Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

Spice, Alex J; Aw, Rochelle; Bracewell, Daniel G.; Polizzi, Karen M.

doi:10.1016/j.synbio.2020.06.003

Cited by 18 publications

(22 citation statements)

References 59 publications

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“…This is where the components required for protein synthesis are added to the cell lysate. The reaction mix outlined below has been improved using Design of Experiments (DOE) to result in increased protein synthesis (Spice et al, 2020a). Alternatively, a standard reaction mix more closely aligned to that used for CFPS with S. cerevisiae and P. pastoris in other published papers (Aw & Polizzi, 2019;Hodgman & Jewett, 2013;Zhang, Liu, & Li, 2020) can be used.…”

Section: Coupled In Vitro Transcription and Translationmentioning

confidence: 99%

“…The protocol above uses an improved reaction mix that was developed using a Design of Experiments approach (Spice et al, 2020a). Alternatively, it is possible to use the alternative recipe, which was used for the initial development of the system.…”

Section: Of 20mentioning

confidence: 99%

“…Later, the P. pastoris CFPS system was used for the production of virus‐like particles (VLPs), specifically those derived from the model hepatitis B core antigen VLP (Spice et al., 2020b). Most recently, the productivity of the system was increased using a minimized Design of Experiments (DOE) approach to improve the composition of the reaction mix (Spice et al., 2020a). The main effects influencing the productivity of the system were elucidated, and protein synthesis of firefly luciferase and the biopharmaceutical human serum albumin (HSA) was increased by 4.8‐fold and 3.5‐fold, respectively, using the improved reaction mix (Spice et al., 2020a).…”

Section: Commentarymentioning

confidence: 99%

“…Most recently, the productivity of the system was increased using a minimized Design of Experiments (DOE) approach to improve the composition of the reaction mix (Spice et al., 2020a). The main effects influencing the productivity of the system were elucidated, and protein synthesis of firefly luciferase and the biopharmaceutical human serum albumin (HSA) was increased by 4.8‐fold and 3.5‐fold, respectively, using the improved reaction mix (Spice et al., 2020a). Although the composition of the reaction mix was modified in our most recent publication, the methodology for extract preparation and reaction setup was consistent throughout.…”

Section: Commentarymentioning

confidence: 99%

“…There are specific advantages of using a eukaryotic system over the E. coli commercial kits available, including the ability to perform post‐translational modifications, such as disulfide bond formation. The system outlined below is capable of producing 116.2 µg/ml of human serum albumin (HSA; Spice, Aw, Bracewell, & Polizzi, 2020a), a complex biopharmaceutical containing numerous disulfide bonds. These yields exceed the reported GFP production in HeLa (Mikami, Masutani, Sonenberg, Yokoyama, & Imataka, 2006) and RRL systems (Kobs, 2008), and luciferase in CHO Brodel et al., 2014), insect (Ezure et al., 2006) and S. cerevisiae platforms (Gan & Jewett, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell‐Free System

Spice

Polizzi

2020

CP Protein Science

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Cell-free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell-free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell-free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post-translational modifications. Here, we describe methods for conducting cell-free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription-translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike.

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