From Cells to Cell-Free Platforms: A Comprehensive Overview

Introduction

Cells have molecular machinery to make proteins, which are essential tools for cellular differentiation and growth. DNA contains genetic instructions needed to make different proteins. Biotechnological tools allow transferring the specific gene from one organism to another, i.e., bacterial cells, which are cultivated on an artificial medium and used to isolate the target proteins. For instance, Escherichia coli and Saccharomyces cerevisiae are mostly used for the production of recombinant human insulin. Moreover, other protein-based medicines, such as vaccines or antibodies, have also been developed using recombinant gene technology for therapeutic application in humans. During in vivo recombinant protein expression, once DNA is transferred, bacterial cells are cultured in liquid culture, at either pilot scale or industrial scale. The desired proteins are isolated from the cells through a complex process, protein purification, and utilized for therapeutic purposes. Some other cellular pathways can be utilized to carry out biochemical reactions to develop various products, including biofuels, bioplastics, etc. However, cell-based systems are highly complex and cannot be used directly due to the maintenance of cell viability and the controlled environment. In vivo recombinant technology for protein manufacturing also has drawbacks, including protein aggregation, degradation, and DNA loss, as well as several time-consuming experimental procedures, such as DNA cloning in the vector, DNA transformation, and overexpression of the target protein (Yoshihiro Shimizu et al., 2006). With the help of recent developments in synthetic biology, it is now possible to design and create biological systems that have the capacity to carry out intricate molecular processes at all levels, from the molecular to the general, to extracellular (Laohakunakorn, 2020). In contrast, another strategy is the use of a Cell-free System (CFS), in which molecular processes are, activated efficiently using crude cellular extracts, instead of whole cells, supplemented with multitudes of accessory ingredients, including amino acids, transcription factors, cofactors, translational regulators, nucleotides, and energy substrates (ATP, GTP). CFS, generally known as Cell-free gene expression (CFE), is an in vitro technique that retains protein synthesis and other native biochemical reactions independently from an intact cell. Hence, simplifying the complicated interconnections encountered when working in an intact cell (Kelwick et al., 2020; Koo et al., 2020). A purified bacterial RNA polymerase is employed in the first step of CFE for mRNA synthesis, whereas a cellular extract supplies the ribosomes and every other necessary element required for protein expression. E. coli, yeast, wheat germ, rabbit reticulocytes, insect cells, Chinese hamster ovary (CHO) cells, and even human cell cultures are just a few of the many different cell-free systems that have been developed (Zemella et al., 2015). CFE offers an unparalleled high degree of controllability and simplicity over the biochemical reactions for gene expression and metabolism by overcoming the restriction of maintaining cell viability.

CFS can prototype cellular function and utilized as a key research technique in biology and biotechnology for more than 120 years. Furthermore, over the past two decades, the multitude of cell-free platforms have undergone tremendous development, and increases in the yield of bioactive recombinant DNA (rDNA) proteins have led to the application of these systems for the in vitro expression of proteins. Recent development in cell-free platforms has to speed up the use of this technology, which can satisfy many of the requirements of biochemical research, clinical purposes, paper-based diagnostic devices, industrial application, biosensors, etc. Therefore, this chapter aims to elaborate on the rapidly advancing field of cell-free platforms. This chapter describes cell-free systems obtained from different model organisms, with their advantages and limitations.