Improved Cell-Free RNA and Protein Synthesis System

Abstract

Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…160 The recombinant nature of the various components allows for an increase in productivity and global efficiency, as elongation factors, ribosome recycling factors, and release factors can be individually adjusted. 161 In the future, it is possible that cell-free translation systems may achieve a high quantitative mode of protein production, and certainly these systems will be of particular interest in the production of recombinant vaccines.…”

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

“…A mirror-image ribosome is also a key stepping stone towards building a fully mirror-image cell (“ D. coli ”) 60 . The E. coli ribosome is ideal for this project because it has been extensively characterized (including detailed protocols for its efficient in vitro assembly 62 ), and it is active without rRNA modifications 63 (which would be difficult to produce in mirror-image). These 65 proteins represent an ideal set with lengths from 38 to 890 residues (21 30S subunits, 33 50S subunits, and 11 key translation accessory factors) (Table S1).…”

Conceptual and methodological advances in cell-free directed evolution

“…A method known as iSAT could enable engineering of the ribosome for greater functionality [48*], improving translation in all cell-free evolution schemes. Introduction of molecular chaperones to cell-free translation systems increases protein yield [49] and prevents aggregation of proteins and their mutants by promoting proper folding [50]; similarly, nanogel artificial chaperones are emerging as an alternative means to help mitigate library losses [51]. …”