Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

Abstract

During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

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

“…The first reported in vitro artificial incorporation of carboxy radio-labeled amino acids into proteins from rat liver cells extracts took place in 1948, 145 and more generally, since the early 1950s, 146 it has been shown that disrupted cells from both eukaryotes 147 or prokaryotes 148,149 are still capable of synthesizing proteins. Based on these observations, "cell-free translation" systems have been developed and used to obtain minute amounts of labeled proteins for identification or characterization in the laboratory setting.…”

“…In general, these systems are derived from cells engaged in a high rate of protein synthesis, and commercially available systems exist for various applications including ribosome display and unnatural amino acid incorporation. 146 The development of cell-free systems depends upon both qualitative and quantitative considerations. Qualitative control may be achieved through use of insect cells, 153,154 or HeLa cells, 155 to overcome improper protein folding or glycosylation.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Typical cell‐free expression systems include rabbit reticulocyte, wheat embryo, and Escherichia coli cell extract enriched with energy constituents and free amino acids (Wiegand, Lee, Ostrov, & Church, 2019). For instance, a bacterial cell‐based CFPS system embodies the bacteria lysate, DNA, and energy modules (creatine phosphate, glucose‐6‐phosphate, phosphoenolpyruvate, glucose) all of which could be optimized to obtain higher efficacy (Chong, 2014). Therefore, such supple system with its efficiency being eminently dependent on active cytoplasmic extracts could be applied as a striking tool for protein production in an uncomplicated batch‐format reaction in vitro (Caschera, 2017).…”

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

“…Sophisticated protocols of bacterial cell-free protein synthesis system have been established as coupled transcription-translation systems with exogenous DNA (7,8). Due to advanced developments, such as a continuous-flow system that provides reaction solutions with essential substrates and energy sources (9) and the PURE (protein synthesis using recombinant elements) system that is reconstituted by purified ribosomes and well-known translation-related factors (10,11), the cellular machinery of protein synthesis can now be prepared and reconstituted from various organisms in the laboratory or obtained from commercial suppliers (3,12).…”