Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort
A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origi...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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KeAi Communications Co., Ltd.
2020-09-01
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| Series: | Synthetic and Systems Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X20300302 |
| _version_ | 1797204517397200896 |
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| author | Alex J. Spice Rochelle Aw Daniel G. Bracewell Karen M. Polizzi |
| author_facet | Alex J. Spice Rochelle Aw Daniel G. Bracewell Karen M. Polizzi |
| author_sort | Alex J. Spice |
| collection | DOAJ |
| description | A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs. |
| first_indexed | 2024-04-24T08:36:29Z |
| format | Article |
| id | doaj.art-2c14f66d13b440f7990cf46e39759377 |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-04-24T08:36:29Z |
| publishDate | 2020-09-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-2c14f66d13b440f7990cf46e397593772024-04-16T16:18:07ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2020-09-0153137144Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effortAlex J. Spice0Rochelle Aw1Daniel G. Bracewell2Karen M. Polizzi3Department of Chemical Engineering, Imperial College London, London, UK; Imperial College Centre for Synthetic Biology, Imperial College London, UKDepartment of Chemical Engineering, Imperial College London, London, UK; Imperial College Centre for Synthetic Biology, Imperial College London, UKDepartment of Biochemical Engineering, University College London, London, UKDepartment of Chemical Engineering, Imperial College London, London, UK; Imperial College Centre for Synthetic Biology, Imperial College London, UK; Corresponding author. Department of Chemical Engineering, Imperial College London, London, UK.A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.http://www.sciencedirect.com/science/article/pii/S2405805X20300302Cell-free protein synthesisPichia pastorisSynthetic biologyDesign of experiments (DOE) |
| spellingShingle | Alex J. Spice Rochelle Aw Daniel G. Bracewell Karen M. Polizzi Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort Synthetic and Systems Biotechnology Cell-free protein synthesis Pichia pastoris Synthetic biology Design of experiments (DOE) |
| title | Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort |
| title_full | Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort |
| title_fullStr | Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort |
| title_full_unstemmed | Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort |
| title_short | Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort |
| title_sort | improving the reaction mix of a pichia pastoris cell free system using a design of experiments approach to minimise experimental effort |
| topic | Cell-free protein synthesis Pichia pastoris Synthetic biology Design of experiments (DOE) |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X20300302 |
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