Improvements to Simplicity and Stability of the Cell‐Free Protein Synthesis Platform to Enable Applications in the Field and the Classroom

Abstract

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.