Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

Abstract

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

Reactivity of human antisera to codon optimized SARS-CoV2 viral proteins expressed in Escherichia coli

“…Cloning and validation can be completed within two weeks for an E. coli expression system [ 17 ]. However, the absence of posttranslational modification represents a major advantage and defect of E. coli expression systems [ 18 19 20 ], and codon bias may lead to truncated products because of premature termination of protein translation [ 21 22 ]. To overcome this issue, we prepared chemically synthesized viral protein coding sequences in which E. coli preferred codons were used.…”

Escherichia coli as an antibody expression host for the production of diagnostic proteins: significance and expression

“…This issue was resolved in this instance by using two mutant strains of BL21(DE3) -C41(DE3) and C43(DE3) [49] which were less prone to the cytotoxic effects. Whilst it can be effective, screening for optimal protein synthesis conditions can be time-consuming, high-cost, and laborious [50].…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Deletion of the prfA gene encoding RF1 is a frequent strategy to improve the ncAA incorporation efficiency in vivo and in vitro ( Johnson et al, 2011 ). Cell extracts from such strains have shown numerous advantages ( Smolskaya et al, 2020 ). However, RF1 is essential for translation termination of over 300 genes in the genome, and the direct deletion of prfA could seriously affect cell growth before cell extract preparation, even leading to cell death ( Chin, 2017 ).…”