined from a cDNA clone generated by reverse transcription and amplification techniques (RT-PCR).
• the cDNA clone was selectively amplified from a total RNA population isolated from developing barley endosperm tissue cv Alexis, 20 days after anthesis, as described by Rechinger, et al. 1993, Theor. Appl. Genet. 85:829-840.
• the mRNA in the RNA population was reverse transcribed with a poly dT primer having a BamHl restriction site sequence at the 5 'end (5' ATGGATCCT ⁇ 3') [SEQ ID NO: 6].
• the reaction mixture (20 ⁇ l) comprising 1 ⁇ g total RNA, 1 ⁇ M poly dT primer, 1 mM deoxynucleoside triphosphates, 28U RNasin (Promega) and 20U M-MuLV reverse transcriptase (Boehringer Mannheim) in a buffer supplied by the manufacturer, was incubated at 40°C for 30 minutes.
• the first strand cDNA was amplified with a sequence-specific, but degenerate primer, based on a determined amino acid sequence located near the N-terminus of the 17 kDa Foam Protein (PQQQMN) [SEQ ID NO: 7], having a BamHl restriction site sequence at the 5 'end (5 'ATGGATCCICAICAICAIATGAA 3') [SEQ ID NO: 8] ("I" denotes the degeneracy).
• PQQQMN 17 kDa Foam Protein
• the reaction mixture (lOO ⁇ l) comprised 4 ⁇ l first strand cDNA reaction mixture, 0.2 ⁇ M sequence-specific and polydT primers, 0.2 mM deoxynucleoside triphosphates and 2.5U AmplitaqTM DNA polymerase (Perkin Elmer) in a buffer supplied by the manufacturer.
• the cDNA was amplified in a Perkin Elmer Thermocyler 480 with 30 heating cycles (95°C for 1 minute / 50°C for 1 minute) and then analysed by agarose gel electrophoresis. A cDNA fragment of approximately 580 nucleotides was isolated, cloned into a plasmid vector and its nucleic acid sequence was determined with an AmpliCycleTM Sequencing Kit (Perkin Elmer). The determined and back- translated nucleic acid sequence [SEQ ID NO: 9] and the determined and deduced amino acid sequence [SEQ ID NO: 10] are shown below in Table 1.
• Nucleotides 1 - 57 were back-translated from the determined N-terminal amino acid sequence of the 17 kDa Foam Protein using the codon bias of the barley 1-3,1-4 ⁇ -glucanase according to Jensen et al., 1996 supra.
• the deduced amino acid sequence encoded by the partial cDNA clone shows complete homology with the sequence of N-terminal and cyanogen bromide peptide fragments determined for the purified 17 kDa foam polypeptide, confirming that the isolated cDNA encodes the 17 kDa Foam Protein.
• Underlined sequences in Table 1 indicate the sequences of the cyanogen bromide fragments.
• PCR primers used to amplify the clone are indicated above the sequence. * Denotes stop codon.
• the cDNA clone encoding the 17 kDa Foam Protein was used to screen a commercially available barley genomic library (Lambda Fix II Barley cv Igri Genomic Library from Stratagene, Catalog No: 946104) in order to obtain the genomic gene sequence.
• barley genomic library Lambda Fix II Barley cv Igri Genomic Library from Stratagene, Catalog No: 946104
• three positive plaques were identified out of 400,000 plaques.
• One characterized lambda genomic clone contained a hybridizing
• This genomic sequence contains the complete coding sequence for a precursor form of the 17 kDa Foam Protein.
• the deduced amino acid sequence of this precursor 17 kDa Foam Protein (SEQ ID NO: 12) is predicted to comprise a 19 amino acid signal peptide with a signal peptide cleavage site between Ala 19 and Gin 20, according to the signal peptide algorithm developed by Nielsen, et al., 1997 Protein Engineering 10: 1-6.
• the N-terminal amino acid of the determined mature 17 kDa Foam Protein amino acid sequence is Leu 21.
• the actual signal peptide cleavage site is Gln20 iLeu21, or alternatively that the N-terminal Gln20 is cleaved off post-translationally to give the amino acid sequence of a mature 17 kDa Foam Protein.
• the deduced amino acid sequences of the 17 kDa Foam Proteins encoded by the genomic and cDNA clones show close homology, with six amino acid missmatches. These sequence differences may reflect heterogeneity between cultivars or the presence of more than one copy of the gene in the haploid genome.
• the gene 3' flanking sequence of 140 nucleotides contains consensus sequences for three polyadenylation signals.
• the gene 5' upstream sequence of 674 nucleotides contains a TATAA box, shown underlined in Table 2, located 85 nucleotides upstream of the translation start codon.
• An endosperm box (TGTAAAG) followed by a GCN4 box (ATGAGTCAT), shown in bold, located 304 nucleotides upstream of the translation start, are transcription regulatory elements found in the promoters of many cereal endosperm storage protein genes (Muller and Knudsen 1993 The Plant Journal A: 343-355).
• the nucleotide sequence of a wheat gene coding for a homologue of the barley 17 kDa Foam Protein was determined from a genomic DNA fragment amplified by PCR. Wheat grain (Triticum aestivum L.) cv Husar was germinated and grown in the dark for 6 days and the etiolated leaves harvested for the preparation of genomic DNA using a Plant DNA Isolation Kit from Boehringer Mannheim. Wheat genomic DNA (0.5 ⁇ g) was PCR amplified with degenerate sense and antisense oligonucleotide primers based on the deduced amino acid sequence of the barley 17 kDa Foam Protein:
• the genomic DNA was amplified with 2.5 pmol of each primer and AmpliTaqTM DNA polymerase (Perkin Elmer) in a reaction mixture provided by the manufacturer, using a ⁇ touch-down' thermocycling program (95°C for 0.45 minutes, 54°C [ -1°C /cycle ( 40°C) ] for 0.45 minutes and 72°C for 2 minutes for 15 cycles; 95°C for 0.45 minutes, 40°C for 0.45 minutes and 72°C for 2 minutes for 25 cycles; 72°C for 6 minutes).
• PCR product of about 500 nucleotides was isolated, cloned in a pCR 2.1-TOPO plasmid vector (Invitrogen) and sequenced with an AmpliCycleTM sequencing Kit (Perkin Elmer) on an Applied Biosystems 373 DNA sequencer.
• the nucleotide sequence of the amplified genomic DNA was found to encode a homolog of the barley 17 kDa Foam Protein.
• the nucleic acid sequence [SEQ ID NO: 20] and deduced amino acid sequence [SEQ ID NO: 21] of the wheat 17 kDa Foam Protein are shown in Table 4.
• Sequence alignment of the nucleic acids encoding wheat and barley 17 kDa Foam Protein homologs indicates that the wheat amplified genomic fragment, including the primers, encodes the last 2 residues of the predicted signal peptide and extends to within 4 amino acids of the C-terminus of the mature barley 17 kDa Foam Protein sequence.
• the high degree of sequence homology between the wheat and barley 17 kDa Foam Protein homologues is consistent with recognition of a wheat 17 kDa polypeptide by the anti-barley 17 kDa Foam Protein antibodies described in Example 19.
• 17 kDa Foam Protein was isolated from beer and from barley as described for Examples 2 and 3. Head Hunter foam assays were performed on the isolated proteins dissolved in distilled water at concentrations of 0.25 or 0.50 mg/ml. The data shown below in Table 5 demonstrates that the protein isolated from beer was able to produce a high and stable foam, whereas the protein isolated from barley produced only a moderate foam with very little stability. The foam capacity of the barley-form of 17 kDa was only slightly affected by the presence or absence of nicks in the peptide chain. Table 5
• Foam assays were also performed on beer supplemented with 17 kDa Foam Protein that had been isolated from beer.
• the 17 kDa Foam Protein was supplemented either alone or in combination with LTPl that had been isolated from beer foam (Bech, et al., 1995, supra).
• a lager beer, Carlsberg Pilsner, naturally containing 30 mg/1 of the foam-type of LTPl and 25 mg/1 of 17 kDa Foam Protein was diluted 1 : 1 with a solution of 4% ethanol in water and used as basis for these experiments.
• the rabbits received 250 ⁇ g LMW foam preparation at each injection.
• a volume of 20 ml serum was obtained from each animal approximately two months after the first injection, and then at monthly intervals. Serum obtained from the second bleeding of one of the animals (batch no. 1897), was used throughout the experiments described below.
• Antibodies of the immunoglobulin G class were purified from other serum components by affinity chromatography on Hi-Trap Protein A- Sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions.
• the pool of antibodies thus obtained also contained antibodies recognizing LTPl of the foam- type, which is a prominent Foam Protein in beer.
• Antibodies recognizing LTPl were removed from the IgG pool by affinity chromatography on a small column containing LTPl of the foam-type covalently attached to CNBr-activated Sepharose according to the manufacturer's instructions. When the remaining antibodies were used in Western blots of beer protein, only 17 kDa Foam Protein was stained as a distinct double band (See Figure 2B, lane A foam-LTP-1; lane B, beer; lane C, 17 kDa Foam Protein from beer). The antibodies specific to 17 kDa Foam Protein were used as coating reagent in ELISA assays, performed as described below for Example 10. Biotinylated antibodies used in this assay were prepared using the complete IgG fraction of serum no. 1897.
• a small affinity column was prepared by covalently coupling antibodies specific to 17 kDa Foam Protein to CNBr-activated Sepharose
• the assay was performed in polystyrene wells arranged in strips of twelve, and the strips were placed in frames each containing eight strips (Nunc Immuno Module C12 MaxisorpTM from Life Technologies, Denmark).
• the specific 17 kDa Foam Protein antibodies were diluted to
• PBS 10 phosphate buffered saline: 10 mM sodium phosphate pH 7.3, 150 mM NaCl. 200 ⁇ l aliquots were added to each well and incubated at 4°C for 16-20 hours. After this, the wells were emptied and washed 5 times with PBST (PBS 10 supplemented with 0.01% Tween 20) (Merck, Darmstadt, Germany). Residual binding sites on the polystyrene surface were then blocked by adding 200 ⁇ l
• BSA/PBST PBS10 supplemented with bovine serum albumin (BSA), 1 g/1, and 0.05% Tween 20
• BSA bovine serum albumin
• Tween 20 bovine serum albumin
• step 3 biotinylated antibodies were diluted to 1 ⁇ g/ml in BSA/PBST. 200 ⁇ l aliquots were incubated in the wells for 10 minutes at ambient temperature. After this, the wells were emptied and washed.
• step 4 a conjugate of streptavidin and horseradish peroxidase (SIGMA) was diluted to 0.25 ⁇ g/ml in BSA/PBST. 200 ⁇ l aliquots were incubated in the wells for 10 minutes at ambient temperature. After this, the wells were emptied and washed.
• step 5 a substrate was prepared containing 3.3 ',5.5'- tetramethylbenzidine (TMB), 100 ⁇ g/ml, and H O , 0.015%o, in phosphate-citrate buffer pH 5.0. 200 ⁇ l aliquots were incubated in the wells for 5 minutes at ambient temperature.
• TMB 3.3 ',5.5'- tetramethylbenzidine
• H O 0.015%o
• Each series of analyses included a set of 17 kDa Foam Protein standards prepared from "Pool 1", a fraction of beer foam rich in this protein.
• Pool 1 a fraction of beer foam rich in this protein.
• One preparation of Pool 1 obtained from a commercial lager beer (Carlsberg Pilsner), was used in the experiments described below.
• the total protein content in this Pool 1 was determined by amino acid analysis, and standards were prepared in the range 0-33 ng Pool 1 protein/ml.
• the final assay was highly specific to 17 kDa Foam Protein. Only very slight reactions with other proteins purified from beer, barley or malt could be observed (See Table 7).
• this protein was purified from malt essentially as described in the literature (Hejgaard, 1982 Physiol. Plant 54:174-182) and used for immunization of rabbits using the methods described for Example 9.
• a non-competitive ELISA procedure of the sandwich-type was then established for protein Z, essentially as described for 17 kDa Foam Protein in Example 10.
• the preparation of protein Z used for immunization was also used as standard in the ELISA assays.
• a set of 22 beers varying in original gravity from 7.3 to 15.9 and with widely different levels of beer proteins were used for testing the correspondence between the two methods of quantification. Some beers were brewed with 30-40% maize grits as adjunct, and some were all-malt beers.
• the ELISA results agreed well with the quantifications based on affinity chromatography. The slope of straight lines obtained as best fit when plotting analytical values obtained by affinity chromatography vs. values obtained by ELISA were 0.78 for the barley-type LTP, 0.90 for foam-type LTP and 0.82 for protein Z. Thus, affinity chromatography gave slightly lower results than ELISA for all three proteins, probably due to a slight loss of material during the chromatographic procedure.
• Foam was produced from 15 liters of lager beer by sparging with nitrogen gas in a foam tower overnight at a rate of 450 ml/minute (S ⁇ rensen et al., 1993 supra). The nitrogen gas was saturated with water vapor before introduction into the foam tower. After collapse, the foam collected at the outlet was diluted to the original volume with distilled water and reintroduced into the foam tower. A second and a third flotation was performed as described above. After each flotation step, aliquots of the foam, termed the flotate, and of the remaining unfoamed liquid, termed the remanent, were collected for analysis.
• the content of 17 kDa Foam Protein in flotates and remanents and in the original lager beer was determined by ELISA assays performed as outlined in Example 10. During the first flotation, at least 75% of the 17 kDa Foam Protein found in the beer was transferred to the foam, whereas only 25% or less remained in the unfoamed liquid. During repeated flotations, only very small amounts of 17 kDa Foam Protein were found in the remanent, whereas the 17 kDa Foam Protein found in foam from a flotation was transferred almost quantitatively to a subsequent foam fraction (see Figure 3).
• Example 13 Selective Removal of 17 kDa Protein Severely Reduces Foam Potential
• a small affinity column was prepared by covalent coupling of antibodies specific to 17 kDa Foam Protein to CNBr-activated Sepharose according to the manufacturer's directions.
• a volume of 90 ml of a standard lager beer from a production plant (Carlsberg Pilsner) was repeatedly passed through the column, and the content of 17 kDa Foam Protein, foam-type LTP, barley-type LTP and protein Z was determined after each passage of the column by means of ELISA assays.
• the foaming capacity of the sample was determined after each passage by use of HeadHunter equipment.
• Example 14 Content of 17 kDa Foam Protein in Beer and Correlation to Foam Half-Life
• the content of 17 kDa Foam Protein in 50 Danish lager beers was determined by means of the ELISA procedure described in Example 10.
• the beers were obtained from a variety of Danish breweries and included both all-malt types and beer brewed with maize grits as adjunct. They were collected during the first three months of 1997 and were at that time all within the day of latest purchase. The samples were further analyzed by ELISA procedures for the foam-type of LTPl, the barley-type of LTPl and for protein Z as described above for Example 10.
• the content of CO 2 was determined as described in the section on analytical procedures.
• the foam half-life (F) of the bottled beer was determined on the Foam Stability Analyzer.
• the average concentration of 17 kDa Foam Protein and other beer proteins in 50 Danish lager beers is shown below in Table 8.
• the content of CO 2 in beer is known to influence foam half-life determinations made on the Foam Stability Analyzer. Therefore, the beers were grouped according to their content of CO 2 before investigating if any relationships existed between foam half-life and content of beer proteins.
• the foaming properties of a beer may be enhanced by the use of barley malt, genetically engineered to contain an elevated content of the 17 kDa Foam Protein, as a raw material in the beer brewing process.
• the 17 kDa Foam Protein is found in endosperm of mature grain and as a member of the hordein storage protein family is synthesized during grain development.
• the accumulation of 17 kDa Foam Protein in the developing endosperm is enhanced, for example, by the insertion of additional copies of the 17 kDa Foam Protein gene into the barley genome, under the transcriptional control of its native promoter or, alternatively, under the control of any one of the various previously characterized endosperm specific promoters, e.g.
• the sequence encoding the mature 17 kDa Foam Protein is cloned downstream of the promoter of the D hordein gene (Hor3, GenBank Accession number: X84368), as shown in Figure 9. Since proteins homologous in sequence to 17 kDa Foam Protein are found in wheat (Table 4) and in rye (see Example 19, below) (Rocher, et al., 1996, Biochem. Biophys. Acta. 1295:13-22), and may be found in other cereals, it is assumed that these homologs could equally be used to enhance the foaming properties of beer.
• the 17 kDa Foam Protein cDNA clone or other probes may be used to screen out gene sequences encoding 17 kDa Foam Protein homologs from cDNA or genomic libraries constructed from cereals such as rye, wheat or rice.
• PCR amplification techniques may be used to amplify homologous 17 kDa Foam Protein gene sequences from genomic DNA prepared from these cereals.
• Similar transformation expression cassettes namely promoter, signal peptide-encoding sequences and terminator, can be used to express transgenes encoding 17 kDa Foam Protein homologues in the developing barley grains or in other cellular hosts.
• the foaming properties of beer may be enhanced by the addition of 17 kDa Foam Protein or its homologues to the wort.
• 17 kDa Foam Protein or a homolog is produced on an industrial scale by expression of the 17 kDa Foam Protein gene in either laboratory yeast (e.g. Saccharomyces cerevisiae), or brewers yeast (e.g. Saccharomyces carlsbergensis).
• yeast e.g. Saccharomyces cerevisiae
• yeast e.g. Saccharomyces carlsbergensis
• the 17 kDa Foam Protein coding sequence, fused to an appropriate sequence encoding a yeast signal peptide is cloned into a self-replicated yeast expression plasmid under the transcriptional control of an inducible promoter and transformed and maintained in yeast under selective pressure.
• the 17 kDa Foam Protein secreted into the yeast growth medium can subsequently be purified according to protocols similar to those described in Example 2.
• the 17 kDa Foam Protein polypeptide expression cassette (yeast promoter + yeast signal peptide coding sequence + 17 kDa Foam Protein coding sequence) can be stably integrated into the yeast genome.
• 17 kDa Foam Protein secreted into the yeast growth medium may similarly be purified as described in Example 2 and subsequently added to the wort. Stable integration of the 17 kDa Foam Protein expression cassette into brewers yeast allows the secretion of the 17 kDa Foam Protein directly into the wort during fermentation.
• FIG. 5 One example of a self-replicating yeast expression vector is shown in Figure 5, which comprises the PRB 1 promoter (derived from the S. cerevisiae PRB 1 gene encoding protease B), the PGK terminator (derived from the S. cerevisiae PGK gene encoding phosphoglycerate kinase) and the mature 17 kDa Foam Protein coding sequence inserted downstream of the sequence encoding the B. macerans (1-3, l-4)- ⁇ -glucanase signal peptide.
• PRB 1 promoter derived from the S. cerevisiae PRB 1 gene encoding protease B
• the PGK terminator derived from the S. cerevisiae PGK gene encoding phosphoglycerate kinase
• the mature 17 kDa Foam Protein coding sequence inserted downstream of the sequence encoding the B. macerans (1-3, l-4)- ⁇ -glucanase signal peptide
• the 17 kDa Foam Protein yeast expression vector is derived from pBl-L-MH(A16-M) (Meldgaard, et al., 1995, Glycoconjugate J. 12: 380-390) using the following cloning steps.
• the plasmid, pBl-L-MH(A16-M) is first linearized with BgM and the site is blunt ended.
• the plasmid is then digested with /JstEII to excise the (1-3, l-4)- ⁇ -glucanase coding sequence, which is then replaced with the 17 kDa Foam Protein coding sequence amplified from the 17 kDa Foam Protein cDNA using the following primers:
• the PCR product is then digested with ifatEII (ifatEII site underlined) prior to the replacement cloning step.
• ifatEII ifatEII site underlined
• the 17 kDa Foam Protein yeast expression vector is transformed into an appropriate Leu- yeast strain and cultivated in fermentors in SC medium without Leu as described by Meldgaard, et al., 1995, supra.
• yeast expression plasmid shown in Figure 5 may be used as a basis.
• the plasmid is restriction digested with EcoRI, and the large fragment is purified.
• pCH216 Hadfield, 1994 In: Molecular Genetics of Yeast. A Practical Approach. Johnston, J.R. (ed.). Oxford University Press, Oxford, UK, pp. 17-48) containing the APTI gene, conferring resistance to the antibiotic G418.
• the APTI gene is transcribed from a yeast PGKl promoter which makes it usable as a dominant selectable marker in brewer's yeasts at a concentration of 30 mg/ml on YPD plates (rich medium).
• the resulting plasmid construct (see Figure 6) is devoid of yeast 2 ⁇ origin of replication sequences, making self-replication in yeast impossible.
• yeast with this construct requires the integration of the whole plasmid into the yeast genome.
• Integration of the plasmid is obtained through employment of the first part ('loop-in') of the method for replacement of chromosome segments in yeast described by Scherer and Davis, 1979, PNAS USA 76:4951-495.
• the plasmid needs to be linearized in one of the yeast DNA sequences that it harbors (Orr— Weaver, et al, 1981 PNAS USA 78:6354-6358). Linearization is, therefore, performed through restriction digestion at the unique Ngo AIV site (with any of the isoschizomers NgoAYV, Nael, NgoMI or r ⁇ I) in the PRBl gene, and the resulting linear D ⁇ A used to transform S. carlsbergensis brewer's yeast to G418-resistance.
• G418-resistant yeast clones are then checked for integration of the plasmid containing the 17 kDa Foam Protein expression cassette at the proper location in the wildtype PRBl gene. The functionality of the latter gene will not be changed, as none of the gene is missing after proper integration.
• the content of 17 kDa Foam Protein varied widely within this set of samples. About 8 times more 17 kDa Foam Protein was extracted from the cultivar Maresi than from Polygena and Optima under the specified extraction conditions
• 17 kDa Foam Protein may be linked to total protein content within a variety.
• One of these samples had about 50% more total protein than the other and also about 50% more 17 kDa Foam Protein.
• 17 kDa Foam Protein there is no correlation between 17 kDa Foam Protein and total protein. The ratio of
• 17 kDa Foam Protein in the samples varies from approximately 0.5-2 mg 17 kDa protein/g total protein (Table 9).
• the barley extract On Western blotting, the barley extract gave a double band with a molecular mass of approximately 17-18 kDa ( Figure 8, lane C).
• the identity of the two bands from the barley extract was investigated by blotting from SDS-PAGE onto a PNDF membrane, staining shortly with Comassie blue stain and cutting out each of the two bands separately.
• the N-terminal sequence of the first 4-8 amino acids of the proteins corresponding to the two bands was then determined as described in Analytical Procedures. Both bands gave a sequence identical to the N- terminal sequence of 17 kDa Foam Protein (Table 1) and therefore probably represent isoforms of 17 kDa Foam Protein.
• the rye extract ( Figure 8, lane A) gave three major bands at approximately 15, 17 and 20 kDa, whereas the wheat extract (lane B) gave two bands at approximately 17 and 20 kDa. Apart from these bands, faint bands were observed at higher molecular mass from the rye and wheat extracts.
• the antibodies to 17 kDa Foam Protein from barley recognize components in wheat and rye with approximately the same molecular mass.
• the amino acid sequence and/or the tertiary structure of these components are expected to be very similar to the 17 kDa Foam Protein from barley.
• a transformation cassette useful for introducing enhanced amounts of 17 kDa Foam Protein into barley is shown in Figure 9.
• the cassette which includes the hor 3-1 promoter; the 17 kDa Foam Protein coding sequence; and the NOS terminator, is transformed into barley.
• Transformed lines identified by PCR screening for the presence of transgene, are grown to maturity.
• Transformed lines expressing enhanced levels of 17 kDa Foam Protein in the grain are identified by using the ELIZA assay described in Example 10, and the selected lines are bred to homozygosity for the transgene.
• Homozygous GMO barley lines expressing enhanced levels of 17 kDa Foam Protein are then crossed with GMO barley expressing enhanced levels of LTPl.
• GMO barley lines transformed with a chimeric LTPl gene cassette (Chi26 promoter-Ltpl or Hor3-l promoter-Ltpl) which accumulate enhanced levels of LTPl in the mature grain.
• Hybrid GMO barley lines carrying both LTPl and 17kDa Foam Protein transgenes are then used as a preferred barley for the production of beer and other foaming products.
• Example 21 Isolated Rye Nucleic Acid Sequences Encoding 17 kDa Foam Proteins
• the nucleic acid sequences of three rye genes encoding homologues of the barley and wheat 17 kDa Foam Protein have been determined from cDNA and genomic clones, generated by RT-PCR and PCR amplification respectively.
• the cDNA was selectively amplified from a total RNA population isolated from developing rye (Secale cereale) endosperm tissue harvested 25 days after anthesis, as described by Rechinger, et al. supra.
• the total RNA was reverse transcribed with two antisense primers, whose sequence was based on conserved nucleotide sequences located at the 3' end of the coding sequences of the barley and wheat 17 kDa Foam Protein genes:
• the reverse transcription reactions with 1 mg RNA were performed at 55°C using the TitanTM One Tube RT-PCR System supplied by Boehringer Mannheim GmbH.
• the PCR amplification step was 30 thermocyles (94°C for 30 seconds / 60°C for 30 seconds / 68°C for 30 seconds) followed by 7 minutes at 68°C and the amplification products were analysed by agarose gel electrophoresis.
• cDNA fragments of approximately 440 and 380 nucleotides were isolated, cloned into a pCR 2.1-TOPO plasmid vector (Invitrogen) and the nucleotide sequence of the inserts determined with an AmpliCycleTM Sequencing Kit (Perkin Elmer) on an Applied Biosystems 373 DNA sequencer.
• the nucleotide sequence of the 369 and 435 nucleotide inserts revealed two distinct cDNA sequences [SEQ ID NO:27] and [SEQ ID NO:28], which share close homology to the barley and wheat 17 kDa Foam Protein coding sequences.
• Rye genomic sequences encoding homologues of the barley 17 kDa Foam Protein were determined from genomic DNA fragments amplified by PCR. Rye grain were germinated and grown in the dark for 6 days and genomic DNA was prepared from the etiolated leaves using a Plant DNA Isolation Kit from Boehringer Mannheim GmbH. Rye genomic DNA (0.1 mg) was amplified with 25 pmol each of the sense primer [SEQ ID NO:26] and the antisense primer [SEQ ID NO:24], using native P/w DNA polymerase (Stratagene) in a reaction mixture recommended by the manufacturer.
• the PCR amplification was performed using a 'touch-down' thermocycling program (95°C for 0.45 minutes, 68 °C [- 1 °C / cycle (54 °C) ] for 0.45 minutes and 72 °C for 2 minutes for 15 cycles; 95 °C for 0.45 minutes, 54 °C for 0.45 minutes and 72 °C for 2 minutes for 25 cycles; 72 °C for 6 minutes).
• the amplification products were analysed by agarose gel electrophoresis and DNA fragments of approximately 440 nucleotides were isolated, cloned into a pCR- Bluntll TOPO vector (Invitrogen) and sequenced as described for the cDNA fragments.
• the nucleotide sequences of the cloned fragments revealed three distinct gene sequences present in the rye genome, which all showed homology to the barley 17 kDa Foam Protein gene.
• Two of the rye gene sequences [SEQ ID NO:29] and [SEQ ID NO:31] showed identity to the cDNA clones, [SEQ ID NO:27] and [SEQ ID NO:28] respectively, while a third had a distinct sequence [SEQ ID NO:33].
• Alignment of the nucleotide sequences of the rye genes with the barley 17 kDa Foam Protein gene indicates that the amplified rye sequences, including the primers, encode homologues of the 17 kDa Foam Protein starting at the second residue of the mature barley 17 kDa Foam Protein and extending to 7 residues short of the C-terminus.
• the amino acid sequences of the deduced rye 17 kDa Foam Protein homologues are aligned with the barley and wheat 17 kDa Foam Protein sequences in Table 10, where the rye gene shown in SEQ ID NO:29 encodes the rye polypeptide shown in SEQ ID NO:30, the rye gene shown in SEQ ID NO:31 encodes the rye polypeptide shown in SEQ ID NO:32, and the rye gene shown in SEQ ID NO:33 encodes the rye polypeptide shown in SEQ ID NO:34.
• Amino acid identity is represented by ellipses (....)
• amino acid deletions are represented by a dashed line ( )
• the alignment end is represented by an asterisk (*).
• the N- terminal amino acid sequence of the deduced rye 17 kDa Foam Proteins shown in SEQ ID NOS:32 and 34 are almost identical to that of two unidentified rye polypeptides of 15 and 18 kDa, previously detected in a rye grain extract (Rocher et al., 1996, BBA 1295: 13-22).
• the rye 17 kDa Foam Protein shown in SEQ ID NO:34 is 11 amino acid residues smaller than the protein shown in SEQ ID NO:32 due to a 33 nucleotide deletion in its gene.
• the conserved cysteine residues found in the barley and wheat 17 kDa Foam Protein homologues are also conserved in all the rye homologues.
• GGTTCCGATT GAGATAACGA GGATGGTGCT TCAGACCCTT CCGTCGATGT GCAGCGTGAA
• GGTTCCGATT GAGATAACGA GGATGGTGCT TCAGACCCTT CCGTCGATGT GCAGCGTGAA

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