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Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.

ABSTRACT: An "endotoxin-free" E. coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand. However, preparation of the most complex CFPS reagent - the cell extract - remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3) strain. Here we report a streamlined procedure for preparing E. coli cell extract from ClearColi™ using auto-induction media. In this work, the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression. Culturing Clearcoli™ cells in autoinduction media significantly reduces the hands-on time required during extract preparation, and the resulting "endotoxin-free" cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase, an FDA approved leukemia therapeutic. It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E. coli-based extract for CFPS.

SUBMITTER: Hunt JP

PROVIDER: S-EPMC6926305 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.

Hunt J Porter JP Zhao Emily Long EL Soltani Mehran M Frei Madison M Nelson J Andrew D JAD Bundy Bradley C BC

Synthetic and systems biotechnology 20191213 4

An "endotoxin-free" E. coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand. However, preparation of the most complex CFPS reagent - the cell extract - remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3) strain. Here we report a streamlined procedure for preparing E. coli cell extract from ClearColi™ using auto-induction media. ...[more]

PMID: 31890926

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Project description:Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits and biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the extract preparation method affects its functionality. A key aspect of extract performance relevant to many applications is the activity of the native host transcriptional machinery that can mediate protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are variable for different extract preparation techniques, and specifically low in some conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial ?70 promoters is constrained by the rate of transcription in crude extracts, and that processing the extract with a ribosomal runoff reaction and subsequent dialysis alleviates this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common extract preparation process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined and detailed protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing using native transcriptional machinery and will encourage the further proliferation of cell-free gene expression technology for new applications.

Dataset Information

Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.

Publications

Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.

Similar Datasets

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