Cell‐free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just‐add‐water cell‐free system

Abstract

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

“…Cell-free expression systems, freeze-dried on paper or other supports, have been recently used to create robust biosensors and in situ bioproduction reagents capable of room temperature storage for extended periods of time (5–7, 20). Upon rehydration, such freeze-dried CFE systems support transcription-translation reactions enabling biosensing or bioproduction programmed by the supplied synthetic gene circuits.…”

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

“…9–11 Recently, cell-free systems have been shown to enable high yielding synthesis of, for example, active metalloenzymes, 12–14 and allow rapid prototyping of biological circuits and pathways. 15–18 For natural products research, CFPS offers flexibility over cell-based expression because the reaction conditions can be more easily controlled: for example, atypical precursors can simply be fed in during the course of a reaction 19,20 or made in situ using all enzymes from a biosynthetic gene cluster. Biosynthetic pathways could potentially be engineered for higher expression without the need to modify a native host strain.…”

The growing impact of lyophilized cell-free protein expression systems

“…Because standard cell-free extracts must be used quickly or stored frozen, and because preparation of the extracts requires several hours, dependency on such has the potential to bottleneck execution of rapid protein expression cycles. Ondemand protein expression systems, that transcribe and translate from rapidly-produced DNA 44 and facilitate activity assessment without product purification, enable parallel synthesis cycles to be executed in as little as 3 to 4 h. 10 …”

“…The resultant protein was produced at high yields and was immediately available for screening without purification. 10 In this article, we will highlight technological advances and future applications enabled by lyophilized, on-demand protein expression systems.…”