Methods for Expression of Recombinant Proteins Using a <i>Pichia pastoris</i> Cell‐Free System

Aw, Rochelle; Spice, Alex J; Polizzi, Karen M.

doi:10.1002/cpps.115

Cited by 10 publications

(10 citation statements)

References 39 publications

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“…Original and unmodified CFE protocols were followed for E. coli (Sun et al, 2013;Sato et al, 2022), S. venezuelae (Moore et al, 2021;Toh et al, 2021) and P. pastoris (Spice et al, 2020;Spice et al, 2022). Luminescence measurements were performed as previously described (Aw et al, 2020). Commercial AAs (RTS Sampler kit-Biotech Rabbit, Germany) were used following the manufacturer's instructions, or individual standards were used as stated within the figure legends.…”

Section: Cfe Reactionsmentioning

confidence: 99%

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Nagappa

Sato

Alam

et al. 2022

Front. Bioeng. Biotechnol.

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Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

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Section: Cfe Reactionsmentioning

confidence: 99%

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Nagappa

Sato

Alam

et al. 2022

Front. Bioeng. Biotechnol.

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“…However, previously published efforts with eukaryotic CFPS have struggled to achieve useful yields in meaningful reaction volumes. In the case of the Hepatitis B core antigen model VLP (HBc), Pichia pastoris and wheat-germ extract (WGE) systems have yielded only 6.4 and 4 μg/mL VLP production, respectively, and neither system was used beyond the microliter scale for HBc [18][19][20] .…”

Section: Expression Of Multisubunit Protein Complexes In Byl With In-...mentioning

confidence: 99%

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2022

Preprint

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Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 uL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

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“…30,32 However, for the problem that the prokaryotic expression system cannot modify the expression products after translation, some researchers regard Pichia pastoris as the host of eukaryotic expression which contributes to the formation of the spatial conformation of the product. 33,34…”

Section: Recombinationmentioning

confidence: 99%

A comprehensive review of oyster peptides: Preparation, characterisation and bioactivities

Hao

Wang

Cao

et al. 2021

Reviews in Aquaculture

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In 1980, bioactive peptides were first described in a study on peptide biosynthesis in the neurointermediate lobe of Xenopus laevis. 1 Since then, bioactive peptides with beneficial physiological properties to living organisms have been widely studied. At present, bioactive peptides, including those obtained from marine and terrestrial environments, are a promising area for development for the international food industry. Marine bioactive peptides have broad potential use in the development of functional foods and as new therapeutics and also play an important role in improving culture efficiency.Oysters, the most cultured shellfish worldwide, are an important marine biological resource to humans. According to statistics reported by the Food and Agriculture Organization of the United Nations (2018; http://www.fao.org/fishe ry/stati stics/ en), global oyster production was around 6.1 million tons. The chemical composition of oysters is (based on dry flesh weight) as follows: protein (39.1%-53.1%); glycogen (21.6%-38.9%); and fat (7.8%-8.7%). 2 Methods commonly used for protein extraction include salting out, enzymatic hydrolysis and organic solvent-or/and pH-based protocols. The most abundant amino acids in oysters are taurine (Tau), glycine (Gly), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu) and proline (Pro). Moreover, since eight essential amino acids account for

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

Cited by 10 publications

References 39 publications

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

A comprehensive review of oyster peptides: Preparation, characterisation and bioactivities

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

Cited by 10 publications

References 39 publications

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

ALiCE®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

A comprehensive review of oyster peptides: Preparation, characterisation and bioactivities

Contact Info

Product

Resources

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ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system