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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.

Project description:BackgroundAndrogenetic alopecia (AGA) is one of the most common hair loss diseases worldwide. However, current treatments including medicine, surgery, and stem cells are limited for various reasons. Cell-free fat extract (CEFFE), contains various cell factors, may have potential abilities in treating AGA. This study aims to evaluate the safety, effectiveness and the underlying mechanism of CEFFE in treating AGA.MethodsSex hormone evaluation, immunogenicity assay and genotoxicity assay were conducted for CEFFE. In vivo study, male C57BL/6 mice were injected subcutaneously with dihydrotestosterone (DHT) and were treated with different concentration of CEFFE for 18 days (five groups and n = 12 in each group: Control, Model, CEFFELow, CEFFEMiddle, CEFFEHigh). Anagen entry rate and hair coverage percentage were analyzed through continuously taken gross photographs. The angiogenesis and proliferation of hair follicle cells were evaluated by hematoxylin-eosin, anti-CD31, and anti-Ki67 staining. In vitro study, dermal papilla cells (DPCs) were incubated with different concentrations of CEFFE, DHT, or CEFFE + DHT, followed by CCK-8 assay and flow cytometry to evaluate cell proliferation cycle and apoptosis. The intracellular DHT level were assessed by enzyme-linked immunosorbent assay. The expression of 5α-reductase type II, 3α-hydroxysteroid dehydrogenase and androgen receptor were assessed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or/and western blot.ResultsIn CEFFE-treated mice, an increase in the anagen entry rate and hair coverage percentage was observed. The number of CD31-positive capillaries and Ki67-positive cells were increased, suggesting that CEFFE promoted the proliferation of DPCs, modulated the cell cycle arrest, inhibited apoptosis caused by DHT, reduced the intracellular concentration of DHT in DPCs, and downregulated the expression of AR.ConclusionsCEFFE is a novel and effective treatment option for AGA through producing an increased hair follicle density and hair growth rate. The proposed mechanisms are through the DHT/AR pathway regulation and regional angiogenesis ability.

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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