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Publication Number
US-11718856-B2
Publication Date
2023-08-08
Expiration Date
Abstract
A method of in vivo assembly of a recombinant micelle including: introducing a plasmid into a plant cell, wherein: the plasmid includes a segment of deoxyribonucleic acid (DNA) for encoding a ribonucleic acid (RNA) for a protein in a casein micelle, the segment of DNA is transcribed and translated; forming recombinant casein proteins in the plant cell, wherein: the recombinant casein proteins include a κ-casein and at least one of an αS1-casein, an αS2-casein, a β-casein; and assembling in vivo a recombinant micelle within the plant cell, wherein: an outer layer of the recombinant micelle is enriched with the κ-casein, an inner matrix of the recombinant micelle include at least one of the αS1-casein, the αS2-casein, the β-casein.
Core Innovation
The described invention concerns in vivo assembly of a recombinant micelle by co-expressing ruminant casein proteins in a plant cell. The method comprises co-expressing at least two ruminant casein proteins, including a κ-casein and at least one of αS1-casein, αS2-casein, or β-casein, and the κ-casein and the selected casein form the recombinant micelle in the plant cell.
A plant-cell in vivo assembly approach is provided in which recombinant caseins are produced inside the plant cell from a plasmid encoding casein proteins. The recombinant micelles include an outer κ-casein-enriched layer and an inner matrix enriched in the selected αS1-casein, αS2-casein, and/or β-casein.
The description addresses problems associated with producing micelle structures with controlled composition and morphology by assembling recombinant caseins in plant cells. It also indicates plant-derived N-terminal signal peptides, post-translational modification such as phosphorylation and glycosylation, and additional transcription units affecting intracellular calcium and phosphate.
Claims Coverage
The document provides two independent claim sets, covering a method of in vivo assembly of a recombinant micelle and a plant cell configured to co-express the caseins to form the recombinant micelle in the plant cell. Across the independent claims, the inventive subject matter is the co-expression of κ-casein together with at least one of αS1-casein, αS2-casein, or β-casein such that these proteins assemble into a recombinant micelle within a plant cell.
In vivo assembly by co-expressing κ-casein plus αS/β caseins in a plant cell
Co-expressing at least two ruminant casein proteins in a plant cell, wherein the at least two ruminant casein proteins comprise a κ-casein and at least one of an αS1-casein, an αS2-casein, or a β-casein, and wherein the κ-casein and the at least one of the αS1-casein, the αS2-casein, or the β-casein form the recombinant micelle in the plant cell.
Plant cell co-expressing κ-casein plus αS/β caseins to form a recombinant micelle
A plant cell co-expressing at least two ruminant casein proteins, wherein the at least two ruminant casein proteins comprise a κ-casein and at least one of an αS1-casein, an αS2-casein, or a β-casein, and wherein the κ-casein and at least one of the αS1-casein, the αS2-casein, or the β-casein form a recombinant micelle in the plant cell.
Overall, the claim coverage is centered on producing recombinant micelles in vivo by co-expressing κ-casein together with at least one of αS1-casein, αS2-casein, or β-casein in a plant cell so that these caseins assemble into a recombinant micelle in situ. The dependent claims further specify micelle architecture with an outer κ-casein layer and inner matrix composition, assembly location in a plant organelle and optionally the vacuole, a soybean context, and expression via plasmid DNA encoding RNA.
Stated Advantages
The description addresses problems associated with producing micelle structures with controlled composition and morphology by assembling recombinant caseins in plant cells.
Documented Applications
Assessing micelle size and morphology by immunogold labeling and transmission electron microscopy.
Assessing protein composition by HPLC and PAGE.
Monitoring changes in seed mineral-level components such as calcium oxalate-related measures.
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