Applications Of Cell-free Protein Synthesis Research Articles

A robust Escherichia coli cell-free expression toolbox driven by sigma factors

The development of various expression systems leads to wide and exciting applications of cell-free protein synthesis (CFPS) in synthetic biology. However, the limited regulatory transcription elements still restrict the applications. This work presents a set of orthogonal expression components with various expression features in the Escherichia coli-based cell-free systems. These expression systems were based on the endogenous sigma factors from E. coli and heterologous sigma factors from Bacillus subtilis that naturally recognize specific promoter sequences with different strength. Sigma factors and their specific promoters were combined and screened to function orthogonally between each other in E. coli-based cell-free systems. Additionally, several promoters were confirmed constitutive expression in cell-free systems, which can be used as the primary transcription in the regulatory cascades for gene circuits, getting rid of the bacteriophage expression system. Promoters were performed and orthogonally regulated by sigma factors in varying expression strength, which enlarges the expression elements for cell-free systems. The sigma factor toolbox contributes to the assembly of more complex synthetic genetic circuits in cell-free systems and could constitute an exciting expansion in synthetic biology.

The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications.

The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy.

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

Cell-Free Protein Synthesis Containing Disulfide Bonds and Its Application to Protein Engineering

The cell-free protein synthesis system, which is sometimes called in vitro expression, has been remarkably improved in the past decade. However, proteins containing disulfide bonds could not been produced as active forms using the conventional cell-free system because it was carried out under reduced conditions. We have demonstrated that modification of the reaction conditions, i.e., no addition of reducing reagent, removal of the reducing reagent from the E. coli S30 extract by dialysis, and the addition of some molecular chaperons, enabled the correct folding of such proteins as Burkholderia cepacia lipase, Streptomyces antibioticus phospholipase D, Phanerochaete chrysosporium manganese peroxide (MnP), and artificial antibodies (scFv and Fab). As an application of cell-free protein synthesis, we have developed a novel high-throughput protein library construction system by combining the amplification of a single DNA molecule by means of PCR with a cell-free protein expression system. This completely cell-free system is termed SIMPLEX (single-molecule-PCR-linked in vitro expression). By applying SIMPLEX, we have achieved (1) inversion of the enatioselectivity of the bacterial lipase, (2) improvement in the H2O2 stability of the fungal MnP, and (3) enhancement of the binding affinity of anti-human-serum-albumin scFv.

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA Asp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.