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Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology
Published on: February 25, 2019
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Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform
Chiara Heide1,2,3, Gizem Buldum1, Ignacio Moya-Ramirez1,3
1Department of Chemical Engineering, Imperial College London, London, United Kingdom.
Frontiers in Bioengineering and Biotechnology
|February 19, 2021
Summary
Researchers developed a cell-free protein synthesis (CFPS) platform using Chinese hamster ovary (CHO) cells. Supplementing accessory proteins GADD34 and K3L significantly boosted protein production yields, enabling broader mammalian CFPS applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- Cell-free protein synthesis (CFPS) offers a powerful tool for protein production.
- Developing robust mammalian CFPS systems, particularly from Chinese hamster ovary (CHO) cells, presents challenges in maintaining translational activity.
- Existing methods often suffer from low yields due to the deactivation of the translational machinery.
Purpose of the Study:
- To establish and optimize a functional cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells.
- To identify and implement strategies to overcome low yields and translational machinery deactivation in CHO CFPS.
- To provide a guideline for developing user-friendly and efficient mammalian CFPS systems.
Main Methods:
- Stepwise development and optimization of cell cultivation and lysate preparation from CHO cells.
- Introduction of accessory proteins GADD34 and K3L to prevent phosphorylation-induced deactivation of the translational machinery.
- Comparison of different strategies for incorporating GADD34 variants to assess impact on yield and implementation.
Main Results:
- Optimized protocols for cell culture and lysate preparation were established.
- Supplementation with GADD34 and K3L proteins increased turbo Green Fluorescent Protein (tGFP) yield by up to 100-fold.
- Different strategies for accessory protein addition showed varying trade-offs between yield enhancement and ease of use.
Conclusions:
- The developed CHO CFPS platform provides a reliable system for mammalian protein synthesis.
- The addition of accessory proteins is crucial for enhancing yield and stability in mammalian CFPS.
- The optimized protocols facilitate broader adoption and application of CHO-derived cell-free protein synthesis.
