Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein
Summary
The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.
Abstract
Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.
Introduction
Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems11,12.
The subcellular localization and modification of recombinant proteins depends on the specific signal peptide encoded at the N-terminus, which targets the protein to its final organelle destination during gene expression. Recombinant proteins are designed to localize to the apoplast, endoplasmic reticulum (ER), chloroplast, vacuole, or cytoplasm, based on their unique properties13. For antigens and antibodies, secreted proteins are preferred. Before secretion, proteins progress through ER and Golgi for correct folding and post-translational modifications14,15,16.
After production in plants, recombinant proteins must be purified from plant extracts. Typically, proteins are purified from total soluble plant extracts, which contain abundant intracellular proteins, misfolded and unmodified products, and cytochromes17. To remove impurities, plant extracts undergo extensive fractionation, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-specific affinity chromatography, phytate precipitation, polyethylene glycol precipitation, and adjustment of temperature and pH18,19. Such impurity-removal steps are laborious and can compromise protein quality.
The plant cell compartment beyond the plasma membrane is defined as the apoplast, encompassing the cell wall, intercellular spaces, and xylem20. The aqueous phase is commonly called apoplast washing fluid (AWF). AWF contains molecules secreted by cells to regulate numerous biological progresses like transport, cell wall metabolism, and stress responses21,22. In contrast to TSP, the molecular constituents of AWF are less complex. Extracting recombinant proteins from AWF may avoid contamination by intracellular proteins, misfolded products, and cytoplasmic remnants. Briefly, extraction lipid enters leaves through the stomata under negative pressure, and the AWF is collected by gentle centrifugation23. While isolation of AWF is widely employed to study the biological progress inside apoplast24,25,26,27, it has not been exploited in plant-based production systems.
Improving plant productivity and obtaining high purity target proteins are central challenges for plant production systems28. Typically, proteins are purified from total soluble plant extracts containing large amounts of intracellular proteins, misfolded and unmodified products, and cytochromes29, which require great costs for subsequent purification30. The use of AWF protein extraction methods for the extraction of exogenous recombinant proteins secreted into apoplast can effectively improve the homogeneity of recombinant proteins and reduce the cost of protein purification. Here, we use a signal peptide PR1a to enable exogenous protein secretion into the apoplast space and introduce a detailed method for recombinant protein purification from the AWF of N. benthamiana.
Protocol
Representative Results
Discussion
Using plants to produce therapeutic proteins has expanded quickly in recent years1,2,3,4,5,6. Protein-encoding genes are cloned into expression vectors and delivered into plant tissues via agroinfiltration. After production by plant cells, proteins are purified for downstream applications. Typically, recombinant proteins are …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by the Youth Innovation Promotion Association CAS (2021084) and the Key Deployment Project Support Fund of the Three-Year Action Plan of the Institute of Microbiology, Chinese Academy of Sciences.
Materials
| 100 mesh nylon fine mesh yarn | Guangzhou Huayu Trading limited company | hy-230724-2 | For AWF protein extraction |
| 50 ml centrifuge tubes | Vazyme | TCF00150 | For centrifuge |
| ClonExpress II one step cloning kit | Vazyme | C112-02 | For clone |
| FastDigest Sac1 | NEB | R3156S | For clone |
| FastDigest XbaI | NEB | FD0685 | For clone |
| ImageJ 1.5.0 | / | / | For greyscale analysis |
| Large filter | Thermo Fisher | NEST | For filtering |
| Laser scanning confocal microscopy | Leica SP8 | For subcellular localization | |
| Ni sepharose excel | Cytiva | 10339806 | For protein purification |
| Precast protein plus gel | YEASEN | 36246ES10 | For SDS-PAGE |
| Small filter | Nalgene | 1660045 | For filtering |
| SuperSignal West Femto Substrate Trial Kit | Thermo Fisher | 34076 | For western blotting |
| Triton X-100 | SCR | 30188928 | To configure the extraction buffer |
| Type rotaryvang vacuum pump | Zhejiang taizhouqiujing vacuum pump limited company | 2XZ-2 | For vacuum infiltration |
| Vertical mixer | Haimen Qilinbel Instrument Manufacturing limited company | BE-1200 | For mixing |
| β-mercaptoethanol | SIGMA | BCBK8223V | To configure the extraction buffer |
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