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Publication Number
US-12227750-B2
Publication Date
2025-02-18
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 disclosed invention relates to an in vivo plant-cell system for assembling recombinant casein micelles. A plasmid encoding kappa-casein and αS1/αS2/β-casein, including plant promoters and N-terminal signal peptides, is introduced into a plant cell to produce recombinant casein proteins and target the recombinant proteins to protein storage vacuoles. Post-translational processing occurs in the plant cell, including signal peptide removal and phosphorylation, to support micelle assembly.
The recombinant micelles form with kappa-casein enriched on an outer layer and αS1/αS2/β-casein positioned in an inner matrix. Mineral availability is described as enhancing micelle assembly through calcium and phosphate, and additional elements include enzymes that modulate intracellular calcium/phosphate to improve micelle formation. The disclosure further describes additional plasmid elements including selectable/screenable markers.
To increase expression of casein proteins in the plant cell, the disclosure suppresses expression of native plant proteins or native plant peptides using RNA interference, including suppressing native seed storage proteins such as glycinin and β-conglycinin. Verification of recombinant micelle formation is described using immunogold labeling and transmission electron microscopy, along with HPLC profiles and relative abundance analysis. Documented construct components are described for Arabidopsis and soybean.
Claims Coverage
The independent claim covers a method for expressing casein protein in a soybean plant by combining casein expression in plant cells, accumulation in a protein storage vacuole, and suppressing expression of a native plant protein or native plant peptide so that casein expression increases. Dependent claims refine which casein proteins are used and which native plant proteins or peptides are suppressed, including RNAi-mediated suppression targeting messenger RNA.
Expressing casein protein in soybean plant cells
Expressing casein protein in a plant cell in a soybean plant.
Accumulating casein protein in a protein storage vacuole
Accumulating casein protein in a protein storage vacuole.
Suppressing native plant protein or peptide to increase casein expression
Suppressing expression of a native plant protein or a native plant peptide in the plant cell, wherein the suppressing expression increases expression of casein protein in the plant cell.
Using a specific bovine casein target
Expressing casein protein that is specified as bovine k-casein, αS1-casein, αS2-casein, or β-casein.
RNAi targeting messenger RNA for suppression
Suppressing expression of the native plant protein or native plant peptide using RNA interference to target the messenger RNA of the native plant protein or the native plant peptide.
Suppressing β-conglycinin
Suppressing expression of a native plant protein or native plant peptide wherein the native plant protein or native plant peptide is β-conglycinin.
Suppressing glycinin
Suppressing expression of a native plant protein or native plant peptide wherein the native plant protein or native plant peptide is glycinin.
Across the independent and dependent claims, the inventive coverage centers on expressing casein protein in soybean plant cells and accumulating it in protein storage vacuoles, while suppressing specific native plant proteins or peptides to increase casein expression, including RNAi-mediated suppression targeting native messenger RNA and focusing suppression on glycinin and β-conglycinin. Dependent claims further refine the casein protein subtype to specific bovine casein forms.
Stated Advantages
Suppressing expression of the native plant protein or native plant peptide increases expression of casein protein in the plant cell.
Documented Applications
Producing recombinant casein micelles using an in vivo plant-cell system, with examples of construct components for Arabidopsis and soybean and verification of micelle formation.
Expressing casein protein in a soybean plant by expressing casein in plant cells, accumulating the casein in a protein storage vacuole, and suppressing expression of native seed storage proteins, including glycinin and β-conglycinin, to increase casein expression.
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