IL313682A

IL313682A - Artificial DNA molecules, cells, transgenic plants, and their use for preparing milk-like micelles

Info

Publication number: IL313682A
Application number: IL313682A
Authority: IL
Inventors: LUTZKY Tal; Tiroler Amir; Greenfeld Elena
Original assignee: Plantopia Ltd; LUTZKY Tal; Tiroler Amir; Greenfeld Elena
Priority date: 2024-06-18
Filing date: 2024-06-18
Publication date: 2026-01-01

Description

ARTIFICIAL DNA MOLECULES, CELLS, TRANSGENIC PLANTS, AND USE OF SAME FOR PREPARING MILK-LIKE MICELLES REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[001] The contents of the electronic sequence listing (PGMT-P-003-IL.xml; size: 102,6bytes; and date of creation: March 5, 2024) is herein incorporated by reference in its entirety.

FIELD OF INVENTION

[002] The present invention relates to artificial DNA molecules encoding recombinant proteins and production of same, such as in plants or cells thereof.

BACKGROUND

[003] Bovine milk attributes a significant portion of the global milk market whereas plant-based alternatives account for $1 billion in the US and an estimated $700 million is estimated for lactose-intolerant milk. Bovine milk is known to have four specific caseins, u0001-s1-casein, u0001-s2-casein, u0002-casein, and u0003-casein. Mammal- or mammalian-produced milk is a very complex fluid that includes several thousand components (e.g., if all triglycerides are identified). Although mammal-produced milk, such as bovine milk, is considered by many to be an ideal source of nutrition, various milk alternatives to mammal- or mammalian-produced milk (e.g., bovine milk), such as plant- or nut-based milks, e.g., soy, almond, or coconut milk, have been pursued for reasons related to mammal- or mammalian-produced milk's allergenicity, lactose intolerance of certain components, personal preference, and the perceived environmental benefits of a reduced dairy industry.

[004] Existing dairy milk alternatives, such as soy, almond, or coconut milk fall short both in flavor and in functionality; moreover, a large part of the industrial and cultural significance of dairy milk stems from its usefulness in derivative products, such as cheese, yogurt, cream, or butter. Non-dairy plant-based milks, while addressing environmental and health concerns (and while providing adequate flavor for a small segment of the population), almost universally fail to form such derivative products.

[005] Current methodologies are defective in terms of their sustainability, scalability, and cost-effectiveness. Thus, there is still a great need for molecular farming as provided herein, such as for the production of a dairy substitute or composition that has desirable flavor and performance characteristics, such as comprising micelle like particles, and methods of using same, such as using artificial DNA molecules, and recombinant proteins encoded by same.

SUMMARY

[006] According to the first aspect, there is provided an artificial DNA molecule comprising: (a) a first nucleic acid sequence encoding an apoplast or a vacuole sorting signal sequence; and (b) a second nucleic acid sequence encoding a casein protein, wherein the first nucleic acid sequence and the second nucleic sequence are operably linked.

[007] According to another aspect, there is provided a plasmid or an expression vector comprising the artificial DNA molecule of the invention.

[008] According to another aspect, there is provided a recombinant protein encoded by the artificial DNA molecule of the invention, or the plasmid or expression vector of the invention.

[009] According to another aspect, there is provided a transgenic or a transfected cell comprising any one of: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; (c) the recombinant protein of the invention; and (d) any combination of (a) to (c).

[010] According to another aspect, there is provided a lysate, homogenate, extract, of the transgenic or a transfected cell of the invention, or any fraction thereof.

[011] According to another aspect, there is provided a transgenic plant or plant tissue comprising any one of: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; (c) the recombinant protein of the invention; (d) the transgenic or transfected cell of the invention; and (e) any combination of (a) to (d).

[012] According to another aspect, there is provided a host cell, comprising an artificial DNA molecule comprising a first nucleic acid sequence encoding an apoplast or vacuole sorting signal sequence and a casein protein, and a second nucleic acid sequence encoding a mammalian kinase.

[013] According to another aspect, there is provided a lysate, homogenate, or extract derived from the host cell of the invention, or any fraction thereof.

[014] According to another aspect, there is provided a composition comprising any one of: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; (c) the recombinant protein of the invention; (d) the transgenic or transfected cell of the invention; (e) the lysate, homogenate, extract, or any fraction thereof, of the invention; and any combination of (a) to (e), and an acceptable carrier.

[015] According to another aspect, there is provided a composition comprising: (a) a first DNA molecule comprising a first nucleic acid sequence encoding an apoplast or a vacuole sorting signal sequence and a second nucleic acid sequence encoding a casein protein; and (b) a second DNA molecule comprising a third nucleic acid sequence encoding a mammalian kinase.

[016] According to another aspect, there is provided a method for preparing a transgenic or transfected plant, the method comprising contacting a plant or a plant part with an effective amount of the plasmid or expression vector of the invention, or the composition of the invention.

[017] According to another aspect, there is provided a method for producing a recombinant casein protein or a plurality thereof, the method comprising the steps: (a) providing a plant or a plant part comprising the artificial DNA molecule of the invention; and (b) culturing the plant or plant part from step (a) such that the recombinant casein protein is encoded by the artificial DNA molecule.

[018] According to another aspect, there is provided an in vitro or ex vivo method for preparing a milk-like micelle, the method comprising mixing the lysate, homogenate, or extract of the invention.

[019] In some embodiments, the first nucleic acid sequence is set forth in any one of SEQ ID Nos: 1-2, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[020] In some embodiments, the casein protein is a single casein protein selected from the group consisting of: u0001-S1 casein, u0001-S2 casein, u0002 casein, u0003 casein.

[021] In some embodiments, the casein protein is selected from the group consisting of: u0001-Scasein, u0001-S2 casein, u0002 casein, u0003 casein, and any combination thereof.

[022] In some embodiments, the second nucleic acid sequence is set forth in any one of SEQ ID Nos: 15-28, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[023] In some embodiments, the plasmid or expression vector comprises a promoter sequence wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked thereto.

[024] In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a combination thereof.

[025] In some embodiments, the constitutive promoter comprises the nucleic acid sequence set forth in SEQ ID Nos: 29-34, and 69-76, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[026] In some embodiments, the recombinant protein comprises the amino acid sequence set forth in any one of SEQ ID Nos: 35-64, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[027] In some embodiments, the recombinant protein comprises the amino acid sequence set forth in any one of SEQ ID Nos: 49-64.

[028] The lysate, homogenate, or extract of the invention, comprising any one of: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; (c) the recombinant protein of the invention; and any combination of (a) to (c).

[029] In some embodiments, the transgenic plant is a leafy green or a grain.

[030] In some embodiments, the transgenic plant is lettuce or oat.

[031] In some embodiments, the host cell is a cell of a transgenic plant, a transfected plant, or of a tissue derived therefrom.

[032] In some embodiments, the encoded casein protein and the encoded mammalian kinase are present in the same compartment of the host cell, and wherein the compartment is the apoplast or vacuole of the host cell.

[033] In some embodiments, the artificial DNA molecule further comprises a third nucleic acid sequence being an internal ribosome entry site (IRES), and wherein the third nucleic acid sequence is positioned between the first nucleic acid and the second nucleic acid of the artificial DNA molecule.

[034] In some embodiments, the nucleic acid sequence and the second nucleic acid sequence are operably linked.

[035] In some embodiments, the first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos: 15-28, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[036] In some embodiments, the second nucleic acid sequence is set forth in SEQ ID Nos: 65-67, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[037] In some embodiments, the first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos: 1-5, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[038] In some embodiments, the host cell further comprises at least one fourth nucleic acid sequence encoding a site-1 protease (S1P) enzyme.

[039] In some embodiments, the fourth nucleic acid sequence is set forth in SEQ ID NO: 68 or a functional analog thereof.

[040] In some embodiments, any one of the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and any combination thereof, is integrated in the genome of the host cell.

[041] In some embodiments, the at least one fourth nucleic acid sequence is integrated in the genome of the host cell.

[042] In some embodiments, the second nucleic acid sequence further encodes an endoplasmic reticulum (ER) sorting signal sequence, an apoplast sorting signal sequence, or both.

[043] In some embodiments, the second nucleic acid sequence further encodes an endoplasmic reticulum (ER) sorting signal sequence, an apoplast sorting signal sequence, or both, and wherein the sequence encoding the mammalian kinase is positioned 3' to the sequence encoding the ER sorting signal sequence, the apoplast sorting signal sequence, or both.

[044] In some embodiments, a protein fraction of the composition consists essentially of the recombinant protein of the invention.

[045] In some embodiments, the composition comprises one or more milk-like micelles.

[046] In some embodiments, the one or more milk-like micelles being characterized by a structure comprising an outer layer and an inner layer, wherein the outer layer comprises plant recombinant u0003 casein protein and a first plant protein and the inner layer comprises at least one plant recombinant: u0001-S1 casein protein, u0001-S2 casein protein, u0002 casein protein, or any combination thereof, and a second plant protein.

[047] In some embodiments, at least one of the plant recombinant u0001-S1 casein protein, u0001-Scasein protein, u0002 casein protein, u0003 casein protein, or any combination thereof, comprises at least one post-translational modification.

[048] In some embodiments, the at least one post-translational modification is phosphorylation.

[049] In some embodiments, the one or more milk-like micelles is essentially similar to milk micelles in size, Z-potential, or both.

[050] In some embodiments, the milk is bovine-derived milk.

[051] In some embodiments, any one of: the first nucleic acid sequence is set forth in any one of SEQ ID Nos: 1-5, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof; the second nucleic acid sequence is set forth in any one of SEQ ID Nos: 15-28, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof; and both (a) and (b).

[052] In some embodiments, each of the first DNA molecule and the second DNA molecule is integrated in a separate plasmid or expression vector, or both are integrated together in a single plasmid or expression vector.

[053] In some embodiments, the method further comprises a step preceding step (a) comprising producing the plant or plant part comprising the artificial DNA molecule, the preceding step comprises transfecting a plant or a plant part with an effective amount of the plasmid or expression vector of the invention, or the composition of the invention.

[054] In some embodiments, the method further comprises a step proceeding step (b) comprising extracting the cultured plant or plant part, thereby obtaining an extract comprising the recombinant casein protein or plurality thereof.

[055] In some embodiments, the method further comprises mixing the recombinant casein protein or a plurality thereof, thereby obtaining one or more micelles. [056] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[057] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[058] Fig. 1 includes a photograph of a western blot analysis showing recombinant expression of u0001S1 casein protein harboring an apoplast sorting signal in ‘Noga’ lettuce. (1) Molecular weight marker; (2) wilt-type (w.t) Noga; (3)-(14) different recombinant plants expressing u0001S1 casein with an apoplast sorting signal; and (15) 10 ng u0001S casein standard.

[059] Fig. 2 includes a photograph of a western blot analysis showing recombinant expression of u0001S2 casein protein harboring a vacuole sorting signal, in ‘Noga’ lettuce. (1) Molecular weight marker; (2) w.t Noga; (3)-(14) different recombinant plants expressing u0001S2 casein with a vacuole sorting signal; and (15) 10 ng u0001S casein standard.

[060] Fig. 3 includes a photograph of a western blot analysis showing recombinant expression of u0002 casein protein harboring an apoplast sorting signal in ‘Noga’ lettuce. (1) Molecular weight marker; (2) w.t Noga; (3)-(10) different recombinant plants expressing u0002 casein with an apoplast sorting signal; (11) 12.5 ng u0002 casein standard; and (12) Marker.

[061] Fig. 4includes a photograph of a western blot analysis showing recombinant expression of u0002 casein protein harboring a vacuole sorting signal in ‘Noga’ lettuce. (1) Molecular weight marker; (2) w.t Noga; (3)-(8) different recombinant plants expressing u0002 casein with an apoplast sorting signal, co-expressing Fam20 kinase protein; (9) 10 ng u0002 casein standard; and (10-12) recombinant plants expression u0002 casein with a vacuole sorting signal.

[062] Fig. 5 includes a photograph of a western blot analysis showing recombinant co-expression of u0002 casein protein and the kinase Fam20 in ‘Noga’ lettuce. (1) Molecular weight marker; (2) w.t Noga; (3)-(4), and (10)-(12) different recombinant plants expressing u0002 casein; (5-9) different recombinant plants co-expressing u0002 casein and Fam20; and (13) 12.5 ng u0002 casein standard.

[063] Fig. 6 includes a photograph of a western blot analysis showing recombinant expression of u0003 casein protein harboring an apoplast sorting signal in ‘Noga’ lettuce. (1) Molecular weight marker; (2) w.t Noga; (3)-(10) different recombinant plants expressing u0003 casein with an apoplast sorting signal; and (11) 10 ng u0003 casein standard.

DETAILED DESCRIPTION DNA molecules and expression vectors

[064] According to the first aspect, there is provided an artificial DNA molecule comprising: (a) a first nucleic acid sequence encoding a plant cell compartment sorting signal sequence; and (b) a second nucleic acid sequence encoding a casein protein.

[065] In some embodiments, the first nucleic acid sequence and the second nucleic sequence are operably linked.

[066] The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, operably linked refers to the first nucleic acid sequence and the second nucleic sequence being linked to the regulatory element(s) in a manner that allows for expression of the first nucleic acid sequence and the second nucleic sequence. In some embodiments, the first nucleic acid sequence and the second nucleic sequence are co-expressed. In some embodiments, the first nucleic acid sequence and the second nucleic sequence are expressed at the same time or together. In some embodiments, when the first nucleic acid sequence is expressed the second nucleic sequence is expressed. In some embodiments, when the second nucleic acid sequence is expressed the first nucleic sequence is expressed.

[067] In some embodiments, a plant cell compartment comprises any cellular compartment of a plant cell. In some embodiments, a plant cell compartment comprises or is a plant cell organelle.

[068] Types of plant cell compartments, as well as signal sorting sequences directing transport and/or secretion of a protein thereto, are common and would be apparent to one of skill in the art of plant cell biology.

[069] In some embodiments, a plant cell compartment and/or an organelle comprises: apoplast, vacuole, chloroplast, leucoplast, chromoplast, glyoxysomes, nucleus, Golgi apparatus, mitochondria, endoplasmic reticulum, peroxisome, and any combination thereof.

[070] In some embodiments, a plant cell compartment comprises apoplast, vacuole, or both. In some embodiments, a vacuole comprises a central vacuole.

[071] In some embodiments, the first nucleic acid comprises a sequence encoding an apoplast or a vacuole sorting signal.

[072] In some embodiments, the first nucleic acid sequence is set forth in SEQ ID Nos: 1-14, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[073] The terms “homology” and/or “identity”, as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a stricter comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[074] The following is a non-limiting example for calculating homology or sequence identity between two sequences (the terms are used interchangeably herein). The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.

[075] In some embodiments, % homology or identity as described herein are calculated or determined using the basic local alignment search tool (BLAST). In some embodiments, % homology or identity as described herein are calculated or determined using Blossum 62 scoring matrix. In some embodiments, % homology or identity as described herein are calculated or determined using any tool known and accessible to a person of skill in the art. Non-limiting examples of such methods and/or tools include, but are not limited to, PAM30, PAM70, BLOSUM 30, BLOSUM 45, BLOSUM 80, to name a few.

[076] In some embodiments, a casein protein is selected from: u0001-S1 casein, u0001-S2 casein, u0002 casein, u0003 casein, a functional analog thereof, or any combination thereof.

[077] In some embodiments, the casein protein is a single casein protein. In some embodiments, the casein protein is a single: u0001-S1 casein protein, u0001-S2 casein protein, u0002 casein protein, u0003 casein protein, or a functional analog thereof. In some embodiments, the casein protein is u0001-S1 casein protein. In some embodiments, the casein protein is u0001-S2 casein protein. In some embodiments, the casein protein is u0002 casein protein. In some embodiments, the casein protein is u0003 casein protein. In some embodiments, a single casein protein is a functional analog of a casein protein.

[078] In some embodiments, u0001-S1 casein is disclosed under UniProt ID 2662.

[079] In some embodiments, u0001-S2 casein is disclosed under UniProt ID 2663.

[080] In some embodiments, u0002 casein is disclosed under UniProt ID 2666.

[081] In some embodiments, u0003 casein is disclosed under UniProt ID 2668.

[082] Sequences, types, and biology of casein protein(s) would be apparent to one of ordinary skill in the art, such as reviewed by Holt et al., 2013 (“Invited review: Caseins and the casein micelle: Their biological function and structured, and behavior in foods”; Journal of Dairy Science 96(10):6127-6146).

[083] In some embodiments, the second nucleic acid sequence is set forth in SEQ ID Nos: 15-28, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[084] According to another aspect, there is provided a plasmid or an expression vector comprising the artificial DNA molecule of the invention.

[085] In some embodiments, the plasmid or expression vector comprises a promoter sequence. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, or both, are operably linked thereto.

[086] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase e.g., RNA polymerase II. Promoters are composed of discrete functional modules, each comprising or consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[087] In some embodiments, a promoter is a constitutive promoter, an inducible promoter, or a combination thereof.

[088] In some embodiments, a promoter is considered “responsive” to an input signal if the input signal modulates the function of the promoter, indirectly or directly. In some embodiments, an input signal may positively modulate a promoter such that the promoter activates or increases, transcription of a nucleic acid to which it is operably linked. In some embodiments, an input signal may negatively modulate a promoter such that the promoter is prevented from activating or inhibits, or decreases, transcription of a nucleic acid to which it is operably linked. In some embodiments, an input signal modulates the function of the promoter directly by binding to the promoter or by acting on the promoter without an intermediate signal.

[089] In some embodiments thereof, a promoter responsive to an input signal and/or regulatory protein is considered an “inducible” promoter. Inducible promoters for use according to the present invention include any inducible promoter described herein or known to one of ordinary skill in the art. Non-limiting examples of inducible promoters include, but are not limited to, chemically-regulated, biochemically-regulated, and/or physically-regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and/or other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO), and/or a tetracycline trans-activator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and/or light-regulated promoters (e.g., light responsive promoters from plant cells), or any combination thereof.

[090] In some embodiments, the promoter comprises a nucleic acid sequence set forth in SEQ ID Nos: 29-34, and 69-76, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[091] In some embodiments, a constitutive promoter comprises a nucleic acid sequence set forth in SEQ ID Nos: 29-34, and 69-76, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[092] The terms “nucleic acid” and “nucleic acid molecule”, which are used herein interchangeably, are well known in the art. A "nucleic acid molecule" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). In some embodiments, a nucleobase includes non-naturally occurring purine or pyrimidine base found in DNA, RNA, or a hybrid thereof.

[093] The term “nucleic acid molecule” includes but not limited to single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNA such as miRNA, siRNA and other short interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, small rRNA, hnRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, nucleic acids of infectious origin, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides.

[094] In some embodiments, a nucleic acid molecule comprises a DNA molecule.

[095] The terms “expression”, “expressing, or “encoding”, being used herein interchangeably, refer to the biosynthesis of a gene product, including the transcription and/or translation of a gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (e.g., polypeptide, a recombinant protein of the invention, etc.).

[096] Expressing a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector. One such example of an expression vector containing p16-Ink4a is the mammalian expression vector pCMV p16 INK4A available from Addgene.

[097] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.

[098] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.

[099] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 58(1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and/or the like.

[0100] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0101] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0102] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0103] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the recombinant protein), the expression construct of the present invention can also include sequences engineered to optimize stability, transcription efficiency and/or rate (e.g., codon optimization), production, purification, yield or activity of the expressed recombinant protein.

[0104] In some embodiments, the artificial DNA molecule of the invention is codon optimized for expression in a target cell.

[0105] In some embodiments, a target cell comprises a cell suitable for or configured to expression, over-expression, production, secretion, or any combination thereof, of the recombinant protein of the invention.

[0106] According to another aspect, there is provided a composition comprising: (a) a first DNA molecule comprising a first nucleic acid sequence encoding a plant cell sorting signal sequence and a second nucleic acid sequence encoding a casein protein; and (b) a second DNA molecule comprising a third nucleic acid sequence encoding a mammalian kinase.

[0107] In some embodiments, the first DNA molecule and the second DNA molecule are integrated in a separate plasmid or expression vector. In some embodiments, the first DNA molecule is integrated in a first plasmid or expression vector and the second DNA molecule is integrated in a second plasmid or expression vector. In some embodiments, the first DNA molecule and the second DNA molecule are integrated together in a single plasmid or expression vector.

Recombinant proteins

[0108] According to another aspect, there is provided a recombinant protein encoded by an artificial DNA molecule of the invention. According to another aspect, there is provided a recombinant protein encoded by a plasmid or an expression vector of the invention. According to another aspect, there is provided a recombinant protein encoded by an artificial DNA molecule integrated in a plasmid or an expression vector of the invention.

[0109] As used herein, the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA, such as the artificial DNA molecule of the invention, and is thus not naturally occurring. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA may be in the form of a vector, plasmid or virus used to express the recombinant protein in a cell.

[0110] In some embodiments, the recombinant protein comprises an amino acid sequence set forth in any one of SEQ ID Nos: 35-64, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0111] In some embodiments, the recombinant protein comprises an amino acid sequence set forth in any one of SEQ ID Nos: 49-64, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0112] The terms “analog” or “functional analog” are used herein interchangeably, and refer to a polypeptide that is similar, but not identical, to the recombinant protein of the invention that still is capable of directing transport or secretion to a selected plant cell compartment (e.g., a plant cell compartment sorting signal), form milk-like micelle(s) (e.g., a casein protein), or both. The analog may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the polypeptide of the invention.

[0113] In some embodiments, the analog or functional analog to the recombinant protein polypeptide of the invention comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the amino acid sequence set forth in SEQ ID Nos: 35-64, or any value and range therebetween. In some embodiments, the analog or functional analog to the recombinant protein polypeptide of the invention comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the amino acid sequence set forth in SEQ ID Nos: 49-64, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0114] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

Cells, plants, and extracts

[0115] According to another aspect, there is a cell comprising: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; (c) the recombinant protein of the invention; (d) any combination of (a) to (c).

[0116] In some embodiments, a cell is or comprises a transgenic cell, a transformed cell, a transfected cell, a transduced cell, or any combination thereof.

[0117] In some embodiments, a cell is or comprises: a unicellular organism, a cell of a multicellular organism, or a cell in a culture (such as of a cell line, a stable line, a primary cell line).

[0118] As used herein, the term "transgenic cell" refers to any cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous polynucleotide, such as an “isolated” or “artificial” (used herein interchangeably) DNA molecule of the invention, introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, the exogenous polynucleotide (e.g., the artificial DNA molecule of the invention) or vector is stably integrated into the cell or particularly the genome of the cell. In some embodiments, the transgenic cell expresses an artificial DNA molecule of the invention. In some embodiments, the transgenic cell expresses a plasmid or an expression vector of the invention. In some embodiments, the transgenic cell expresses a recombinant protein of the invention. In some embodiments, the transgenic cell, prepared using a cell that is initially devoid of an artificial DNA molecule of the invention that has been transformed or genetically modified so as to include the artificial DNA molecule of the invention.

[0119] In some embodiments, a multicellular organism comprises or is a plant. In some embodiments, the cell is a plant cell.

[0120] In some embodiments, a unicellular organism comprises a fungus or a bacterium. In some embodiments, the fungus is a yeast cell.

[0121] In some embodiments, the cell comprises a plurality of artificial DNA molecules as disclosed herein. In some embodiments, the plurality of artificial DNA molecules comprise a plurality of different nucleic acid sequences. In some embodiments, each different nucleic acid sequence encodes one or more recombinant proteins as disclosed herein.

[0122] According to another aspect, there is provided a lysate, a homogenate, or an extract of the cell, including any combination thereof.

[0123] In some embodiments, a lysate, homogenate, or an extract, comprises any fraction thereof.

[0124] In some embodiments, the lysate, homogenate, or extract comprises: (a) an artificial DNA molecule of the invention; (b) a plasmid or expression vector of the invention; (c) a recombinant protein of the invention; or (d) any combination of (a) to (c).

[0125] In some embodiments, the lysate, homogenate, or extract comprises a recombinant protein of the invention. In some embodiments, the lysate, homogenate, or extract comprises a plurality of recombinant proteins of the invention. In some embodiments, the lysate, homogenate, or extract comprises a recombinant casein protein. In some embodiments, the lysate, homogenate, or extract comprises a plurality of recombinant casein proteins. In some embodiments, the lysate, homogenate, or extract comprises a recombinant: u0001-S1 casein protein, u0001-S2 casein protein, u0002 casein protein, u0003 casein protein, or any combination thereof.

[0126] Methods and/or means for extracting, lysing, homogenizing, fractionating, or any combination thereof, a cell or a culture of same, are common and would be apparent to one of ordinary skill in the art of cell biology and biochemistry. Non-limiting examples include, but are not limited to, pressure lysis (e.g., such as using a French press), enzymatic lysis, soluble-insoluble phase separation (such for obtaining a supernatant and a pellet), detergent-based lysis, solvent (e.g., polar, or nonpolar solvent), liquid chromatography mass spectrometry, or others.

[0127] According to another aspect, there is provided a transgenic plant, a plant part, a plant tissue, or any combination thereof, comprising: (a) an artificial DNA molecule of the invention; (b) a plasmid or expression vector of the invention; (c) a recombinant protein of the invention; (d) a transgenic or transfected cell of the invention; or (e) any combination of (a) to (d).

[0128] In some embodiments, the transgenic plant comprises a leafy green or a grain.

[0129] In some embodiments, leafy green comprises lettuce. In some embodiments, the transgenic plant is a lettuce plant. In some embodiments, a grain comprises oat.

[0130] In some embodiments, a plant or plant material comprises any one of: leaf, fruit, root, tuber, bulb, stem, seed, and any combination thereof.

[0131] According to another aspect there is provided a host cell, comprising an artificial DNA molecule comprising a first nucleic acid sequence encoding an apoplast or vacuole sorting signal sequence and a casein protein, and a second nucleic acid sequence encoding a mammalian kinase.

[0132] In some embodiments, the host cell is or comprises a cell of a transgenic plant, a transfected plant, or of a tissue derived therefrom.

[0133] In some embodiments, the encoded casein protein and the encoded mammalian kinase are present in the same compartment of the host cell. In some embodiments, the compartment is the apoplast or vacuole of the host cell.

[0134] In some embodiments, the artificial DNA molecule further comprises a third nucleic acid sequence. In some embodiments, the third nucleic acid sequence is or comprises being an internal ribosome entry site (IRES). In some embodiments, the third nucleic acid sequence is positioned between the first nucleic acid and the second nucleic acid of the artificial DNA molecule. In some embodiments, the 5' end to 3' end organization of the first, second, and third nucleic acid sequences of the artificial DNA molecule is: first nucleic acid sequence, third nucleic acid sequence, and second nucleic acid sequence.

[0135] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked. In some embodiments, the first, second and third nucleic acid sequences are simultaneously transcribed, e.g., in polycistronic-like manner.

[0136] In some embodiments, the first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos: 15-28, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[0137] In some embodiments, the second nucleic acid sequence is set forth in SEQ ID Nos: 65-67, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[0138] In some embodiments, the first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos: 1-5, a functional analog thereof having at least 80% sequence homology or identity thereto, and any combination thereof.

[0139] In some embodiments, the host cell further comprises at least one fourth nucleic acid sequence. In some embodiments, the at least one fourth nucleic acid sequence encodes a protease, peptidase, or the like. In some embodiments, the fourth nucleic acid sequence encodes a site-protease (S1P) enzyme. In some embodiments, the protease, peptidase, or the like is or comprises S1P. In some embodiments, the fourth nucleic acid sequence is set forth in SEQ ID NO: 68 or a functional analog thereof.

[0140] In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, or any combination thereof, is integrated in the genome of the host cell.

[0141] According to another aspect, there is provided a transgenic plant, a transfected plant, a part thereof, or a tissue derived therefrom, comprising a first polynucleotide encoding a casein protein, and a second polynucleotide encoding a kinase. In some embodiments, the kinase is derived from a mammal. In some embodiments, the kinase is a mammalian kinase (e.g., harboring a sequence of, characterizing, or known to present and/or identified in a mammal). In some embodiments, the kinase comprises a kinase known to or capable of phosphorylating a casein protein. In some embodiments, the kinase comprises a kinase known to or capable of phosphorylating a bovine casein. In some embodiments, the phosphorylated casein comprises u0001-S1 casein, u0001-S2 casein, u0002 casein, u0003 casein, or any combination thereof.

[0142] In some embodiments, the first polynucleotide comprises a nucleic acid sequence set forth in SEQ ID Nos: 15-28, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0143] In some embodiments, the second polynucleotide comprises a nucleotide sequence set forth in SEQ ID Nos: 65-67, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0144] In some embodiments, the second polypeptide encodes mammalian kinase known as “one of family with sequence similarity 20”, “member C (Fam20)”, “DMP-4”, “DMP4”, “GEF-CK”, “RNS”, “G-CK”, “Golgi associated secretory pathway kinase”, or “FAM20C Golgi associated secretory pathway kinase”.

[0145] In some embodiments, the second nucleic acid sequence further encodes an endoplasmic reticulum (ER) sorting signal sequence, an apoplast sorting signal sequence, or both.

[0146] In some embodiments, the sequence encoding the mammalian kinase is positioned 3' to the sequence encoding the ER sorting signal sequence, the apoplast sorting signal sequence, or both.

[0147] In some embodiments, the sequence encoding the ER sorting signal sequence, the apoplast sorting signal sequence, or both is positioned 5' to the sequence encoding the mammalian kinase.

[0148] In some embodiments, the second nucleic acid sequence is organized from 5' to 3' as follows: ER sorting signal sequence, apoplast sorting signal sequence, or both, and mammalian kinase.

[0149] In some embodiments, the sequence encoding the ER sorting signal sequence is set forth in SEQ ID NO: 8.

[0150] In some embodiments, the transgenic plant, transfected plant, plant part, tissue derived therefrom, or any combination thereof, further comprises at least one third polynucleotide. In some embodiments, the at least one third polynucleotide comprises at least 2 polynucleotide. In some embodiments, the at least one third polynucleotide comprises one or more polynucleotides. In some embodiments, the at least one third polynucleotide comprises a nucleic acid sequence encoding: (i) a plant cell compartment sorting signal sequence; (ii) a protease or a proteolytic enzyme (e.g., peptidase), or both (i) and (ii). In some embodiments, the protease is mammalian-kinase specific protease. In some embodiments, the protease has a mammalian kinase substrate. In some embodiments, the substrate of peptization of the protease is or comprises a mammalian kinase. In some embodiments, the protease is or comprises site-1 protease (S1P).

[0151] In some embodiments, the at least one third polynucleotide encoding a plant cell compartment sorting signal sequence comprises a nucleotide sequence set forth in SEQ ID Nos: 1-14, a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0152] In some embodiments, the at least one third polynucleotide encoding S1P enzyme comprises a nucleotide sequence set forth in SEQ ID NO: 68 or a functional analog thereof having at least 70%, 80%, 90%, 95%, 99% sequence homology or identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention..

[0153] In some embodiments, the first polynucleotide, the second polynucleotide, the at least one third polynucleotide, or any combination thereof, are integrated in the genome of the transgenic plant. In some embodiments, the first polynucleotide, the second polynucleotide, the at least one third polynucleotide, or any combination thereof, are integrated in an artificial DNA molecule residing the genome of the transgenic plant.

[0154] According to another aspect, there is provided a transgenic plant, a transfected plant, a part thereof, or a tissue derived therefrom, derived or obtained from the transgenic plant, transfected plant, part thereof, or tissue derived therefrom, of the invention, including any fraction thereof.

[0155] The terms “derived from” and “obtained from” are used herein interchangeably.

Compositions

[0156] According to another aspect, there is provided a composition comprising any one of: (a) an artificial DNA molecule of the invention; (b) a plasmid or expression vector of the invention; (c) a recombinant protein of the invention; (d) a transgenic or transfected cell of the invention; (e) the lysate, homogenate, extract, or any fraction thereof, of the invention; or (f) any combination of (a) to (e), and an acceptable carrier. [0157] Ins some embodiments, the composition comprises proteins. In some embodiments, the composition comprises a protein fraction. In some embodiments, the composition the proteins or the protein fraction of the composition consists essentially of the recombinant protein of the invention.

[0158] As used herein, the term “consists essentially of” denotes that a given compound or substance constitutes the vast majority of a particular portion or fraction of the composition.

[0159] In some embodiments, consists essentially of means that: the recombinant protein of the invention, constitute at least 80%, 90%, 95%, at least 98%, at least 99%, or at least 99.9% by weight, of the proteins or the protein fraction of the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0160] In some embodiments, the composition comprises one or more micelles. In some embodiments, the composition comprises a plurality of micelles. In some embodiments, the one or more micelle(s) is or comprises milk-like micelle(s)

[0161] As used herein, the term “plurality” refers to any integer being equal to or greater than 2.

[0162] As used herein, the terms “micelle” and “particle” are interchangeable.

[0163] In some embodiments, the one or more micelles comprises an outer layer and an inner layer. In some embodiments, the outer layer comprises plant recombinant u0003 casein protein and a first plant protein. In some embodiments, the inner layer comprises at least one plant recombinant: u0001-S1 casein protein, u0001-S2 casein protein, u0002 casein protein, or any combination thereof, and a second plant protein. In some embodiments, the first plant protein and the second plant protein are the same protein. In some embodiments, the first plant protein and the second plant protein are two different proteins. In some embodiments, the first, the second, or both, plant proteins are a plurality of first proteins and second proteins. In some embodiments, the first and second plant proteins are not recombinant proteins, e.g., native plant proteins, such as expressed or produced by wildtype or genetic reference plant from endogenous plant gene(s).

[0164] In some embodiments, at least one of the plant recombinant: u0001-S1 casein protein, u0001-Scasein protein, u0002 casein protein, u0003 casein protein, or any combination thereof, comprises at least one post-translational modification.

[0165] In some embodiments, the least one post-translational modification is phosphorylation.

[0166] In some embodiments, the micelle of the composition of the invention is essentially similar to a micelle of milk. In some embodiments, milk comprises or consists of bovine milk. In some embodiments, the micelle of the composition of the invention is essentially similar to a micelle of milk, optionally wherein milk is or comprises bovine milk.

[0167] As used herein, the term “essentially similar” refers to being at least 70%, 80%, 90%, 95%, or 99% identical, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0168] In some embodiments, essentially similar refers to the micelle of the composition of the invention having a size, average size, or maximal size, diameter, average diameter, or maximal diameter, taste, flavor, scent, organoleptic properties, or any combination thereof, essentially similar to a micelle of milk. In some embodiments, the one or more micelle(s) is essentially similar to milk micelles in size, Z-potential, or both.

[0169] Methods and means for determining and/or characterizing micelles are common and would be apparent to one of ordinary skill in the art. Non-limiting example for such methods includes, but are not limited to a transmission electron microscopy (TEM), dynamic light scattering (DLS), to name a few.

[0170] In some embodiments, the composition is an edible composition. In some embodiments, the composition is a dairy substitute. In some embodiments, the composition is a pre dairy substitute composition/product/ingredient, e.g., being used as an ingredient and/or starting material for the preparation of a dairy substitute or substituent.

[0171] In some embodiments, the composition is a dry composition. In some embodiments, the composition is dried. In some embodiments, the composition is lyophilized or a lyophilized composition.

[0172] Any means and methods of drying that are known to a person of ordinary skill in the art are contemplated by current invention.

Methods of use

[0173] According to another aspect, there is provided a method for preparing a transgenic or transfected plant.

[0174] According to another aspect, there is provided a method for producing a recombinant casein protein or a plurality thereof.

[0175] According to another aspect, there is provided a method for preparing a milk-like micelle.

[0176] In some embodiments, the method comprises contacting a plant or a plant part with an effective amount of the plasmid or expression vector of the invention.

[0177] In some embodiments, the method comprising a step (a) comprising providing a plant or a plant part comprising the artificial DNA molecule of the invention. In some embodiments, the method comprises a step (b) comprising culturing the plant or plant part from step (a). In some embodiments, the culturing provides conditions such that the recombinant casein protein is encoded by the artificial DNA molecule of the invention. In some embodiments, the recombinant protein comprises the recombinant protein of the invention.

[0178] In some embodiments, the method further comprises a step comprising producing the plant or plant part comprising the artificial DNA molecule of the invention. In some embodiments, the step comprises transfecting a plant or a plant part with an effective amount of a plasmid or expression vector of the invention, or a composition of the invention. In some embodiments, the step comprises introducing an effective amount of a plasmid or expression vector of the invention or a composition of the invention, to a plant or a plant part. In some embodiments, the step is preceding step (a).

[0179] In some embodiments, the method further comprises a step comprising extracting the cultured plant or plant part, thereby obtaining an extract comprising a recombinant casein protein or plurality thereof. In some embodiments, the step (the culturing) is proceeding step (b).

[0180] In some embodiments, the method further comprises mixing the recombinant casein protein or a plurality thereof, thereby obtaining one or more micelles. In some embodiments, the one or more micelles are essentially similar to milk micelles. In some embodiments, milk comprises or is bovine-derived milk.

[0181] Methods for introducing exogenous DNA, such as comprising a plasmid or expression vector of the invention or a composition of the invention, are common and would be apparent to one of ordinary skill in the art.

[0182] In some embodiments, introducing comprises contacting a plant or a plant part with an effective amount of Agrobacterium-based vector. In some embodiments, introducing comprises contacting a plant or a plant part with an effective amount of TRV-based vector. In some embodiments, introducing comprises contacting a plant or a plant part with an effective amount of gold particles. In some embodiments, introducing comprises contacting a plant or a part thereof with an effective amount of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system.

[0183] In some embodiments, the introducing is Agrobacterium mediated transformation, TRV mediated transient expression, gold particles bombardment, and/or CRISPR-inserted DNA.

General definitions

[0184] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0185] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0186] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0187] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0188] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0189] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0190] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

[0191] The nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0192] To this end, any amino acid chain of any sequence or length will inherently undergo agglomeration at some point and conditions, depending on the concentration, pH etc., thereby giving rise to agglomerate-like structure. Accordingly, co-expressing a plurality of proteins in a cell, such as casein proteins, is likely to provide agglomerated structures, including the plurality of the casein proteins. Nonetheless, it would be apparent to a skilled artisan that agglomerated structures spontaneously in vivo formed in a plant cell are not the intricate and complexed “milk-like” micelles.

[0193] Accordingly, in order to form milk-like micelles further including plant material, the inventors devised the expression of each individual casein protein in a separate plant, to be isolated and mixed ex vivo or in vitro in the presence of plant material, to achieve controllable and proper interaction. Therefore, the inventors have titrated separately expressed u0001Casein (S1 and Scombined), u0002-Casein, and u0003-Casein, in the presence of plant material and found that particular weight per weight ratio of the casein proteins (ranging from 2:2:1 to 6:6:1) is required to achieve properly sized micelles further including plant material (data not shown). [0194] Further, to emphasize the importance of ex vivo or in vitro mixing individually expressed casein proteins in the presence of plant material, the inventors further showed that exogenous supplementation of calcium ions, and specifically calcium chloride at a concentration of 2 mM to mM is crucial so to obtain “milk-like” micelles, e.g., of about 450 nm on average, and characterized by a zeta potential of -20 mV and -5 mV. The inventors found that at least 2 mM of CaCl2 is required to initiate a reaction, whereas above 60 mM sedimentation of calcium was highly evident. Further, in a dynamic light scattering (DLS) analysis mostly large insoluble particles likely including protein aggregates, but not micelles, were observed. [0195] In view of the above findings showing the necessity of individual expression of single casein proteins in a plant so as to obtain a milk-like micelle ex vivo or in vitro, the inventors have pursued molecular optimization of nucleic acid sequences required for in plant expression, e.g., protein expression/secretion to a particular plant cell compartment, post-translation modification, etc., as further disclosed hereinbelow.

Materials and Methods Creation of DNA sequence

[0196] To construct the modified plant, the inventors utilizes a transgenic construct cloning process. The process begins with amplifying or synthesizing the desired genes and DNA sequences. The desired genes and DNA sequences are then cloned into an initial backbone - pUPD2 to be verified by digestion and sequencing. After successful validation, these sequences are then used for assembling full and functional transcriptional units (TUs) (few or all of the listed): promoter, UTR, sorting signal, CDS, and a terminator into the final GoldenBraid3.backbone plasmid (Alpha or Omega) (Sarrion-Perdigones, A. et al., 2011, Vazquez-Vilar, M. et al, 2017).

Infiltration

[0197] To produce recombinant, non-animal, casein in Romaine lettuce that is bovine equivalent, the inventors applied the Agrobacterium tumefaciens-mediated transformation (AGRO) method (Gasser, C. S. & Fraley, R. T., 1989). The inventors utilizes AGL0, a modified and non-pathogenic strain of AGRO, to facilitate the transgene transformation (Weir B. et al., 2001, Aida R. et al., 1999). All reagents and chemicals used for transformation were of high purity and analytical grade, to meet the standards of plant cell tissue culture applications. In the first step of the plant transformation process, lettuce seeds are treated with bleach to provide disinfection. The sterile seeds are sprouted in a petri dish on a meager bed until the cotyledons emerge. The cotyledons are then cut and embedded in media containing Agrobacterium with the desired construct. The cotyledons are further dried and returned to a petri dish for incubation in the dark. When incubation is over, the cotyledons are transferred to an antibiotic selection plate with hormones, where the cotyledons are grown to a callus stage. The inventors use antibiotics (carbenicillin) to exterminate any leftover Agrobacterium at this stage, as part of the selection process. Each callus grown from the treated cotyledons comes from a single successfully transformed plant cell. Further, after cellular differentiation and plant regeneration, plants are grown, and the transgene becomes a permanent part of the transgenic plant’s genome. The successful transformation events undergo hardening conditions in the ground, and the modified plants are self-pollinated to generate seeds for homozygous lines.

Protein extraction

[0198] Caseins are extracted from the plant tissue via mechanical cell breakage and solubilization in an extraction buffer or a diluted sample buffer. The presence of the protein of interest from the insert is confirmed using specific antibodies on a western blot membrane. Then, the plant lysate is further analyzed by liquid chromatography, followed by tandem mass spectrometry (LC-MS/MS) for the validation of the sequence of the protein of interest from the insert, as well as post-translational modifications.

Results

[0199] The inventors have generated numerous constructs comprising nucleic acid sequences encoding various casein proteins, for expression in lettuce, under the regulation of several promoters, as disclosed herein. [0200] To this end, the inventors have expressed the various casein proteins, e.g., u0001 casein, u0002 casein, and u0003 casein fused to diverse repertoire of plant cell compartment sorting sequences. Specifically, the sequences direct expressed proteins to the: endoplasmic reticulum (ER), Golgi apparatus, chloroplast, mitochondria, apoplast, or vacuole. In this regard, the inventors found that directing plant recombinant casein protein(s), to any one of the ER, Golgi apparatus, chloroplast, and mitochondria, resulted in negligible or undetected amounts of plant recombinant casein proteins (data not shown). [0201] Surprisingly, the inventors showed that in sharp contrast to directing casein expression to the ER, Golgi apparatus, chloroplast, and mitochondria, u0001S1 casein was found to be sufficiently expressed in any one of the apoplast and vacuole of a plant cell, and be extracted therefrom ( Figs. 1 and 2 , respectively). [0202] Similarly, the inventors showed that u0002 casein can also be expressed in any one of the apoplast and vacuole of a plant cell, and be extracted therefrom ( Figs. 3 and 4 , respectively). [0203] As was the successful cases of u0001 casein and u0002 casein expression under direction to the apoplast, the inventors also showed that u0003 casein is sufficiently expressed in the apoplast of a plant cell and be extracted therefrom ( Fig. 6 ). [0204] Further, the inventors postulated that directed expression to the vacuole and/or apoplast, may have further attributes to post-translation modifications (PTM), such as phosphorylation. [0205] In this regard, co-expression of Fam20 with u0002 casein resulted in phosphorylation of the latter, which was evident by the shift in MW ( Fig. 5 ). Phosphorylation of u0001S1 casein (expressed with a vacuole sorting signal) co-expressed with Fam20 was determined by the inventors, using LC-MS/MS (data not shown). To this end, the expression of Fam20 in conjunction with plant cell compartment sorting signal sequence directing the recombinant Fam20 to the ER and/or apoplast (the nucleic acid sequence encoding the Fam20 further included plant cell compartment sorting signal sequences to the ER and apoplast) was found to be advantageous in relation to proper phosphorylation sites, types of phosphorylation, amount of the phosphorylated recombinant casein protein, or any combination thereof (data not shown). [0206] All in all, the inventors have shown that major casein proteins were successfully manipulated to be expressed in specific sites in the plant cell, e.g., apoplast and/or vacuole. This expression was regulated by selected promoters, as disclosed herein. The inventors have also shown that in order to achieve phosphorylation of the casein proteins, the casein protein is to be directed to the apoplast or vacuole, in conjunction to being co-expressed with a designated exemplary kinase, in a plant cell. Advantageously, the expression of the kinase is directed to the ER and/or apoplast of the plant cell. Posttranslational modification, e.g., phosphorylation of casein(s) is known to be of significance for casein activity and/or functionality. [0207] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS What is claimed is :

1. An artificial DNA molecule comprising : ( a ) a first nucleic acid sequence encoding an apoplast or a vacuole sorting signal sequence ; and ( b ) a second nucleic acid sequence encoding a casein protein , wherein said first nucleic acid sequence and said second nucleic sequence are operably linked .

2. The artificial DNA molecule of claim 1 , wherein said first nucleic acid sequence is set forth in any one of SEQ ID Nos : 1-2 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof .

3. The artificial DNA molecule of claim 1 or 2 , wherein said casein protein is a single casein protein selected from the group consisting of : α - S1 casein , a - S2 casein , u0000 casein , к casein .

The artificial DNA molecule of claim 1 or 2 , wherein said casein protein is selected from the group consisting of : α - S1 casein , α - S2 casein , u0000 casein , к casein , and any combination thereof . ß A.

5. The artificial DNA molecule of any one of claims 1 to 4 , wherein said second nucleic acid sequence is set forth in any one of SEQ ID Nos : 15-28 , a functional analog thereof having at least % sequence homology or identity thereto , and any combination thereof .

6. A plasmid or an expression vector comprising the artificial DNA molecule of any one of claims 1 to 5 .

7. The plasmid or expression vector of claim 6 , comprising a promoter sequence wherein said first nucleic acid sequence and said second nucleic acid sequence are operably linked thereto .

The plasmid or expression vector of claim 7 , wherein said promoter is a constitutive promoter , an inducible promoter , or a combination thereof . .

9. The plasmid or expression vector of claim 8 , wherein said constitutive promoter comprises the nucleic acid sequence set forth in SEQ ID Nos : 29-34 , and 69-76 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof . 30

10. A recombinant protein encoded by the artificial DNA molecule of any one of claims 1 to , or the plasmid or expression vector of any one of claims 6 to 9 .

11. The recombinant protein of claim 10 , comprising the amino acid sequence set forth in any one of SEQ ID Nos : 35-64 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof .

12. The recombinant protein of claim 10 or 11 , comprising the amino acid sequence set forth in any one of SEQ ID Nos : 49-64 .

13. A transgenic or a transfected cell comprising any one of : ( a ) the artificial DNA molecule of any one of claims 1 to 5 ; ( b ) the plasmid or expression vector of any one of claims 6 to 9 ; ( c ) the recombinant protein of any one of claims 10 to 12 ; and ( d ) any combination of ( a ) to ( c ) .

14. A lysate , homogenate , extract , of the transgenic or a transfected cell of claim 13 , or any fraction thereof .

15. The lysate , homogenate , or extract of claim 14 , comprising any one of : ( a ) the artificial DNA molecule of any one of claims 1 to 5 ; ( b ) the plasmid or expression vector of any one of claims 6 to 9 ; ( c ) the recombinant protein of any one of claims 10 to 12 ; and ( d ) any combination of ( a ) to ( c ) .

16. A transgenic plant or plant tissue comprising any one of : ( a ) the artificial DNA molecule of any one of claims 1 to 5 ; ( b ) the plasmid or expression vector of any one of claims 6 to 9 ; ( c ) the recombinant protein of any one of claims 10 to 12 ; ( d ) the transgenic or transfected cell of claim 13 ; and 31 ( e ) any combination of ( a ) to ( d ) .

17. The transgenic plant of claim 16 , being a leafy green or a grain .

18. The transgenic plant of claim 16 or 17 , being lettuce or oat .

19. A host cell , comprising an artificial DNA molecule comprising a first nucleic acid sequence encoding an apoplast or vacuole sorting signal sequence and a casein protein , and a second nucleic acid sequence encoding a mammalian kinase .

20. The host cell of claim 19 , being a cell of a transgenic plant , a transfected plant , or of a tissue derived therefrom .

21. The host cell of claim 19 or 20 , wherein said encoded casein protein and said encoded mammalian kinase are present in the same compartment of said host cell , and wherein said compartment is the apoplast or vacuole of said host cell .

22. The host cell of any one of claims 19 to 21 , wherein said artificial DNA molecule further comprises a third nucleic acid sequence being an internal ribosome entry site ( IRES ) , and wherein said third nucleic acid sequence is positioned between said first nucleic acid and said second nucleic acid of said artificial DNA molecule .

23. The host cell of any one of claims 19 to 22 , wherein said first nucleic acid sequence and said second nucleic acid sequence are operably linked .

24. The host cell any one of claims 19 to 23 , wherein said first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos : 15-28 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof .

25. The host cell of any one of claims 19 to 24 , wherein said second nucleic acid sequence is set forth in SEQ ID Nos : 65-67 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof .

26. The host cell of any one of claims 19 to 25 , wherein said first nucleic acid sequence comprises a sequence set forth in SEQ ID Nos : 1-5 , a functional analog thereof having at least % sequence homology or identity thereto , and any combination thereof .

27. The host cell of any one of claims 19 to 26 , further comprising at least one fourth nucleic acid sequence encoding a site - 1 protease ( S1P ) enzyme . 32

28. The host cell of claim 27 , wherein said at last one fourth nucleic acid sequence is set forth in SEQ ID NO : 68 or a functional analog thereof .

29. The host cell of any one of claims 19 to 28 , wherein any one of said first nucleic acid sequence , said second nucleic acid sequence , said third nucleic acid sequence , and any combination thereof , is integrated in the genome of said host cell .

30. The host cell of claim 27 or 28 , wherein said at least one fourth nucleic acid sequence is integrated in the genome of said host cell .

31. The host cell of any one of claims 19 to 30 , wherein said second nucleic acid sequence further encodes an endoplasmic reticulum ( ER ) sorting signal sequence , an apoplast sorting signal sequence , or both , and wherein said sequence encoding said mammalian kinase is positioned 3 ' to said sequence encoding said ER sorting signal sequence , said apoplast sorting signal sequence , or both .

32. A lysate , homogenate , or extract derived from the host cell of any one of claims 19 to 31 , or any fraction thereof .

33. A composition comprising any one of : ( a ) the artificial DNA molecule of any one of claims 1 to 5 ; ( b ) the plasmid or expression vector of any one of claims 6 to 9 ; ( c ) the recombinant protein of any one of claims 10 to 12 ; ( d ) the transgenic or transfected cell of claim 13 ; ( e ) the lysate , homogenate , extract , or any fraction thereof , of any one of claims 14 , , and 32 ; and ( f ) any combination of ( a ) to ( e ) , and an acceptable carrier .

34. The composition of claim 33 , wherein a protein fraction of said composition consists essentially of the recombinant protein of any one of claims 9 to 11 . 33

35. The composition of claim 33 or 34 , comprising one or more milk - like micelles .

36. The composition of claim 35 , wherein said one or more milk - like micelles being characterized by a structure comprising an outer layer and an inner layer , wherein said outer layer comprises plant recombinant к casein protein and a first plant protein and said inner layer comprises at least one plant recombinant : a - S1 casein protein , α - S2 casein protein , u0000 casein protein , or any combination thereof , and a second plant protein .

37. The composition of claim 36 , wherein at least one of said plant recombinant α - S1 casein protein , α - S2 casein protein , u0000 casein protein , к casein protein , or any combination thereof , comprises at least one post - translational modification .

38. The composition of claim 37 , wherein said at least one post - translational modification is phosphorylation .

39. The composition of any one of claims 33 to 38 , wherein said one or more milk - like micelles being essentially similar to milk micelles in size , Z - potential , or both .

40. The composition of claim 39 , wherein said milk is bovine - derived milk .

41. A composition comprising : ( a ) a first DNA molecule comprising a first nucleic acid sequence encoding an apoplast or a vacuole sorting signal sequence and a second nucleic acid sequence encoding a casein protein ; and ( b ) a second DNA molecule comprising a third nucleic acid sequence encoding a mammalian kinase .

42. The composition of claim 41 , wherein any one of : ( a ) said first nucleic acid sequence is set forth in any one of SEQ ID Nos : 1-5 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof ; ( b ) said second nucleic acid sequence is set forth in any one of SEQ ID Nos : 15-28 , a functional analog thereof having at least 80 % sequence homology or identity thereto , and any combination thereof ; and 34 ( c ) both ( a ) and ( b ) .

43. The composition of claim 41 or 42 , wherein each of said first DNA molecule and said second DNA molecule is integrated in a separate plasmid or expression vector , or both are integrated together in a single plasmid or expression vector .

44. The composition of any one of claims 41 to 43 , wherein said second nucleic acid sequence further encodes an ER sorting signal sequence , an apoplast sorting signal sequence , or both .

45. A method for preparing a transgenic or transfected plant , the method comprising contacting a plant or a plant part with an effective amount of the plasmid or expression vector of any one of claims 6 to 9 , or the composition of any one of claims 41 to 44 .

46. A method for producing a recombinant casein protein or a plurality thereof , the method comprising the steps : ( a ) providing a plant or a plant part comprising the artificial DNA molecule of any one of claims 1 to 4 ; and ( b ) culturing said plant or plant part from step ( a ) such that said recombinant casein protein is encoded by said artificial DNA molecule .

47. The method of claim 46 , further comprising a step preceding step ( a ) comprising producing the plant or plant part comprising said artificial DNA molecule , said preceding step comprises transfecting a plant or a plant part with an effective amount of the plasmid or expression vector of any one of claims 6 to 9 , or the composition of any one of claims 1 to 44 .

48. The method of claim 46 or 47 , further comprising a step proceeding step ( b ) comprising extracting said cultured plant or plant part , thereby obtaining an extract comprising said recombinant casein protein or plurality thereof .

49. The method of claim 48 , further comprising mixing said recombinant casein protein or a plurality thereof , thereby obtaining one or more micelles , optionally wherein said one or more micelles being essentially similar to milk micelles in size , Z - potential , or both , and optionally wherein said milk is bovine - derived milk .

50. An in vitro or ex vivo method for preparing a milk - like micelle , the method comprising mixing the lysate , homogenate , or extract of any one of claims 14 , 15 , and 31 . 35