Efficient Purification of RuBisCO from Complex Cellular Material and leaf Lysates using Liquid-Liquid Phase Separation

Technology

A solution containing RuBisCO is prepared from one of three sources: plant cells, host prokaryotic or eukaryotic cells expressing recombinant RuBisCO large (RbcL) and small (RbcS) subunits, or a cell-free expression system producing RbcL and RbcS. A RuBisCO linker protein is then added to the solution under low-salt conditions. The linker protein contains at least two RuBisCO recognition domains separated by a flexible peptide linker and binds to at least two separate molecules of RuBisCO, inducing phase separation and forming a dense RuBisCO-linker precipitate. The precipitate is resuspended in a high-salt buffer to disrupt the linker-mediated network, releasing RuBisCO, which is then isolated and purified in its native form (8 RbcL and 8 RbcS subunits).  

Problem Addressed

RuBisCO is a highly attractive plant protein for human nutrition because it offers a complete essential amino acid profile, excellent digestibility, is tasteless and colorless when pure, and is highly abundant - comprising up to 50% of leaf protein. The mass of Rubisco harvestable from fast growing plants can be greater than the protein yield of existing grain crops on an equivalent land area. However, its commercial use has been limited by major challenges: extraction from plant leaves require large-scale biomass processing, and extraction is complicated by the need to preserve the native structure while removing chlorophyll and bitter-tasting compounds. Current methods are labor-intensive, costly, and prone to denaturation and browning, resulting in low yields and poor product quality that limit scalability and consumer acceptance. This technology addresses the need for a scalable, cost-effective strategy for extracting highly pure RuBisCO by leveraging a novel LLPS-based purification inspired by natural biochemical features of the protein, facilitating functional protein recovery for sustainable food and biotechnology markets. While proof of concept has been successfully demonstrated in microbial systems, this approach holds strong potential for application to plant leaf extraction.

Advantages

• Enables scalable, cost-effective RuBisCO purification by using LLPS instead of multi-step chromatography, reducing time and expense
• Preserves RuBisCO’s native structure and activity under mild, reversible conditions, avoiding denaturing or browning during extraction and purification
• Improves purity and sensory quality by selectively removing chlorophyll and bitter-tasting compounds
• Supports versatile and sustainable production from both plant biomass and recombinant systems